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Form-determining function of the genes required for the assembly of the head of bacteriophage T4
Abstract
The physiological manifestation of conditional lethal mutations in the genes involved in the formation of the T4 head have been re-examined. If the product of genes 20 or 40 is defective, open-ended tubular structures (polyheads) are synthesized. Infection with mutants in gene 22 leads to the formation of polyheads, about 70% of which consist of two or more concentric layers (multilayered polyheads). Cells infected with mutants in gene 21 and 24 yield a mixed burst of capsid-like particles (τ-particles) and polyheads, many of which have hemispherical caps.
... The blood sample (20 ml each bleed) was drawn via aseptic venipuncture into tubes containing ethylenediamine tetraacetic acid (EDTA) and the volunteers were bled once a week throughout the study period the P. falciparum was being cultured to provide fresh erythrocytes. Before use, the blood samples were centrifuged at 3000 rpm for 10 minutes, the plasma and buffy coat discarded and the pelleted cells resuspended IeJSME 2011: 5 (2): [18][19][20][21][22][23][24][25][26][27][28] in 15 ml RPMI 1640 + gentamycin. The cell suspension was again centrifuged, supernatant discarded and the cells washed again. ...
... SDS-PAGE analysis was performed using a discontinuous system as described by Laemmli et al. 26 . Samples of the uninfected and infected erythrocytes from different ABO blood groups were thawed and denatured by heating at 95°C for 5 minutes in Laemmli sample buffer containing 0.06% Tris-HCl, pH 6.8, 10% glycerol, 0,02% SDS. ...
... For each gel, a positive control (horseradish peroxidase) and a negative control (soybean trypsin inhibitor) were included. After electrophoresis, the SDS-PAGE gels were stained according to manufacturer's instructions using the IeJSME 2011: 5 (2): [18][19][20][21][22][23][24][25][26][27][28] GelCode ® Glycoprotein Staining Kit (Pierce, USA) to detect glycoproteins. Figure 1 shows the SDS-PAGE patterns obtained for uninfected and infected red blood cell extracts from the 4 healthy human subjects with different ABO blood groups viz. 1 subject with blood group AB (AB c and AB t ), 2 subjects with blood group A (A1 c and A1 t ; A2 c and A2 t ) and 1 with blood group B (B1 c and B1 t ). Figure 2 shows the SDS-PAGE patterns obtained for uninfected and infected red blood cell extracts from 3 other healthy subjects, 1 more subject with blood group B (B2 c and B2 t ) and 2 with blood group O (O1 c and O1 t ; O2 c and O2 t ). ...
... The extent of the redundancy is about 2% of the genome size (106). (107), incomplete processing (cleavage) of head proteins (123,198); may function to protect phage DNA following injection ( (61,122), protease for cleavage of head structural proteins (123, 151, 196) 104 am, ts L Head assembly (61,122), major protein of assembly core in r particle; degraded (120,121,123,151,170,196) (61,122); interacts with host in organization of capsid subunits (41,76,77,191,192,218) Q DNA-binding protein (3,4); translational repressor of its own synthesis (163) [Q] ...
... The extent of the redundancy is about 2% of the genome size (106). (107), incomplete processing (cleavage) of head proteins (123,198); may function to protect phage DNA following injection ( (61,122), protease for cleavage of head structural proteins (123, 151, 196) 104 am, ts L Head assembly (61,122), major protein of assembly core in r particle; degraded (120,121,123,151,170,196) (61,122); interacts with host in organization of capsid subunits (41,76,77,191,192,218) Q DNA-binding protein (3,4); translational repressor of its own synthesis (163) [Q] ...
... The extent of the redundancy is about 2% of the genome size (106). (107), incomplete processing (cleavage) of head proteins (123,198); may function to protect phage DNA following injection ( (61,122), protease for cleavage of head structural proteins (123, 151, 196) 104 am, ts L Head assembly (61,122), major protein of assembly core in r particle; degraded (120,121,123,151,170,196) (61,122); interacts with host in organization of capsid subunits (41,76,77,191,192,218) Q DNA-binding protein (3,4); translational repressor of its own synthesis (163) [Q] ...
... Infection with a phage containing a mutation in a group A gene generally results in the accumulation of a characteristic aberrant head-related particle in the infected cell (40,81). Gene 23 codes for the major head protein (41,67,101), and infection with an amber mutant in this gene fails to produce head-related structures, whereas temperature-sensitive mutants grown at nonpermissive temperatures result in the formation of deformed heads (41,81). ...
... Infection with a phage containing a mutation in a group A gene generally results in the accumulation of a characteristic aberrant head-related particle in the infected cell (40,81). Gene 23 codes for the major head protein (41,67,101), and infection with an amber mutant in this gene fails to produce head-related structures, whereas temperature-sensitive mutants grown at nonpermissive temperatures result in the formation of deformed heads (41,81). Certain missense mutations in gene 23 result in the random formation of petite (39) and giant head particles (38). ...
... Defects in the internal protein genes are not lethal, although these proteins are required for maximal efficiency ofthe assembly of the phage particles (11,103). The absence of the internal proteins causes the accumulation of some polyheads, and at least one internal protein, IPHI, is required for the formation of multilayered polyheads that arise in gene 22-defective infected cells (81). In addition, some internal proteins are required for the assembly of giant phage particles (32). ...
... MORPHOGENETIC PATHWAYS Phage T4 General considerations. At least 22 gene products are required for normal T4 head assembly ( Fig. 2) (86,93,210). Seven of these, 20, 21, 22, 23, 24, 31, and 40, are known as group A genes (93,211) and, together with genes I, II, and III, (25,301), which code for three internal proteins, are known to be involved in the morphogenesis of the competent prohead. ...
... Infection with T4 phage containing a mutation in the group A genes (genes 23, I, II, and III excepted) results in the intracellular accumulation of aberrant head-related particles (93,210,322). Gene 23 codes for the major head protein (94,177,292), and infection of nonsuppressing cells with a nonsense mutant in this gene results in a failure to produce head-related structures. Genes I, II, and III code for three internal proteins (25) that form, in association with gp22, the assembly core of T4 (301). ...
... Mutations in either gene 20 or gene 40 result in the accumulation of single-layered open tubes called polyheads (94,206,210,322). Polyheads are composed of gp23 and the internal proteins gp22, gpIII, gpII, and possibly gpI (211,212,301). ...
... Infection of cells with nonsense or ts mutations in genes for structural proteins often led to accumulation of morphogenetic intermediates in virus assembly. Laemmli, Kellenberger and their coworkers in Geneva had characterized the capsid-related structures accumulating in cells infected with mutants defective in head assembly ( [8]). As a graduate student at Caltech, and working with Bob Edgar and later Bill Wood, I had identified the intermediates in tail assembly and tail fiber assembly [9] [10]. ...
... Using this gel system he was able to show that T4 heads were assembled from more than six different proteins, and identify them as the products of specific T4 genes [1]. Since the phenotype of mutant-infected cells as visualized in the electron microscope had already been determined, he was able to define the pathway of T4 head morphogenesis ( [8]). One striking feature was the presence of defined proteolytic cleavages of a number of the structural proteins, within the organized lattice, coupled to the stages of icosahedral lattice transformation. ...
One of the most transformative experimental techniques in the rise of modern molecular biology and biochemistry was the development of high resolution Sodium Dodecyl Sulfate (SDS) poly acrylamide gel electrophoresis, which allowed separation of proteins – including structural proteins – in complex mixtures according to their molecular weights. Its development was intimately tied to investigations of the control of virus assembly within phage-infected cells. The method was developed by Ulrich K. Laemmli working in the virus structural group led by Aaron Klug at the famed Medical Research Council Laboratory for Molecular Biology (LMB) at Cambridge, UK. While Laemmli was tackling T4 head assembly, I sat at the next bench working on T4 tail assembly. To date, Laemmli’s original paper has been cited almost 300,000 times. His gel procedure and our cooperation allowed us to sort out the sequential protein-protein interactions controlling the viral self-assembly pathways. It is still not fully appreciated that this control involved protein conformational change induced by interaction with an edge of the growing structure. Subsequent efforts of my students and I to understand how temperature sensitive mutations interfered with assembly were important in revealing the intracellular off-pathway aggregation processes competing with productive protein folding. These misfolding processes slowed the initial productivity of the biotechnology industry. The article below describes the scientific origin, context and sociology that supported these advances in protein biochemistry, protein expression, and virus assembly. The cooperation and collaboration that was integral to both the LMB culture and phage genetics fields were key to these endeavors.
... The very first step in phage T4 morphogenesis is the assembly of gp20 portal protein into a dodecamer on the cytoplasmic surface of the inner E. coli membrane 9,26 . Early genetic studies showed that the portal dodecamer nucleates head assembly by interacting with the major capsid protein gp23 and the major scaffolding core protein gp22 27,28 . This nucleating nexus then goes on to build the capsid shell around the scaffolding core which also incorporates several other core proteins including the capsid maturation protease gp21 29 . ...
... Early genetic studies have established that the unique portal vertex is essential to nucleate capsid assembly 27,28 . The same vertex, later, is also essential for other key viral assembly processes including capsid maturation, genome packaging, and neck/ tail attachment 39 . ...
Large biological structures are assembled from smaller, often symmetric, sub-structures. However, asymmetry among sub-structures is fundamentally important for biological function. An extreme form of asymmetry, a 12-fold-symmetric dodecameric portal complex inserted into a 5-fold-symmetric capsid vertex, is found in numerous icosahedral viruses, including tailed bacteriophages, herpesviruses, and archaeal viruses. This vertex is critical for driving capsid assembly, DNA packaging, tail attachment, and genome ejection. Here, we report the near-atomic in situ structure of the symmetry-mismatched portal vertex from bacteriophage T4. Remarkably, the local structure of portal morphs to compensate for symmetry-mismatch, forming similar interactions in different capsid environments while maintaining strict symmetry in the rest of the structure. This creates a unique and unusually dynamic symmetry-mismatched vertex that is central to building an infectious virion.
... The relative molecular weight (MW) of the purified enzyme was estimated by SDS-PAGE, which was performed using a SE600 Ruby Complete Dual Cooled Vertical Slab Unit (GE Healthcare Technologies, USA) with 12.5% (w/v) acrylamide gel according to Laemmli's method [26]. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. ...
... The above assay mixture was incubated at 40°C for 30 min followed by measuring the changes in the concentration of substrates and products by HPLC [26]. The hydrolysis rate (%) was calculated using the formula: (S 1 -S 2 ) × 100/S 1 , where S 1 represents the initial concentration of substrates before enzymatic hydrolysis and S 2 represents the residual concentration of substrates after enzymatic hydrolysis. ...
Naringinase plays a rather important role in reducing the bitterness of juice by hydrolyzing naringin. A novel extracellular naringinase was purified from Aspergillus oryzae 11250 cultured in the presence of orange peel. A 26.78-fold purification rate was achieved by salt-induced precipitation, followed by anion-exchange and gel filtration chromatography with 32% recovery and specific activity of 2194.62 units per mg protein (U/mg). The optimum pH and temperature for naringinase activity were 5.0 and 45 °C, respectively. This enzyme was stable at 30 °C for 5 h. The Km and Vmax of naringinase toward naringin determined by Lineweaver-Burk method were 1.60 ± 0.13 mM and 126.21 ± 5.52 μmol/(min mg), respectively. The enzyme activity was inhibited completely by Ag⁺ at 10 mM. Naringinase is capable of hydrolyzing naringin, neohesperidin, and some other glycosides. A supplement of 6 U/mL of this naringinase in citrus juice sufficiently removed naringin to relieve the bitterness of citrus juice. These properties make the enzyme an ideal candidate for commercial application in the debitterization of orange juice.
... For protein analysis, isolated transformants were cultivated in sixwell microtiter plates until mid-logarithmic growth phase, 4 9 10 7 (Laemmli et al., 1970). Gels were stained using colloidal Coomassie Brilliant Blue G-250 (Dyballa & Metzger, 2009) ...
In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycis-tronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-a-bisabolene production of 7.95 mg L À1 from a single open reading frame, 18.13 fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.
... Cleaned samples of 100 mg of each were dissolved in 1.0 ml of sample loading buffer (Tris-HCl, Glycerol, bromophenol blue, β-mercaptoethanol and SDS) by boiling for 30 min. All samples containing about 150 µg of proteins were electrophoresed in 10% polyacrylamide gel of thickness 1.5 mm according to the method described by Laemmeli et al. [29] . All the extracted proteins from the silk glands of different stages were separately loaded on to the gel. ...
The mango leaf webber, Orthaga exvinacea makes silken webs and galleries during its larval stage. Silk is produced from the silk gland of this insect. Quantitative and qualitative analysis of proteins from the silk glands of last three larval stages and pre-pupae were conducted. Significant differences were observed among different stages. SDS-PAGE of proteins from anterior, middle and posterior regions of silk glands of final instar larvae revealed that the posterior region had several proteins than the other two regions of silk glands. Anterior region had no visible protein bands. The proteins of molecular weights 269 kDa, 251 kDa, 175 kDa, 125 kDa and 66 kDa were common to posterior region of silk glands, web and cocoon of the mango leaf webber. The regenerated protein obtained from the web had a molecular weight of 125 kDa, which may be a fibroin like web and cocoon except 125 kDa molecular weight protein may be sericins, which are the members of a glue protein family. The regenerated liquid silk had two UV absorption peaks at 214 nm and 272 nm. The anti-UV properties of silk protein of Orthaga exvinacea can be exploited in cosmetic industry. Introduction Silks are protein polymers produced by various species of insects and spiders. It is used for different purposes which include construction of protective shelter, structural support for developing eggs and egg sacs, reproduction, foraging and dispersal [1-3]. In the insect order Lepidoptera, Bombycidae and Saturniidae are the two important families utilized for commercial silk production. These two families are characterized by low silk production in early larval stage and enormous silk production in the last instars. During the production of cocoon, around 20% of the body mass is converted to silk. The tasar silkworm, Antheraea mylitta has the highest silk producing capacity among all silk spinning insects [4]. Silk glands in insect larvae are ectodermal in origin, which is anatomically and physiologically divided into three distinct regions, viz., anterior, middle and posterior regions [5]. The anterior, middle and posterior region of silk glands of B. mori larvae consist of 200 cells, 255 cells and 520 cells respectively [6]. The morphogenesis [7] of silk glands are completed within eight days after egg laying. Silk is a natural fiber, up to 95% of which is composed of fibroin and sericin and the remaining 5% constituted by other proteins, waxes, fats, salts and ash [8]. Fibroin is the major structural protein formed by two different polypeptide chains, i.e., heavy (H) and light (L) chains of molecular weights 350 kDa and 25 kDa respectively. These two chains are linked together by di-sulfide bonds [3, 9]. A glycoprotein, P25, has also been associated with H-L complex by non-covalent interactions [10-13]. In B. mori, fibroin was identified as the product of the posterior region of the silk gland, whereas sericin is produced in the middle region that serves as silk reservoir [14]. In the posterior region of silk gland the concentration of fibroin protein is around 12-15% by weight, while fibroin and sericin is 30% by weight in the middle region of silk gland [15]. Sericin accounts for 20-30% by weight of B. mori cocoon fibers [16, 17]. Sericins include sericin P (150 kDa), sericin M (400 kDa) and sericin A (250 kDa) identified in the distal, central and anterior of the middle regions of silk gland respectively [18]. In the lumen of gland silk proteins accumulate as a concentrated gel. During spinning, the liquid silk is subjected to stress and elongation, thus forming silk fibers. The unique properties of silk are mainly due to long storage of silk in the form of gel followed by its rapid conversion to silk filament [19]. Tasar, muga, eri, fagaria and shashe silks are produced by the non-mulberry silkworms A. mylitta, A. assama, Philosamia ricini, Attacus atlas and Gonometa postica respectively.
... Cytoplasmic and nuclear extracts were prepared from liver homogenates using a specific lysis buffer and protease inhibitors [35]. The supernatant fraction was collected and stored in aliquots at -80 °C for further analysis. ...
Background:
Cirrhosis is an important health problem characterized by a significant change in liver parenchyma. In animals, this can be reproduced by an experimental model of bile duct ligation (BDL). Melatonin (MLT) is a physiological hormone synthesized from serotonin that has been studied for its beneficial properties, including its antioxidant potential.
Aim:
To evaluate MLT's effects on oxidative stress, the inflammatory process, and DNA damage in an experimental model of secondary biliary cirrhosis.
Methods:
Male Wistar rats were divided into 4 groups: Control (CO), CO + MLT, BDL, and BDL + MLT. MLT was administered (20 mg/kg) daily beginning on day 15 after biliary obstruction. On day 29 the animals were killed. Blood samples, liver tissue, and bone marrow were collected for further analysis.
Results:
BDL caused changes in biochemical and histological parameters and markers of inflammatory process. Thiobarbituric acid (0.46 ± 0.01) reactive substance levels, superoxide dismutase activity (2.30 ± 0.07) and nitric oxide levels (2.48 ± 0.36) were significantly lower (P < 0.001) n the groups that received MLT. DNA damage was also lower (P < 0.001) in MLT-treated groups (171.6 ± 32.9) than the BDL-only group (295.5 ± 34.8). Tissue damage and the expression of nuclear factor kappa B, interleukin-1β, Nrf2, NQO1 and Hsp70 were significantly lower in animals treated with MLT (P < 0.001).
Conclusion:
When administered to rats with BDL-induced secondary biliary cirrhosis, MLT effectively restored the evaluated parameters.
... Interleukin-1β (IL-1β) was expressed in pg/mL, as described previously [24]. SOD, inducible nitric oxide synthase (iNOS), and activation of nuclear factor kappa B (NF-kB) were assessed using Western blot analysis, as described by Laemmli, et al. [25] and Towbin, et al. [26]. Nuclear extracts were prepared from lung homogenates and the supernatant was collected and stored at -80 °C. ...
The objective was to assess the antioxidant effect of melatonin (MLT) on liver and lung tissues of animals with bile duct ligation (BDL)-induced hepato-pulmonary syndrome (HPS). A model of BDL-induced biliary cirrhosis was used in male Wistar rats. Results suggest that MLT has an antioxidant effect on liver and lung tissues in animals with BDL-induced HPS by higher activity of antioxidant enzymes in the group HPS treated with MLT and the histological analysis of lung parenchyma showing decreased damage in this same group, including other analysis described below.
... To identify the different protein expression based on molecular weight, the SDS-PAGE was adopted to separate the protein 287 . SDS Gel is composed of two different layers which are stacking gel to line up all the protein samples loaded on the gel, and separating gel. ...
Coat protein complex I (COPI) vesicles coated with the heptameric complex coatomer mediate retrograde cargo trafficking from Golgi to endoplasmic reticulum as well as intra-Golgi transport. Whether paralogous subunits of coatomer have different functions is currently unclear. In this thesis, we reveal distinct roles of paralogous coatomer subunits γ1-COP and γ2-COP during the neuronal differentiation of mouse pluripotent cells. Following genome editing experiments, our work shows that γ1-COP specifically facilitates neurite extension and underlines a paralogue-specific function of the COPI pathway and offers evidence for the role of COPI coated vesicles in neuronal polarization. Furthermore, to explore the mechanism of γ1-COP specific functions during pluripotent cells differentiation into neurons, the combination proximity-dependent biotinylation with affinity purification and mass spectrometry (AP-MS) was applied to analyze whether the interactome of γ-COPs reveals paralogue-specific cargos or regulators. In the light of label free quantification (LFQ) analysis, various neurogenesis-related proteins were significantly enriched in the γ1-COP interactome indicating that γ1-COP may preferentially traffic such proteins during the process of pluripotent cells neuronal differentiation. Zusammenfassung Mit dem heptameren Komplex-Coatomer beschichtete Coat Protein Complex I (COPI) -Vesikel vermitteln den retrograden Frachthandel von Golgi zum endoplasmatischen Retikulum sowie den Intra-Golgi-Transport. Ob paraloge Untereinheiten des Coatomers unterschiedliche Funktionen haben, ist derzeit unklar. In dieser Arbeit zeigen wir unterschiedliche Rollen der paralogen Coatomer-Untereinheiten γ1-COP und γ2-COP während der neuronalen Differenzierung pluripotenter Mauszellen. Nach Experimenten zur Bearbeitung des Genoms zeigen unsere Arbeiten, dass γ1-COP die Neuritenverlängerung spezifisch erleichtert, eine paralogspezifische Funktion des COPI-Signalwegs unterstreicht und Hinweise auf die Rolle von COPI-beschichteten Vesikeln bei der neuronalen Polarisation liefert. Um den Mechanismus der γ1-COP-spezifischen Funktionen während der Differenzierung pluripotenter Zellen in Neuronen zu untersuchen, wurde die kombinationsnäherungsabhängige Biotinylierung mit Affinitätsreinigung und Massenspektrometrie (AP-MS) angewendet, um zu analysieren, ob das Interaktom von γ-COPs Paralog zeigt. spezifische Ladungen oder Regulierungsbehörden. Im Lichte der Analyse der markierungsfreien Quantifizierung (LFQ) wurden verschiedene Neurogenese-verwandte Proteine im γ1-COP-Interaktom signifikant angereichert, was darauf hinweist, dass γ1-COP solche Proteine während des Prozesses der neuronalen Differenzierung pluripotenter Zellen bevorzugt transportieren kann.
... Tissue extracts and immunoprecipitated complex were solubilized in Laemmli sample buffer (150 mm Tris-HCl (pH 6.8), 1.67 % 2-mercaptoethanol, 1.67 % sodium dodecyl sulfate, 10% glycerol and bromophenol blue) and boiled for 5 min (Laemmli et al., 1970). Equal amounts of samples were resolved in 10 % SDS polyacrylamide gels. ...
Diabetic retinopathy is the most common cause of vision loss among diabetic patients. Although hyperglycemia produces retinal oxidative stress in long-standing diabetes, the pathogenesis mechanism is unknown. The Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in cell responses against oxidative damage. We used adult Long Evans rats where diabetes was induced by streptozotocin. Normal and treated rats were sacrificed at 7, 20, and 45 days after streptozotocin injection. We analyzed Nrf2 and Keap1 expression in retinal homogenates, cytoplasmic, and nuclear retinal fractions. Normal retina showed Nrf2 expression in all retina nuclear layers. We found a transitory decrease of Nrf2 mRNA and protein expression at 7 and 20 days after the streptozotocin injection that recovered later on: moreover, the protein level increased after 45 days. Keap1 immunoprecipitation revealed similar levels as Nrf2 in normal and diabetic rat retinas, indicating that the diabetic condition did not lead to dissociation of the Keap1-Nrf2 complex. Indeed, glutathione levels and superoxide dismutase activity were not altered in the treated rat retinas. These results do not support oxidative stress in the retina shortly after diabetes induction.
... The detection of bacteriophage lytic enzymes was done after equilibration of the proteins where the resulting focused bands were separated according to size using modifi ed classical methods (SDS-PAGE) and other reducing agents discovered by [33,35,36]. The proteins' bands migration were plotted on 2D and 3D graphs for distance and density properties to predict the size of the proteins and similarity in the phage and bacterium, [37] reported that the calibration curve of SDS-MW size standards plotting the molecular weight towards mobility values estimated from migration distance in gels. ...
Introduction: The invasion of bacteriophage on the associated host bacterium depends on their receptors' orientation that adsorb them to cell surface. During phage replication a valuable number of proteins acts as lytic enzymes for host puncher at the beginning of the infection and other for burst after lytic cycle compilation. Accordingly, the proteomic relationship among phage and bacterium proteins could easily be studied by their protein profi les analysis. Objective: To detect bacteriophages functional enzymes during lytic cycle. Methods: The isolation and identifi cation of Escherichia coli and their parasitic T7 phage group was done using bacterial culture and common plaque assay techniques. The investigations and protein-protein interactions' assays were inveterate by proteins profi le of phage and bacterium using Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) to fi nd out their molecular weights, where the scaled location of each mobile band was compared to the standards of identifi ed proteins weights in the molecular ladder. Thereafter, Protein model's assembly and bands migration was done by computer analytical software. Results: Mobilization of the phage' proteins inside the Two Dimensions (2D) gel ranged between 60 and 12 kDa where a model of 4 main bands with molecular weights of (46, 35, 24 and 14 kDa) is corresponded to the host ones, where pure 9 bands with molecular weight ranged between 96-24 kDa. The computational model analysis showed common shared molecular masses of 47, 34 and 16 kDa on plot area of the phage and the bacterium. Model interpretation confi rmed that proteins ranged from 47.7 to 34.3 kDa resembles 43.3% of whole phage's proteins that assembled the capsid head and the coil, while the molecular weight mass of 22.5 formed the tail's proteins. The lytic enzymes' molecular weight was ranged between 18-14 kDa according to the function of the enzyme. The study revealed that the 34 kDa band has the common shared peak between T7 phage group and associated Escherichia coli host. Conclusion: Functional models of analysed proteins during phage assembly, ensures lytic enzymes are built in the capsid head and the lysozyme in the tail, they facilitate the enzymatic decay for bacterial host. This enzymatic function is related to the lytic cycle of the bacteriophages and their phenomenon in employing the bacterial DNA in proteins manufacturing during their replication inside host.
... At suitable pH and ionic strength, the capsid protein of cowpea chlorotic mosaic virus (CCMV) forms multi-shelled capsids 93,94 , likely instigated by the opposing charge carried by the inner and outer surfaces of the capsid shells. Mutated forms of the major bacteriophage T4 prohead component, gp22, result in formation of multi-layered proheads 95 , and the protein will assemble into multi-layered tubes when other critical prohead components are inactivated or absent 96 . Non viral proteins can also assemble in this fashion. ...
During a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.
... The SDS-PAGE of the crude proteins was performed in polyacrylamide gel to fractionate proteins according to their molecular weight by using mass standard. This will be a useful indicator to help in determining the similarity between strains of fungus [16]. ...
Use of nanoparticles (NPs) in several commercial products has led to emergence of novel contaminants of air, soil and water bodies. The NPs may exhibit greater ecotoxicity due to nano-scale dependent properties over their bulk counterparts. The present investigation explores the effect of in vitro supplementation of TiO 2 , silica and silver NPs on radial growth and ultrastructural changes in the hyphae and spores of two mushroom genera, Ganoderma lucidum and Volvariella volvaceae. A concentration dependent decrease in radial growth on NP amended potato dextrose agar medium was recorded. However, in comparison to control , there was decrease in radial diameter on supplementation with TiO 2 NPs while an increase was recorded for silica and silver NPs amendments as compared to their bulk salts at same concentrations after 48 h of incubation. Optical microscopy studies showed decrease in the number of spores while increase in spore diameter and thinning of hyphal diameter on NPs supplementation. Scanning electron microscopy analysis of fungal growth showed presence of deflated and oblong spores in two fruiting strains of Ganoderma while Volvariella exhibited decreased sporulation. Further, hyphal thinning and branching was recorded in response to NP amendments in both the test mushrooms. Enhancement of protein content was observed on NP compared to bulk supplementation for all cultures, concentrations and hours of incubation except for TiO 2 NPs. Likewise, bulk and NP supplementations (at 100 mg L À1) resulted in enhanced laccase activity with occurrence of laccase specific protein bands on SDS-PAGE analysis. ARTICLE HISTORY
... For protein analysis, isolated transformants were cultivated in 6-well microtiter plates until mid-logarithmic growth phase, 4x10 7 cells were harvested by centrifugation (3000 xg, 3 min) and pellets were resuspended in 200 μL 2xSDS sample buffer (60 mM Tris pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.01% (w/v) bromophenol blue). Equal protein amounts were separated in 10% polyacrylamide gels during Tris-glycine-SDS-PAGE [87]. Gels were stained using Colloidal Coomassie Brilliant Blue G-250 [88] or were subjected to Western blotting (semi-dry method, Trans-Blot Biorad, blotting buffer: 25 mM Tris, 192 mM glycine, 20% methanol) on 0.45 μm Protran Nitrocellulose membranes (Amersham). ...
Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.
... The molecular weight of the purified enzyme was determined through SDS-PAGE analysis according to the protocol described by Laemmli et al. [20]. It was conducted using a discontinuous polyacrylamide gel (10% w/v) containing sodium dodecyl sulfate using the BIORAD apparatus. ...
Enzymatic hydrolysis of naringin by the action of naringinase is one of the standard practices adopted in the citrus fruit juice industry for debittering. In the present study, a submerged fermentation condition was optimized for producing naringinase from Aspergillus niger van Tieghem MTCC 2425. As per Placket–Burman design, pH (3–5), incubation temperature (26–30 °C), and inducer concentration (12–18 g·L−1) were the most important factors influencing the naringinase production. Naringin from citrus waste was used as an inducer. A rotatable central composite design was employed on these three variables and the numerical optimization predicted that fermentation at 29.8 °C, pH 4.7, and inducer concentration of 14.9 g L−1 would yield a maximum naringinase activity of 545.2 IU g−1. During partial purification, ion exchange chromatography led to a 9.92-fold increase in enzyme activity resulting a specific activity of 5460 IU g−1 with an activity recovery of 17%. As reflected by SDS–PAGE profile, the partially purified naringinase showed the molecular weight bands of 10–20, 65, and 80 kDa, respectively. The purified form of enzyme showed optimum stability at pH 5 and 50 °C. The naringinase activity was completely retained up to 150 days when stored at 4 °C.
... Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli et al. [26]. After electrophoresis, the proteins were colored with 0.25% Coomassie Brilliant Blue G-250 (Sigma, Milan, Italy). ...
Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.
... However, the abnormally high levels of HK97 major capsid protein achieved via plasmid expression may allow normal initiation to be bypassed by the formation of a pseudo-initiator composed entirely of the HK97 major capsid protein (i.e., a pentamer surrounded by five hexamers). This is in stark contrast to what occurs during phage T4 infections, where the T4 portal is essential for the initiation of the assembly of normal T4 capsid shells [21]. In the absence of the portal, T4 capsid assembly is initially blocked, but at later times after infection, the major capsid protein reaches high levels and spontaneously assembles into long tubes of capsid protein and not into capsidlike particles [22]. ...
The portal proteins of tailed bacteriophage and Herpesvirus capsids form dodecameric rings that occupy one capsid vertex and are incorporated during the assembly of capsid precursors called procapsids or proheads. Portals are essential and serve as the pore for DNA transit and the site of tail attachment; however, bacteriophage HK97 capsid proteins assemble efficiently without a portal when expressed from plasmids. Following portal co-expression, portals were incorporated into about half of the proheads that were made. In the absence of active capsid maturation protease, uncleaved proheads formed dimers, trimers, and tetramers of proheads during purification, but only if they had portals. These appeared bound to membrane-like fragments by their portals and could be disaggregated by detergents, supporting a role for membranes in their formation and in capsid assembly. The precursors to prohead oligomers were detected in cell extracts. These were able to bind to Octyl-Sepharose and could be released by detergent, while uncleaved proheads without portal or cleaved proheads with portal did not bind. Our results document a discrete change in the HK97 portal's hydrophobicity induced by cleavage of the procapsid shell in which it is embedded. Additionally, we detected an increase in the rate of expansion induced by the presence of a portal complex in cleaved HK97 proheads. These results suggest that portals and capsids influence each other's conformation during assembly. The formation of prohead oligomers also provides a rapid and sensitive assay for identification and analysis of portal incorporation mutants.
... Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed, as described by [28], in a BioRad Mini-Protean gel apparatus (BioRad). For zymograms, 0.2% autoclaved and lyophilized L. lactis NZ9000 cells were included in 16% polyacrylamide gels for the detection of bacteriolytic activity, as previously described [29]. ...
The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.
... Sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein based on the size (Laemmli et al. 1970 was used. Analysis of the blots was performed using the Image Studio Software (LICOR) ...
COPI vesicles mediate retrograde Golgi to ER transport and intra-Golgi transport within the secretory pathway. COPI vesicles are Golgi derived vesicles, which are coated with heptameric complex known as coatomer. Coatomer is made up of seven subunits alpha, beta, beta prime,delta, gamma,zeta and epsilon-COP. COPI coatomer is recruited to the Golgi membrane with the help of small GTPases Arf to stimulate the vesicle formation and capture cargo proteins to deliver them to the targeted membrane. In mammals the -COP subunit has two paralogs. Whereas in the related COPII system, paralogs of coat subunits were shown to expand the cargo repertoire of COPII vesicles, no paralog specific function had been described to date for COPI paralog subunits. In this work we have set out to investigate such specific functions. Guided by RNAseq data in differentiating mES that showed that Copg1 is upregulated during neuronal differentiation. We generated Copg1 and Copg2 KO P19 pluripotent cells and studied if they could differentiate. Strikingly Copg1 KO cells fail to form tight embryonic bodies (EBs) and to form long neurites though they could differentiate into neurons. This work shows for the first time strong evidence for paralog-specific function of the COPI pathway in mammalian cells.
... Molecular weight of FP was determined by 10% SDS-PAGE [38] using 66-6.5 kDa protein ladder. Absorbance spectra of GNP, FP, FP-GNP was acquired between 200 and 700 nm using a UV-visible spectrophotometer (Shimadzu, Lambda 25, Perkin Elmer; Japan) [39,40,41]. Secondary structure of native protein and change in conformation was determined in a 0.1 cm quartz cuvette for CD spectropolarimeter at 25 • C (Jasco J-810) [42,43]. ...
Aim:
To provide multilayered combination therapies encompassing nanoparticles and organic peptides and to assess their efficacy in the treatment of arthritis.
Materials & methods:
Fish oil protein (FP) was isolated from fish oil glands and tagged with spherical gold nanoparticles (GNPs). Tagged GNPs were encapsulated in DPPC liposomes (FP-GNP-DPPC) and characterized.
Results & conclusion:
FP increased the hydrophilicity of GNP, while encapsulation of FP-GNP within liposomes increased the hydrophobicity. In vitro release studies of FP-GNP-DPPC exhibited sustained release of FP in simulated synovial fluid. FP-GNP-DPPC injected into intra-articular joints of rats displayed anti-osteoarthritic effects in osteoarthritic rat model. This is the first study to report the anti-osteoarthritic activity of FP and DPPC encapsulated FP-GNP liposomes.
... Eif4e (eukaryotic translation initiation factor 4E) functions as a protooncogene; its product has been suggested to regulate expression of proteins that are crucial for cell cycle progression, cell survival, and motility. A growing body of evidence implicates this translational factor in cell transformation, tumorigenesis, or tumor progression, e.g., in case of prostate cancer, lymphomas, CML, or lung cancers [83]. ...
Tyrosine kinases play crucial roles in cellular development and tumorigenesis. Tyrosine kinase inhibitors (TKIs) are effective and widely used drug molecules in targeted cancer therapies. Altered expressions of protooncogenes and tumor suppressor genes after DMBA (7,12-dimethylbenz[a]anthracene) treatment have been described as early markers of tumor induction; however their tissue-specific effects remain still unclear. Our study was aimed at examining the short-term possible antineoplastic and chemopreventive effects of a TKI compound (imatinib mesylate) on a DMBA-induced mouse tumor model. In addition, we also investigated the tissue-specific expressions of Hras, Kras, Myc, and Trp53 genes in the brain, bone marrow, spleen, liver, abdominal lymph nodes, thymus, lungs, and kidneys, respectively. 24 hours after the imatinib mesylate injection, we observed significant Kras downregulation in the bone marrow and lung of the DMBA-treated mice. Moreover, the mRNA expression of Myc was also found to be decreased significantly in the spleen. Interestingly, while Trp53 expression was significantly increased in the lung, it was decreased in the other tissues. However, there was also a tendency in the decreased Myc level in the bone marrow, brain, kidneys, lungs, and lymph nodes and in the decreased Hras level in the bone marrow, kidneys, and lungs, although no significant differences were observed. Our findings indicate rapid tissue-specific impact of imatinib mesylate on DMBA-induced gene expression in vivo, supporting the chemopreventive potential of imatinib mesylate in cancer.
... Briefly, SDS-PAGE buffers (monomer, resolving, stacking, treatment and tank buffer) were prepared according to Laemmli et al., (1970). The Monomer solution was prepared by dissolving 29.29g acrylamide and 0.89g N-N-bismethylene acrylamide in 100ml distilled water. ...
In this work, the cellulolytic enzymes that extracted from endophytic T. harzianum strain SYA.F4 were evaluated via submerged fermentation mode by using a variety of agro-lignocellulosic wastes such as rice straw, wheat straw, wood chips and sugarcane bagasse that individually used as cellulases inducer and nitrogen/carbon sources. Among these wastes, the sugarcane bagasse that treated by using HPAC method produced the highest cellulases activities (β-glucanase; 95.5, β-glucosidase; 52.3 and CMCase; 18.6 IU/ml) at maximum biomass (20.5g/l) after 15 days of the incubation period. The statistical Plackett-Burman and Box-Behnken experimental designs were applied to study the effects of the medium ingredients and the culturing conditions on the SYA.F4 mass weight. Via low-cost production line, the maximum biomass production, the highest CMCase, β-glucosidases and β-glucanases activities were detected as 159.86 g/l, 237.7, 314.6 and 429.1 IU/ml respectively after ~80 hr of the incubation period. Finally, the endophytic T. harzianum strain SYA.F4 may be used as a potential safe candidate for the production of cellulolytic enzymes that applied as an anti-phytopathogens agent in vitro.
... Proteins were separated by Tris-glycine-SDS-PAGE. 63 Gels were stained using Colloidal Coomassie Brilliant Blue G-250 64 or subjected to Western blotting on nitrocellulose membranes (Amersham, GE Healthcare) prior to immunodetection using either a HRPlinked mouse-anti-StrepII monoclonal antibody (1:5000, in TBS including 5% (w/v) BSA and milk powder (blocking buffer), Iba Life Science, 2−1509−001) or a rabbit-anti-gLuc antibody (1:5000 in blocking buffer, NEB, E8023S) followed by a HRP-linked goat-antirabbit IgG (1:10 000 in blocking buffer, Agrisera AB, AS09602). Visualization was performed using the Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and the Fusion Fx7 CCD-camera (peQLab GmbH, VWR). ...
... Cells were resuspended in 2× protein sample buffer (60 mM Tris pH 6.8, 4% (w/v) SDS, 20 % (v/v) glycerol, 0,01% (w/v) bromophenol blue) and proteins were separated by Tris-glycine-SDS-PAGE using 12%-PA-Gels (44). Separated proteins were stained using colloidal Coomassie Brilliant Blue G-250 (45) or after subjection to Western blotting on nitrocellulose membranes (Amersham, GE Healthcare) were analyzed by immunodetection using a HRP-linked rabbit-anti-GFP antibody (1:5000, in 1xTBS containing 5% (w/v) BSA and milk powder, Thermo Scientific, A10260) and Pierce™ ECL Western Blotting substrates (Thermo Scientific). ...
Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene ex-
pression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions
of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy.
... Protein detection was performed by chemiluminescence using a commercial ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK). The density of the specific bands was quantified using Scion Image software (Scion Corp., Frederick, MD, USA) (29,30). ...
Introduction:
Intestinal ischemia-reperfusion (I/R) injury may cause cell and tissue damage, reaching also other organs such as the liver. Because of the involvement of free radicals in I/R injury, treatment options with antioxidants have been studied and tested.
Objective:
To evaluate the effect of glutamine (Gln) in the liver of animals with intestinal I/R injury.
Methods: We used 20 male Wistar rats divided into four groups: sham-operated (SO); glutamine + sham-operated (G+SO); intestinal ischemia-reperfusion (I/R); glutamine + intestinal ischemia-reperfusion (G+I/R). The superior mesenteric artery was clamped for 30 minutes and reperfused for 15 minutes. Gln (25 mg/kg/day) diluted in 1 ml of saline was administered intraperitoneally on the two days before I/R induction.
Results:
Levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), lipid peroxidation (LPO) and expressions of interleukin-6 (IL-6) and nuclear factor kappa B (NF-kB) showed a significant reduction in the G+I/R group as compared with the I/R group. The activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) showed an increase in the G+I/R group as compared with the I/R group.
Conclusion:
Pretreatment with Gln reduced oxidative, tissue damage and showed a decrease expression of inflammatory mediators.
... At suitable pH and ionic strength, the capsid protein of cowpea chlorotic mosaic virus (CCMV) forms multi-shelled capsids 93,94 , likely instigated by the opposing charge carried by the inner and outer surfaces of the capsid shells. Mutated forms of the major bacteriophage T4 prohead component, gp22, result in formation of multi-layered proheads 95 , and the protein will assemble into multi-layered tubes when other critical prohead components are inactivated or absent 96 . Non viral proteins can also assemble in this fashion. ...
During a proteolytically-driven maturation process, the orthoretroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at near-physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.
... The fraction was desalted and concentrated by centricon (MWCO 3k, Millipore). Purified NKCT1 was checked for homogeneity by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis 15 . ...
Background & objectives: Increased severity of osteoarthritis (OA) and adverse side effects of its treatment
led to the search for alternative therapies. It was previously reported that snake venom protein toxin
Naja kaouthia cytotoxin 1 (NKCT1) and gold nanoparticle (GNP) individually have potential against
excremental arthritis. In this study, we analyzed the protective activity of GNP conjugated protein toxin
NKCT1 (GNP-NKCT1) against experimental OA.
Methods: Gold nanoparticle conjugation with NKCT1 (GNP-NKCT1) was done and its physiochemical
properties were studied. OA was induced in male albino rats by intra-articular injection of bacterial
collagenase and treatment was done with NKCT1/GNP-NKCT1/standard drug (indomethacin).
Physical parameter (ankle diameter), urinary markers (hydroxyproline, glucosamine, pyridinoline,
deoxypyridinoline), serum and synovial membrane pro-inflammatory markers [tumour necrosis factoralpha
(TNF-α), interleukin-1β (IL-1β), IL-17, vascular endothelial growth factor (VEGF)] and matrix
metalloproteinase 1 (MMP1) were measured. Joint histopathology and scanning electron microscopy
imaging of articular cartilage surface were also done.
Results: Physical parameters, urinary markers, serum and synovial membrane pro-inflammatory
makers and MMP1 were increased in arthritic rats and significantly restored after GNP-NKCT1/
NKCT1 treatment. Joint histopathology and scanning electron microscopy imaging of articular cartilage
surface also indicated the protective effect of GNP-NKCT1 against inflammatory response and cartilage
degradation in osteoarthritic rats.
Interpretation & conclusions: In this study restoration of the arthritic markers and bone degradation
by GNP-NKCT1 treatment indicated the anti-osteoarthritic property of GNP-NKCT1. Further studies
need to be done to confirm these findings.
... The fraction was desalted and concentrated by centricon (MWCO 3k, Millipore). Purified NKCT1 was checked for homogeneity by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis 15 . ...
Background & objectives: Increased severity of osteoarthritis (OA) and adverse side effects of its treatment led to the search for alternative therapies. It was previously reported that snake venom protein toxin Naja kaouthia cytotoxin 1 (NKCT1) and gold nanoparticle (GNP) individually have potential against excremental arthritis. In this study, we analyzed the protective activity of GNP conjugated protein toxin NKCT1 (GNP-NKCT1) against experimental OA.
Methods: Gold nanoparticle conjugation with NKCT1 (GNP-NKCT1) was done and its physiochemical properties were studied. OA was induced in male albino rats by intra-articular injection of bacterial collagenase and treatment was done with NKCT1/GNP-NKCT1/standard drug (indomethacin). Physical parameter (ankle diameter), urinary markers (hydroxyproline, glucosamine, pyridinoline, deoxypyridinoline), serum and synovial membrane pro-inflammatory markers [tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-17, vascular endothelial growth factor (VEGF)] and matrix metalloproteinase 1 (MMP1) were measured. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface were also done.
Results: Physical parameters, urinary markers, serum and synovial membrane pro-inflammatory makers and MMP1 were increased in arthritic rats and significantly restored after GNP-NKCT1/NKCT1 treatment. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface also indicated the protective effect of GNP-NKCT1 against inflammatory response and cartilage degradation in osteoarthritic rats.
Interpretation & conclusions: In this study restoration of the arthritic markers and bone degradation by GNP-NKCT1 treatment indicated the anti-osteoarthritic property of GNP-NKCT1. Further studies need to be done to confirm these findings.
... Expression of Nrf2/Keap1, NQO1, SOD and HSP70, GRP78, and ATF-6 in intestine and liver Western blot analysis was performed on nuclear and cytosolic extracts prepared from intestine and liver homogenates as previously described (Laemmli et al. 1970;Towbin et al. 1992). Briefly, the supernatant was collected and stored at −80°C in 200-μL aliquots until further analysis. ...
Intestinal ischemia and reperfusion (I/R) causes cellular and tissue damage to the intestine and remote organs such as the liver. Increased production of ROS and nitric oxide and dysregulation of cytoprotective enzymes may be involved in intestinal I/R. The aim was to evaluate the protective effects of glutamine on the intestine and liver of rats with intestinal I/R injury. Twenty male Wistar rats (300 g) were divided into four groups: sham-operated (SO), glutamine + SO (G + SO), I/R, and glutamine + I/R (G + I/R). Occlusion of the SMA for 30 min was followed by 15-min reperfusion. Glutamine (25 mg/kg/day) was administered once daily 24 and 48 h before I/R induction. Blood and tissue of were collected for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, histopathological analysis, immunohistochemistry of IL-1β and TNF-α, thiobarbituric acid reactive substance (TBARS) and nitric oxide, Nrf2/keap1, superoxide dismutase (SOD), NADPH quinone oxidoreductase1 (NQO1), inducible nitric oxide synthase (iNOS), heat shock protein (HSP70), glucose-regulated protein 78 (GRP78), and activating transcription factor 6 (ATF-6) by western blot. Statistic analysis by ANOVA-Student-Newman-Keuls test (mean ± SE) significantly was p < 0.05. Tissue damage, AST, ALT, IL-1β, TNF-α, TBARS, NO, Keap1, iNOS, GRP78, and ATF-6 expression were significantly lower in the G + I/R group as compared to the I/R group. Expression of Nrf2, SOD, NQO1, and HSP70, was significantly higher in the G + I/R group as compared to I/R group. Pre-treatment with glutamine provided protection against oxidative damage in the intestine and liver in an experimental model of intestinal I/R.
... Chromatography fractions, isolated toxins and the recombinant inhibitor were monitored by SDS-PAGE (Laemmli et al., 1970). Molecular masses were estimated by the interpolation of a linear logarithmic curve of relative molecular mass of standard proteins (14.4e116 kDa, unstained low range standard, Thermo Scientific) versus the distance of migration of the analyzed proteins in the gel. ...
Phospholipase A2 inhibitors (PLIs) are important targets in the search and development of new drugs. This study aimed at evaluating the potential of an alpha-type phospholipase A2 inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake in its recombinant form (rBaltMIP) to complement the conventional antivenom therapy. Biochemical experiments showed that rBaltMIP presented pI 5.8 and molecular masses of ∼21 kDa by SDS-PAGE and 19.57 kDa by MALDI/TOF MS. After tryptic peptides sequencing, the results were compared with other PLIs available in databases, showing 100% identity between rBaltMIP and its native inhibitor BaltMIP and from 92% to 96% identity with other inhibitors. Myotoxic activities of BthTX-I and BthTX-II toxins were measured via plasma CK levels, showing myotoxic effective concentrations (EC50) of 0.1256 μg/μL and 0.6183 μg/μL, respectively. rBaltMIP neutralized the myotoxicity caused by these two toxins up to 65%, without promoting primary antibody response against itself. Nevertheless, this recombinant PLI was immunogenic when standard immunization protocol with Freud's adjuvant was used. In paw edema assays, EC50 of 0.02581 μg/μL and 0.02810 μg/μL, respectively, were observed with edema reductions of up to 40% by rBaltMIP, suggesting its use as an additional antivenom. In addition, myotoxicity neutralization experiments with the myotoxin BthTX-I showed that rBaltMIP was more effective in inhibiting muscle damage than the conventional antivenom. Thus, considering the severity of envenomations due to Bothrops alternatus (Rhinocerophis alternatus) and the low neutralization of their local effects (such as myotoxicity) by the current antivenoms, rBaltMIP is a promising molecule for the development of novel therapeutic strategies for clinical applications.
... The soluble protein was separated by 10 % SDS-PAGE. One-dimensional SDS-PAGE as described by Laemmli et al. (1970) with some minor amendments, such as 5 % acrylamide, was used for the stacking gel, while 12 % acrylamide was used for the resolving gel. The protein 1 3 separation analysis was performed on a BioRad Mini-Gel electrophoresis apparatus following staining gel with silver (AgNO 3 ). ...
The whitefly Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) is an important pest of cotton in many countries including Pakistan. Even though it has gained resistance for various registered insecticides such as the neonicotinoid insecticide thiamethoxam, the cellular changes occurring in plants during whitefly infestation and resistance are poorly understood. In particular, the proteomic analysis of interaction between cotton plant and devastating whiteflies is poorly understood. To reveal the physiological response as well as the molecular interaction during the incursion of B. tabaci, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D SDS-PAGE) following liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was conducted, combined with in silico protein characterizations and protein–protein interaction following validation of LC–MS/MS results by quantitative real-time PCR. The shotgun proteomic analysis of cotton leaves infested with whitefly resulted in the identification of 38 proteins, and 24 under control (non-infested) conditions. Of them, 11 proteins were identified as shared proteins, 13 were differentially induced in non-infested conditions, and 27 were induced only under whitefly-infested conditions. The major proteins like MAP kinases, COBRA-like protein family, and NBS disease resistance protein were expressed under infested conditions; while the heat shock protein 90 and cyclic nucleotide-gated ion channel 5-like major proteins were expressed under whitefly non-infested conditions. The current manuscript presents new insights into the mechanism of whitefly infestation and our understanding of the roles of COB and COBLs in host-pest interaction. To the best of our knowledge, this is the first ever report to reveal the role of several proteins, including COB and COBLs in host-pest leaf interaction.
Keywords: Cotton plant Leaf tissues LC–MS/MS COB-like proteins Cell morphogenesis
In studying bacteriophage T4—one of the basic models of molecular biology for several decades—there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device—one of the most complex multiprotein components—has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.
Diabetic retinopathy (DR) is a complication of diabetes. Several studies have implicated oxidative stress as a fundamental factor in the progression of the disease. The nuclear factor erythroid-2-related factor 2 (Nrf2) is one of the main regulators of redox homeostasis. Glia Müller cells (MC) maintain the structural and functional stability of the retina. The objective of this study was to evaluate the effect of high glucose concentrations on reactive oxygen species (ROS) production and Nrf2 expression levels in rat MC. MC were incubated with normal (NG; 5 mM) or high glucose (HG; 25 mM) for different times. Incubation with HG increased ROS levels from 12 to 48 h but did not affect cell viability. However, exposure to 3 h of HG caused a transient decrease Nrf2 levels. At that time, we also observed a decrease in the mRNA expression of Nrf2 target genes, glutathione levels, and catalase activity, all of which increased significantly beyond initial levels after 48 h of incubation. HG exposure leads to an increase in the p65 subunit of nuclear factor-κB (NF-kB) levels, and its target genes. These results suggest that high glucose concentrations lead to alteration of the redox regulatory capacity of Nrf2 mediated by NF-kB regulation.
C. Albicans infections have been particularly critical among nosocomial infections in immune-compromised and hospitalized patients. The interaction of microbes with various types of nanoparticles is a crucial stage in determining the biological consequences. The present study aimed to synthesize and evaluate nanomaterial-based vaccines using C. albicans ghosts. The silver nanoparticles (Ag-NPs) and gold nanoparticles (Au-NPs) were environmentally prepared using green biological chemistry using C. albicans ghosts prepared using a modified sponge-like protocol. The interaction of Ag-NPs or Au-NPs with the cell membrane of C. albicans ghost was confirmed by utilizing Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM), and X-Ray Diffraction (XRD) tools. Experimental rats were injected with different doses of C. albicans ghost mixed with Ag-NPs or Au-NPs to evaluate their effects on the immune system. The ions of Ag-NPs or Au-NPs are entirely converted to zero-valent atoms due to the reduction and stabilization effect of the C. albicans ghost. The prepared nanoparticles exhibited small nanosized with good monodispersity and were stable without noticeable agglomeration at room temperature for two months. Enhancement of both humoral and cellular immune responses was noticed. Au-NPs or Ag-NPs to C. albicans ghost resulted in activation of the immune system and increased serum levels of Interferon Gamma (IFN-γ), a lymphocyte-derived pro-inflammatory cytokine.
Background
Besides being a common food component broadly consumed worldwide, egg yolk immunoglobulin Y (IgY) has essential therapeutic potentials. In fact, in a time of ever-increasing risk of antibiotic resistance, it is crucial to find new ways to battle infection, and oral administration of preformed specific antibodies represents one of the most attractive approaches against infection. Infectious diseases of bacterial and viral origin in humans and animals can be controlled and passively cured by orally applied IgYs isolated from chicken egg yolks. Despite multiple obvious advantages of oral administration of IgY, harvesting IgY from egg yolk in a pure form is a challenging task.
Results
In this study, we developed a fast, simple, cost-effective, and efficient protocol for IgY isolation from chicken egg yolks. First, egg yolk was collected and diluted with 5 volumes of cold distilled water, homogenized, pH adjusted, and centrifuged. Next, the supernatant was collected, to which caprylic acid at concentration of 2% v/v was added, followed by pH adjustment to pH 5.0, centrifugation at 4oC, and collection of the resulting supernatant. This step was repeated twice, with adding 2% v/v of caprylic acid each time. The final supernatant was concentrated using ultrafiltration, and the IgY purity and activities were checked by SDS-PAGE, Western blotting, and ELISA. The sequential (2%, 2%, 2%) addition of caprylic acid yielded IgY with a purity of 63.5%, 90.6%, and 95.8%, respectively, and reached 97.9% after ultrafiltration at pH 9.0. The IgY activity increased exponentially to reach 99% after the ultrafiltration step.
Conclusions
The proposed caprylic acid-based protocol of IgY purification from the yolk of chicken eggs seems to be simple, fast, direct, and very cheap. This indicates that this protocol has great potential for scale-up processing.
Tailed, double-stranded DNA bacteriophages provide a well-characterized model system for the study of viral assembly, especially for herpesviruses and adenoviruses. A wealth of genetic, structural, and biochemical work has allowed for the development of assembly models and an understanding of the DNA packaging process. The portal complex is an essential player in all aspects of bacteriophage and herpesvirus assembly. Despite having low sequence similarity, portal structures across bacteriophages share the portal fold and maintain a conserved function. Due to their dynamic role, portal proteins are surprisingly plastic, and their conformations change for each stage of assembly. Because the maturation process is dependent on the portal protein, researchers have been working to validate this protein as a potential antiviral drug target. Here we review recent work on the role of portal complexes in capsid assembly, including DNA packaging, as well as portal ring assembly and incorporation and analysis of portal structures.
Expected final online publication date for the Annual Review of Virology Volume 6 is September 30, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
In der Sepsis kommt es zu einer mikrovaskulären Schrankenstörung. Die genauen Mechanismen hierfür sind noch nicht bekannt. Im klinischen Alltag gibt es noch keine spezifische diagnostische Möglichkeit für den (frühen) Nachweis oder Therapie für die mikrovaskuläre Schrankenstörung. Ein wichtiger Bestandteil der endothelialen Barriere und deren Integrität ist das Adhärenskontaktprotein VE-Cadherin. Es wird vermutet, dass Shedding durch ADAM-10 eine wichtige Rolle bei der Permeabilitätssteigerung spielt. In der vorliegenden Arbeit konnte gezeigt werden, dass es unter Inflammationsbedingungen erstens zu einem Zusammenbruch der Endothelbarriere kommt, zweitens, dass korrespondierend dazu vermehrt sVE-Cadherin im Überstand nachweisbar ist und drittens, dass eine Stabilisierung der Endothelbarriere und Reduktion von sVE-Cadherin sowohl durch einen PDE-4 Inhibitor, als auch durch spezifische ADAM-10 Inhibition möglich ist. Erstmalig konnte dargestellt werden, dass der ADAM-10-Inhibitor GI254023X zu einer wirksamen Barrierestabilisierung und verringerten sVE-Cadherin-Werten führt. Dies lässt darauf schließen, dass Shedding eine Rolle beim Zusammenbruch der Endothelbarriere spielt. Möglicherweise ist sVE-Cadherin selbst ein Faktor, der zum Zusammenbruch der Endothelbarriere führt. Auch bei Sepsispatienten mit schwerer mikrosvaskulärer Schrankenstörung konnten erhöhte sVE-Cadherin-Werte im Serum nachgewiesen werden. Zusammenfassend kann daher die Vermutung aufgestellt werden, dass die Bildung von sVE-cadherin bei der Entzündung eine wichtige Rolle sowohl für die Detektion, als auch bei der Induktion der mikrovaskulären Schrankenstörung spielt. Der Nachweis von sVE-cadherin bei Sepsispatienten könnte als geeigneter Biomarker für den Nachweis einer mikrovaskulären Schrankenstörung in der klinischen Anwendung eine Bedeutung erhalten.
Chlorpyrifos (CPF), an organophosphate insecticide has a wider application throughout the world to protect agricultural crops and vegetables from insects. Polyphenolic compounds are considered as beneficial against toxicities induced by organophosphates. The present study was conducted to understand the neuroprotective role of quercetin in chlorpyrifos‐induced apoptotic events in rats. Twenty‐four male Sprague Dawley rats weighing 170 to 200 g were divided into four groups viz: Control, chlorpyrifos treated (13.5 mg/kg body wt. alternate day), quercetin treated (50 mg/kg body wt. every day) and combined chlorpyrifos + quercetin treated. All the treatments were carried out for a total duration of 60 days. Protein carbonyl content and acetylcholinesterase activity were estimated in serum along with cerebrum and cerebellum to ascertain neurotoxicity. Further, for appraisal of neurodegeneration as a consequence of apoptosis, protein expressions of Bcl‐2, Bax, cytochrome c, caspase‐8, and caspase‐9 were assessed. The results showed that protein carbonyl contents were markedly increased in both serum and brain tissues (cerebrum and cerebellum) of chlorpyrifos‐treated rats when compared with control group and were appreciably improved upon simultaneous supplementation with quercetin. Further, chlorpyrifos treatment revealed a significant decrease in the enzyme activity of acetylcholinesterase in serum as well as in cerebrum and cerebellum, which however was increased upon concomitant treatment with quercetin. In chlorpyrifos‐treated animals, we have observed a significant decrease in the protein expression level of Bcl‐2, but a remarkable increase in the expression levels of Bax, cytochrome c, caspase‐8, and caspase‐9 in both cerebrum and cerebellum. Interestingly, when chlorpyrifos‐treated animals were supplemented with quercetin, a significant increase in the expression of Bcl‐2 and an appreciable decline in the expression levels of Bax, cytochrome c, caspase‐8, and caspase‐9 was observed. In conclusion, the present study advocates that quercetin may prove to be a useful candidate in containing the oxidative‐induced apoptotic events during chlorpyrifos exposure.
Aims
This study aimed to explore the possibility of using the Gluconacin from G. diazotrophicus strain PAL5 in the biological control of diverse sugarcane pythopathogenic bacteria.
Methods and Results
An in silico analysis was first employed to determine the phylogenetic relationship between Gram negative/positive bacteriocin producers and Gluconacin. The analysis showed that this trait is widespread among tested bacterial species and a well‐conserved gene within the Acetobacteraceae family. The bacteriocin gene (GDI_0415) present in the genome of strain PAL5 was than cloned in pDEST™17 and expressed in Escherichia coli BL21‐AI™. A bioassay showed growth inhibition of Xanthomonas albilineans by the recombinant bacteriocin. Subsequent bioassays indicated different levels of antagonistic activity against the majority of the sugarcane phytopathogenic bacteria (X. axonopodis pv. vasculorum, A. avenae subsp. avenae, P. syringae pv. syringae, X. vasicola pv. vasculorum). In addition, the bacteriocin was also antagonistic to some beneficial bacterial strains belonging to G. diazotrophicus and endophytic Bacillus species, which also colonize sugarcane plants.
Conclusions
The GDI_0415 gene, responsible for the production of Gluconacin, is well conserved within the Acetobacteraceae family and presented antagonistic activity against phytopathogenic and a few beneficial sugarcane bacteria.
Significance and Impact of the Study
The production of a recombinant protein, named Gluconacin, opens new avenues for the agro‐biotechnology application in agriculture, mainly with regard to the sugarcane crop.
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The surface epithelium of the bursa of Fabricius consists of interfollicular (IFE) and follicle-associated epithelium (FAE). The IFE comprises (i) cylindrical-shaped secretory cells (SC) and (ii) cuboidal basal cells (BCs). The FAE provides histological and two-way functional connections between the bursal lumen and medulla of the follicle. We used a carbon solution and anti-caveolin-1 (Cav-1) to study the endocytic activity of FAE. Carbon particles entered the intercellular space of FAE, but the carbon particles were not internalized by the FAE cells. Cav-1 was not detectable in the FAE cells or the medulla of the bursal follicle. The absence of Cav-1 indicates that no caveolin-mediated endocytosis occurs in the FAE cells, B cells, bursal secretory dendritic cells (BSDC), or reticular epithelial cells. Surprisingly, a significant number of Cav-1 positive cells can be found among the SC, which are designated SC II. Cav-1 negative cell are called SC I, and they produce mucin for lubricating the bursal lumen and duct. Occasionally, BCs also express Cav-1, which suggests that BC is a precursor of a SC. Transmission electron microscopy confirmed the existence of type I and II SC. The SC II are highly polarized and have an extensive trans-Golgi network that is rich in different granules and vesicles. Western blot analysis of bursa lysates revealed a 21-23 kDa compound (caveolin) and Filipin fluorescence histochemistry provided evidence for intracellular cholesterol. High amount of cholesterol in the feces shows the cholesterol efflux from SC II. The presence of Cav-1 and cholesterol in SC II indicates, that the bursa is a complex organ in addition to possessing immunological function contributes to the cholesterol homeostasis in the chickens.
Plastics cause adverse to ecosystem because of their recalcitrant and non-biodegradable nature. In this concern, biodegradation is the tool to remove the plastics from the environment by microbes. The present study deals with the isolation of efficient high density polyethylene degrading fungi from the polluted environment around Salem District, Tamil Nadu, India. Totally, 14 fungal strains were isolates and among that SF4 showed high amount of HDPE degradation. SF4 was identified as Aspergillus terreus by 28S rRNA sequencing. A. terreus was subjected to immobilization by sodium alginate and treated the HDPE for 30 days. The HDPE degradation was enhanced by immobilization of fungal cells. The protein content was extracted after 30 days of treatment from the supernatant and subjected to SDS-PAGE for molecular weight determination.
An intracellular naringinase from Bacillus amyloliquefaciens11568 isolated from soil was purified, identified, and characterized. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified enzyme gave a single protein band corresponding to a molecular mass of 32 kDa. The optimum pH and temperature for naringinase and its α-L-rhamnosidase and β-D-glucosidase activities were pH 7.5 and 45°C, respectively. The enzymes were stable below 45 °C between pH 3.5 and 8.5. The Km and the Vmax of the isolated naringinase were 0.95 mmol/L and 3847.3 mmol/(L·min), respectively. The isolated naringinase was capable of hydrolyzing naringin, neohesperidin, and other glycosides. Additionally, a concentration of 4 U/mL of the enzyme in citrus juice was sufficient to remove the naringin and alleviate the bitterness of the juice. These results provide an in-depth insight into the structure of the naringinase and the hydrolysis of naringin and other flavonoids.
The study aimed to evaluate cancer-targeting potential of a newly synthesised radiopharmaceutical, (99m) Tc-resveratrol in vivo, using colon cancer model. Colon cancer was induced in 20 male Sprague-Dawley rats by subcutaneous administration of 1,2-dimethylhydrazine (DMH), dissolved in 1 mM EDTA-normal saline, at a dose of 30 mg/kg body weight twice a week for first 4 weeks and once a week for next 12 weeks. A control group containing normal rats was used for result comparison. Colon cancer in DMH-treated group was confirmed by gross analysis of the colon, by histopathological analysis and molecular marker study in tumour tissue. At the end of the treatment period, the animals from the two groups were used for bio-distribution evaluation of (99m) Tc-resveratrol at different time intervals. High uptake of (99m) Tc-resveratrol was recorded in rat liver, spleen and kidneys, and the ratio of colon tumour uptake to normal colon uptake in DMH-treated rats increased significantly (P ≤ 0.01) with time, to reach a maximum value at 2 h but decreased thereafter. High uptake at the tumour site as compared to normal colon tissue was observed; however, the uptake by cancer cells at the target site was limited by high reticulo-endothelial uptake and rapid metabolism.
The product of gene 31 (P31) of bacteriophage T4 is required for the formation of the phage capsid and its related structures. In the absence of active P31, product P23, the major component of the phage capsid, aggregates into “lumps” which sediment with the cell envelope. Temperature-shift experiments with ts-mutants in gene 31 demonstrate that the P23 aggregates can be dissolved by activated P31 and the dissolved P23 is normal, in that it can be used for incorporation into active phage. It is possible that P31 acts catalytically.Two different ts-mutants in gene 31 produce two different temperature-sensitive proteins. One is irreversibly inactivated if produced at the restrictive temperature; but when synthesized at the permissive temperature, it becomes heat stable and remains functional at the restrictive temperature. The other is reversibly affected by temperature, activated following shift to permissive temperature and inactivated if restrictive temperature is established.
Maturation of T4 can proceed in the presence of 5-methyltryptophan or puromycin. Puromycin inhibits phage protein synthesis within a few seconds, thus limiting the amount of phage precursor material available for maturation. After the arrest of protein synthesis, maturation continues unabated until the limiting protein precursor is depleted. At least one protein, the tail fiber protein, is depleted. Phage protein maturable in the presence of puromycin appears about 5 minutes before maturation begins. The level of the maturable material reaches a maximum at the beginning of maturation. This level is taken as a measure of the size of the pool of complete sets of maturable phage protein. The bulk precursor protein begins to accumulate 3 minutes earlier than serum blocking proteins (SBP) and forms a pool about twice as large as the pool of SBP.
The formation of bacteriophage T4 has been studied by characterizing the phage components accumulating in cells infected—under restrictive conditions—with mutants blocked at different stages of the assembly process. Three structures which appear to be intermediates in tail assembly have been isolated by centrifugation: baseplates (S20,w ≅ 80 s), core-baseplates (S20,w ≅ 80 s), and core-baseplates with surrounding sheath (S20,w ≅ 130 s). The functions of genes 19, 48 and 54 are required for the conversion of the baseplate to the core-baseplate. The functions of genes 3, 15 and 18 are required for the formation of the sheath. Gene 18 codes for a major structural protein of the sheath. Genes 3 and 15 specify products required for the stabilization and completion of the sheath.Tail assembly is sequenced; the baseplate is completed first and the core forms on the baseplate. The gene 18 product polymerizes on the core-baseplate and then the 3 and 15 gene products fix the sheath subunits in the polymerized form. After sheath formation, a segment appears to be added to the core at the terminus of the sheath, permitting subsequent attachment of the head. The tail fibers attach only after the particle formed by head-tail union has been acted upon by the gene 9 product. Particles which have not been acted upon by the gene 11 or 12 product adsorb to bacteria but do not kill them.Electron microscopic observations on the state of phage heads in mutant lysates are also presented. Mutations in three genes result in the accumulation of head membranes empty of DNA.Phage heads and phage tails are formed independently of each other.
Conditional lethal mutations in gene 20 lead to the production of polyheads. Observed intracellularly in sections, polyheads are tubular structures. The cross sections are frequently pentagonal. Polyheads are filled with an unidentified internal substance which is different—at least in concentration—from the contents of normal phage. In a lysate most polyheads are open, but a few are sealed at one or both ends; most open polyheads appear empty upon examination. Serological studies show that polyheads and normal heads have at least one antigenic site in common. The normal heads have at least one more antigenic site than the polyheads.To produce polyheads, the normal functions of two “head” genes are necessary: (a) gene 23 which is believed to produce the protein subunit of the capsid, and (b) gene 31, the function of which has not yet been identified.A temperature-sensitive mutation L65 in gene 23 produces abnormal phage heads. This abnormality is also observed in polyheads produced by the double mutant tsL65 (in gene 23) -amN50 (in gene 20).
The agar-filtration method for preparing particle suspensions for the electron microscope is described and its application to particle counting is studied. The coefficient of variation of this method is found to be about 15%. The precision of the absolute determination of titers is, however, limited by the precision of the determination of the latex sphere diameter and is estimated to be 20 to 30%.
Developments have been made in the use of spraying methods in the preparation of specimens for examination in the electron microscope. At the same time, two wholly volatile diluents, containing electrolytes and adjusted to normal pH, have been developed for use in forming the spray drops. The use of volatile electrolytes makes it unnecessary to wash the specimen after the droplet patterns have been formed on the specimen screens.
There are several qualitative and quantitative uses and advantages of the spray technique as described. The qualitative ones are these: (1) the brief drying time of the droplets helps to preserve the shapes of particles upon drying, and also allows studies to be made of rapidly reacting systems; (2) since the drop patterns are reproducibly representative samples of the suspension under investigation, qualitative assays of the particulate composition of the suspension can be made, and a comparison of the particulate composition of two closely similar suspensions can be made under ideal control conditions; (3) the droplet patterns are discretely bounded by blank areas of substrate film, and this fact makes practicable the detection of small and subtle differences between the fine structure of the specimen material and that of the substrate.
The quantitative uses of the spray technique are: (1) by use of reference particles of known numbers in a suspension it is readily possible to determine the volumes of droplets issuing from a spray‐making device; (2) by use of reference particles it is also possible to make an assay of the number of other particles, such as a virus, per unit volume of mixed suspension; (3) if a suspension is highly purified, the particle weight of the material in suspension can be determined. Experiments involving the counting of about 10,000 particles show that the quantitative precision is as good as would be expected from the statistics of random sampling.
The construction and use of a spray gun are described, as well as the characteristics of two volatile diluents: ammonium acetate and ammonium carbonate.
Studies are reported on the properties and in vitro assembly of bacteriophage T4 components that accumulate in mutant-infected cells under restrictive conditions. On the basis of these and earlier observations, a pathway for the assembly of T4 is proposed. It is concluded that the assembly of T4 occurs in stepwise fashion and that most of the steps are under gene control. Components that accumulate in mutant-infected cells are, in general, the precursors to the blocked step. The head, tail and fibers of the phage are assembled independently of one another prior to their union. Genes 16 and 18 control the assembly of the sheath on a precursor tail consisting of core plus baseplate. Genes 13 and 14 control a terminal step in the assembly of the head prior to its union to the tail. This union seems to be spontaneous and not under gene control. Genes 11 and 12 control early steps in tail assembly. When gene 11 or 12 is defective, however, these steps are bypassed, leading to the formation of inactive phage particles.
By applying analytical acrylamide electrophoresis to degraded purified capsid-related material, we found that (1) normal T4 capsids contain a major component, identified as a product of gene 23, plus at least two minor components, k and l, which we could not yet identify genetically; (2) capsids of the short-headed variant contain the same components as the normal ones; and (3) polyheads contain mainly the product of gene 23 and very little, if any, of the minor components k and l.The minor components can be extracted from capsids by treatment with 8 M urea at 45°. A residual capsid is left behind which, in the electron microscope, is not significantly different from the normal one.It is discussed why k and l are not likely to have a morphopoietic role, since they are identifiable neither with the product of genes 66 and 20, whose morphopoietic functions are known, nor with that of other genes known to be necessary for the production of stable heads.
Two structures related to the head of bacteriophage T4, polyheads and τ-particles, are shown by electron microscopy to contain an internal component defined as a core. These cores can show a high degree of organization.Multilayered polyheads are described which contain a normal sized core.The dark polyhedral bodies seen in sections and previously referred to as DNA “condensates” were reinvestigated, and were found to have a visible membrane. However, this membrane is very fragile and does not survive lysis of the cell in a recognizable form.The appearance of thin sections of T4 heads obtained after different fixation methods suggests that the “less dense areas,” which can be observed when fixed under conditions found to be inadequate for DNA fixation, are likely to be artifacts and therefore cannot be used alone as proof for the presence of a core.We conclude that the present information from morphological, genetic, and biochemical experiments strongly suggests that the observed cores have a morphopoietic role. We discuss the structure and possible mechanism of action of such a core.
The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T4amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.