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Antimicrobial activity, cytotoxic effect and characterization of marine bivalve extracts Cerastoderma glaucum
Abstract
Cerastoderma glaucum, a marine bivalve inhabiting Lake Timsah, is surrounded by different pathogenic organisms. The present study evaluates the antimicrobial activities, cytotoxicity and characterization of C. glaucum extracts. Chloroform, methanol and acidic tissue extracts were prepared from C. glaucum collected during winter and summer seasons. Winter acidic extract exhibits potent antimicrobial activities against 21 bacterial, 2 yeast and 2 viral strains. The inhibition zone of this extract ranges from 10 mm against Klebsiella oxytoca, Pseudomonas stutzeri, Globicatella sulfidifaciens and Bacillus (B. badius, B. amyloliquefaciens and B. pumilus) to 24 mm against Shigella flexneri. Also, the inhibition viral activities of this extract at a concentration of 62.5 µg ml−1 against Hepatitis A virus and Herpes simplex virus type 1 (HSV-1) are 62.383% and 57.035%, respectively, with low cytotoxicity of 24.030%. Furthermore, winter acidic extract of C. glaucum has the highest total protein contents (9.8 mg ml−1) compared with the other extracts. Moreover, the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) indicates the presence of four clear low molecular weight peptides bands; 8.588, 7.237, 4.423 and 2.692 kDa. Fourier transform-infrared (FT-IR) analysis indicates the presence of 12 functional groups of proteins in winter acidic extract of C. glaucum at appropriate wavelengths.
A high intake of synthetic food preservatives leads to a variety of
health problems and diseases. Antibacterial compounds have been
discovered in marine invertebrates. As a result, four marine bivalve
molluscs species, three giant clams, Tridacna maxima, Tridacna gigas,
Tridacna squamosa, and (pearl oysters) Pinctada margritifera were
collected from the Red sea and assayed for antibacterial activity against the
reference food spoilage pathogenic strain Salmonella enterica. Among the
four bivalves, T. squamosa aqueous extract demonstrated a significant
antibacterial effect on S. enterica, showing a MIC of 12.5mg/ ml. Phenolic
and flavonoid total content of T. squamosa water extract were 18.5±1.63mg/
g and 2.67±1.48mg/ g, respectively. The antioxidant activity of T. squamosa
water extract was demonstrated by a DPPH radical scavenging IC50 of
58.61±1.02 μg/ml. HPLC analysis confirmed the presence of polyphenol
components, including gallic acid, chlorogenic acid, and methyl gallate. The
IC50 value of T. squamosa aqueous extract for growth inhibition on normal
PBMCs cells was 199.2±2.24μg/ ml, determining its safety. In addition, it
might be employed as innovative dietary supplements and effective natural
food preservatives instead of chemical ones.
Objective: Molluscs are considered as a significant natural source to derive many narrative biological active compounds. Animal extract are usually complex mixtures of bioactive molecules, including proteins and peptides. Freshwater gastropods are found to be a vital source of valuable bioactive substances. These bioactive compounds are involved in various biological functions such as communication, infection, reproduction and self-defense. Discovery of freshwater gastropods bioactive potential and their curious roles are still limited. Experimental approach: Consequently, the present study was evaluated on the bioactive compound extraction; identification and In-vitro antioxidant activity were gritty by total antioxidant activity, DPPH (diphenylpicrylhydrazyl) assay, hydroxyl radical scavenging activity, superoxide radical scavenging, reducing power and ferrous ion chelating from active fraction of Pila virens tissue methanolic extract at various concentrations (20-250μg/ml). Findings: The results of freshwater gastropod methanol tissue extracts exhibited significant total antioxidant activity, DPPH (diphenylpicrylhydrazyl) assay, hydroxyl scavenging and superoxide scavenging activity which predicted as 67.09%, 74.83%, 60.21% and 59.89%. Conclusion: These results concluded that, the tissue extract of Pila virens has novel bioactive compound, antioxidant potential and it has to further characterize to improve the pharmacological active freshwater natural products.
Bacteriocins secreted by some bacteria such as Enterococcaceae has a protected role against Staph. aureus pneumonia. This study identified a new Enterococcus sp. that produces efficient bacteriocin which attenuates Staph. aureus pneumonia in rats. Identification of Enterococcus sp. was achieved according to Bergey’s manual of systematic bacteriology using 16S rRNA analysis. Bacteriocin was extracted, partially purified and characterized. Antagonistic activity of the bacterial cells or its bacteriocin was estimated in vitro against different pathogenic bacteria and in vivo with an animal experiment, where histopathological and hematological examinations were evaluated. The isolate was identified as Ent. faecium ER-3M, with accession number KY774316. It exhibits broad antimicrobial spectrum against Gram-positive and Gram-negative organisms in vitro. Treatment of rats with bacterial cells or bacteriocin (500 Au mg⁻¹), previously infected with Staph. aureus, significantly decreased the symptoms, WBCs, neutrophils, and liver functions. Histopathological examination of rat lung tissues revealed that treatment with probiotic bacteria and/or its bacteriocin can attenuate the pneumonic lesions caused by Staph. aureus bacteria. Partially purified bacteriocin was characterized by a large molecular weight of approximately 40 kDa a proteinous nature according to both Fourier transform infrared spectrometry (FTIR) spectroscopy and 1H-Nuclear magnetic resonance (NMR) analysis and belongs to class III of bacteriocins termed enterolysin. Based on the results, the successful treatment of Ent. faecium ER-3M or its bacteriocin may be one of the promising treatments against Staph. aureus pneumonia.
Objectives: This study aimed to screen Thais savignyi whole body extracts for antibacterial activity against clinical isolates of some human pathogenic bacteria and analyze its biochemical compounds, including carbohydrate, proteins, alkaloids, cholesterol, unsaturated fatty acids, as well as functional groups and elements. Methods: Well agar diffusion was used for screening of acetone and methanol extracts against clinical isolates of E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus. Biuret and SDS-PAGE methods were applied for quantitative and qualitative protein evaluation. Anthrone method was used for quantitative and qualitative analysis of carbohydrates. Wagner and Mayer's reagents were employed for alkaloid screening. The Liebermann-Burchard test was performed for the detection of cholesterol, infrared for identifying functional groups, and Dumas method for elemental analysis. Results: Acetone and methanol crude extracts at 0.2 mg/mL concentration inhibited the growth of all the test bacteria; however, the methanol extract was more effective than the acetone extract. The maximum zone of inhibition of the methanol extract was observed against B. cereus (16 mm). Minimal inhibitory concentration of the most effective fraction ranged between 12.5 and 100 µg/mL. Elemental analysis of the bioactive fraction showed 41.55% carbon, 9.49% hydrogen, 6.13% nitrogen, and 1.64% sulfur. IR spectra confirmed the presence of aliphatic, alkenes, secondary amines, and disulfide groups in the most effective fraction of methanol extract of Thais savignyi. Carbohydrate and protein analysis revealed 4.5% carbohydrate and 9% protein. SDS-PAGE disclosed 5 separate bands with 70, 53, 41, 30, and 22 kilodaltons. Wagner and Mayer test showed the presence of alkaloids; mercuric and iodine solutions indicated the presence of unsaturated fatty acids; cholesterol was detected by Liebermann-Burchard reaction and Salkowski test. Conclusions: The findings of this study suggest that Thais savignyi has antibacterial potential and can be recommended as a source of bioactive compounds of medicinal value. Copyright © 2017, Jundishapur Journal of Natural Pharmaceutical Products. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
A variety of bivalve mollusks (phylum Mollusca, class Bivalvia) constitute a prominent commodity in fisheries and aquacultures, but are also crucial in order to preserve our ecosystem’s complexity and function. Bivalve mollusks, such as clams, mussels, oysters and scallops, are relevant bred species, and their global farming maintains a high incremental annual growth rate, representing a considerable proportion of the overall fishery activities. Bivalve mollusks are filter feeders; therefore by filtering a great quantity of water, they may bioaccumulate in their tissues a high number of microorganisms that can be considered infectious for humans and higher vertebrates. Moreover, since some pathogens are also able to infect bivalve mollusks, they are a threat for the entire mollusk farming industry. In consideration of the leading role in aquaculture and the growing financial importance of bivalve farming, much interest has been recently devoted to investigate the pathogenesis of infectious diseases of these mollusks in order to be prepared for public health emergencies and to avoid dreadful income losses. Several bacterial and viral pathogens will be described herein. Despite the minor complexity of the organization of the immune system of bivalves, compared to mammalian immune systems, a precise description of the different mechanisms that induce its activation and functioning is still missing. In the present review, a substantial consideration will be devoted in outlining the immune responses of bivalves and their repertoire of immune cells. Finally, we will focus on the description of antimicrobial peptides that have been identified and characterized in bivalve mollusks. Their structural and antimicrobial features are also of great interest for the biotechnology sector as antimicrobial templates to combat the increasing antibiotic-resistance of different pathogenic bacteria that plague the human population all over the world.
p>Quantification of cell viability and proliferation form the fundamental for numerous in vitro assays in response to external factors. An MTT assay is a colorimetric assay based on assessing the cell metabolic activity. A549 Lung adenocarcinoma cell line was used to see the cytotoxic potential of a new drug for initial screening of apoptosis or necrosis. The biochemical mechanism behind the MTT assay involves NAD(P)H-dependent cellular oxidoreductase enzyme that converts the yellow tetrazolium MTT [3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] into insoluble (E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan (formazan). The formed formazan can be dissolved with dimethyl sulfoxide (DMSO) to give a purple color with characteristic absorption at 540 nm. Intensity of purple color is directly proportional to the cell number and thus indicating the cell viability.
Video Clip of Methodology: 3 min 56 sec Full screen If Failed </p
Protein aggregation results in the formation of long, unbranched-sheet-rich structures, commonly known as amyloid fibrils, which are believed to be a defense mechanism against the small and most toxic aggregates of misfolded proteins. Disulfide bonds limit the way in which a protein or a peptide can aggregate into these fibrils via steric restraint. There is great interest in understanding the influence of disulfide bonds on the stability of therapeutic proteins such as insulin. Therefore, the identification and characterization of protein disulfide bonds is an essential step to thoroughly understand their biological function. However, the determination of disulfide linkages can be a challenging task. Several methods such as crystallography, NMR and mass spectrometry are used for the determination and identification of disulfide bond linkage; however, their application is limited by large sample requirements, protein size, ambiguous results and complexity of data analysis. Raman spectroscopy has several advantages over other spectroscopic techniques or chemical analyses e.g. ease of measurements, its versatility in application to aqueous solutions, nonaqueous liquids, fibers, films, powders, gels and crystals without destruction of the samples. Therefore, Raman spectroscopy was used in the present research to identify and obtain quantitative information of therapeutic proteins. Solid samples of cysteine and cystine were used to identify and quantitate the ratio of disulfide and S-H bonds. The information obtained was used to identify and characterise disulphide linkage in hirudin, insulin and lysozyme. Raman data provide new insights about therapeutic protein surface organization, which may serve as a basis for the design of therapeutic proteins.
The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, is mainly responsible for this situation; thus, resistance has compelled the scientific community to search for novel therapeutics. In this scenario, antimicrobial peptides (AMPs) provide a promising strategy against a wide array of pathogenic microorganisms, being able to act directly as antimicrobial agents but also being important regulators of the innate immune system. This review is an attempt to explore marine AMPs as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds.
Objectives: The crude protein extracts of edible marine invertebrates Pitar erycina and Donax cuneatus collected from the coastal area of South India were evaluated for its antimicrobial potency against broad spectrum of bacterial pathogens. Methods: The crude proteins were extracted from the flesh with 5% cold acetic acid and buffers at acidic, neutral and basic pH. The crude was partially purified by ammonium sulfate precipitation method and the precipitate was stored at-20°C still evaluation. The antimicrobial property was assessed by well diffusion method. Both the edible oyster extracts showed inhibitory effects against the tested bacterial and fungal strains. Results: The maximum zone of 24mm was observed with Donax cuneatus acidic extract against Bacillus subtilis and a minimum of 17mm zone was observed against Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri. The Pitar erycina acidic extract showed the maximum activity against E.coli and Shigella flexneri with 17mm and 15mm zone respectively. The antimicrobial potency of buffer extracts of the two bivalves was comparingly low with that of the acidic extracts. 1.23mg/ml protein was estimated in Donax cuneatus acidic extract. Conclusion: The crude extracts of protein from edible bivalves showed potent bactericidal property against highly pathogenic species, which may helps to find a safe novel protein/peptide antibiotic.
Objective: To isolate and screen antibiotic producing actinomycetes from potential soil samples
of Gondar town, Ethiopia.
Methods: Fifteen soil samples were collected, serially diluted and spread on starch casein
and oat meal agar supplemented with amoxicillin and cyclohexamide for inhibition of bacteria
and fungi, respectively. Cross streak method was used to check antagonistic activity of isolated
actinomycetes against test organisms. Solid state fermentation and crude extraction were used for
the production of antibiotics from isolates. Agar well diffusion was used for antimicrobial activity
of crude extracts against test organisms.
Results: Three isolates (Ab18, Ab28 and Ab43) have been shown high antagonistic activity during
primary screening. Inhibition zones obtained from crude extracts showed significance differences
when compared with standard antibiotics tested against test organisms (P
A study was carried out in two zones in the ria de Sada (Galicia, NW Spain) with mussels (Mytilus galloprovincialis Lmk) coming from the same stock and cultured in rafts, from seed sized to the first split (individuals with sizes between 15 and 65 mm), in order to evaluate a) the environmental differences in each location and b) their influence on growth (length and dry weight) and on chemical composition (protein, carbohydrates, glycogen, lipid classes, and fatty acids of total lipids). Allometric relations regarding those parameters for mussels on both locations were also determined. The mussels cultured in the inner zone (Arnela), with a higher concentration in chlorophyll a during the period studied and a smaller number of cultured mussels, present the highest performance in dry weight and a nearly doubled amount of glycogen than those in the outer one (Lorbe). There are also significant differences in saturated, monounsaturated, nonmethylene interrupted dienoic, and total fatty acids and sterols, the lowest content being shown by those cultured at Arnela. The mussel is an important dietetic source of polyunsaturated ω-3 fatty acids, as eicosapentaenoic acid and docosahexaenoic acid: both of them are beneficial for health (i.e., the treatment and prevention of cardiac ischemia). The values found range from 1 to 2% of dry weight in both locations. An indicator of the greatest importance if the product is to be used for human food is the ratio of ω-6/ω-3 fatty acids. Habitual diets with a ratio of ω-6 to ω-3 fatty acids of 12-50 are associated with a greater risk. The value observed in this study was about 0.2 in both locations.
Molluscs, one of the most successful phyla, lack clear evidence of adaptive immunity, yet thrive in the oceans, which are rich in viruses. There are thought to be nearly 120,000 species of Mollusca, most living in marine habitats. Despite the extraordinary abundance of viruses in oceans, molluscs often have very long lifespans (10-100s yrs). Thus, their innate immunity must be highly effective at countering viral infections. Antiviral compounds are a crucial component of molluscan defenses against viruses, and have diverse mechanisms of action against a wide variety of viruses, including many that are human pathogens. Antiviral compounds found in abalone, oyster, mussels and other cultured molluscs are available in large supply, providing good opportunities for future research and development. However, most members of the phylum Mollusca have not been examined for the presence of antiviral compounds. The enormous diversity and adaptations of molluscs implies a potential source of novel antiviral compounds for future drug discovery.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Antimicrobial peptides (AMPs) are a heterogeneous group of small amino acidic molecules produced by the innate immune system of a variety of organisms encompassing all orders of life from eukaryotes to amphibians, insects and plants. Numerous AMPs have been isolated from natural sources and many others have been de novo designed and synthetically produced. AMPs have antimicrobial activity in the micromolar range and compared with traditional antibiotics, they kill bacteria very rapidly. They act, principally, by the electrostatic attraction to negatively charged bacterial cells and consequently membrane disruption, but their antibacterial activity may also involve interference with metabolic processes or different cytoplasmic targets. AMPs are a group of unique and incredible compounds that may be directed to a therapeutic use either alone or in combination with existing antibiotics.
In the present work, gold nanoparticles (Au-NPs) have been successfully synthesized by the marine picoeukaryote, Picochlorum sp. The alga culture was used as a reductant for HAuCl4·3H2O resulting in the phytosynthesis of Au-NPs within 48 h. The algal biomass turned purple in color due to the phytogenic Au-NPs at intracellular level. The phytogenic Au-NPs are characterized by surface plasmon band observed close to 520 nm; the phytogenic Au-NPs accumulated in plastids with an average size of 11 nm. The strong signals of gold were reported in their corresponding EDX spectra. FTIR analysis revealed that polysaccharide and protein biomolecules in the algae cell do dual function of reducing the Au3+ ions and stabilizing the phytogenic Au-NPs. In combination with ampicillin (10 µg), gentamicin (10 µg), amphotericin B (25 µg), the phytogenic Au-NPs exerted an outstanding antimicrobial effect and biocidal action against the tested Gram-positive, Gram-negative bacteria and the tested fungal pathogens. This study suggested that the different bioactive compounds reported in the FTIR profile of the phytogenic Au-NPs along with the presence of different fatty acids may play an important role in the synergistic antimicrobial effect of the phytogenic Au-NPs produced by Picochlorum sp. These results not only provide a green approach for the synthesis of Au-NPs but also open a door for new pharmaceutical leads.
Antimicrobial peptides (AMPs) represent the most universal immune effectors. Molluscs constitute the second largest animal phylum, after Arthropods, in term of number of species. Only a negligible number has been investigated regarding AMPs. The choice of the species to be studied relied on their economical importance and availability. First studies on molluscan AMPs dated from 1996 and were based on biological activities of biochemical-purified fractions. Such approach released all the original structures we know, with biological activity sometimes different from one isoform to another. Then, molecular biology techniques were applied to molluscan AMPs starting in 1999. Complete screening of genome expression in various situations became available, as well as some exotic Molluscs, the ones collected from deep-sea hydrothermal vent, for instance. Full sequences of active peptides and precursors, and gene organizations were established. A breakthrough consisted in the discovery of numerous AMP variants, even within the same animal. In addition, computer homology revealed the existence of already known AMPs in new studied molluscan species.
Molluscs are highly delicious seafood and they are also very good source for biomedically imported products. Among the molluscs some have pronounced pharmacological activities or other properties which are useful in the biomedical area. In the present study antimicrobial activity of gill extraction of Perna viridis was investigated. In antibacterial activity the maximum zone of inhibition (19 mm) was observed against Staphylococcus aureus and minimum activity (11 mm) was observed in Salmonella paratyphi. In antifungal activity maximum zone of inhibition was observed in Aspergillus flavus (13mm) and minimum zone of inhibition was recorded in Mucor sp (11mm). The gill extraction samples showed antimicrobial activity was subjected to SDS-PAGE to estimate the molecular weight of proteins present after electrophoresis only one clear band were detected in the gel which represented proteins of molecular weight 9.7 kDa. In the present study indicated that the gill extraction of P.viridis may potential source for antibiotics.
Levels of the heavy metals Chromium (Cr), Copper (Cu), Iron (Fe),Manganese (Mn), Lead (Pb), and Zinc (Zn) were determined in water, sediment and soft tissues of the gastropod Thais carinifera and thebivalveVenerupisaureus inTimsah Lake-Suez Canal.The concentrations of heavymetals in water were 0.5, 0.9, 2.5, 0.22, 0.41and 0.25 for Cr, Cu, Fe, Mn, Pb and Zn µg/L, respectively. The corresponding concentration values in the sediments 12.8, 10.2, 18.8, 14.5, 12 and 45 µg/g dry weight for Cr, Cu, Fe, Mn, Pb and Zn, respectively. While the heavy metals concentrated in molluscs was 17.5, 17.4, 933.5, 20.9, 30.25 and 89 (µg/g dry weight) for Cr, Cu, Fe, Mn, Pb and Zn, respectively. The results indicated that the accumulation of heavy metals is predominant in molluscs than in sediment and water. The gastropod (T. carinifera) and the bivalve (V. aureus) accumulate different amount of metals in their tissues. The accumulation of metals was more pronounced inV. aureus thanT. carinifera.A positive correlation between metal concentration and size of the gastropod T. carinifera, which means that the largest individuals contained the highest levels of metals. While, in V. aureusthe smaller the bivalve, the higher the heavy metal concentration.
Objective: The study of marine organism for their bioactive potential, being a significant part of marine ecosystem has picked up the regularity in recent years with the growth recognition of their importance in human life as well as animals. Methods: In this present study methanol tissue extract of edible green mussel (P. viridis) was assayed for the antibacterial activity against six bacterial pathogens and antioxidant activities also determined. Results: The antibacterial activity of P. viridis tissue extract, showed maximum zone of inhibition (15mm) against Vibrio cholerae and minimum activity (5 mm) was observed in Klebsiella pneumoniae. Molecular size of green mussel protein was determined using Sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). FTIR analysis reveals the presence of bioactive compounds signals at different ranges. The antioxidant activity of crude protein tissue extract from the P. viridis were measured in different system of assay such as DPPH assay (76.9%), total reducing power (27.8 μg), total antioxidant activity (174 μg), hydrogen peroxide scavenging (88.12%) and nitrous oxide scavenging activity (62.5%) at 100μg/ml. Conclusion: crude tissue extract from P. viridis could be effectively used as alternative source of antimicrobial and antioxidant with subsequent health benefits.
The marine invertebrates become one of hot spot for the lead of antimicrobial compounds. Two species of commercially available and edible bivalves (M. meretrix and M. casta) were assayed for antimicrobial activity against 10 bacterial pathogens and 6 fungal pathogens and its biochemical composition. The bivalves were extracted with three different solvent systems respectively methanol, ethanol and acetic acid. All the three extracts of both the species M. meretrix and M. casta showed highest antibacterial activities against S. aureus, E. coli, B. substillus, K. pneumonia, P. fleuroscence and V. cholera. In present investigation the methanolic extract of the two bivalve species of M. meretrix and M. casta was showed inhibition activities against all pathogenic fungal forms. The two bivalve extracts showed high amounts of protein content, which made the variation up to 160-180 microg mg(-1) (wet weight). Both samples had low amount of carbohydrates 4.77-5.77 microg mg(-1) and lipids 0.11-0.17 microg mg(-1), respectively. The results of thin layer chromatography were revealed that presence of pink color spots it clearly indicates the presence of amino acid or peptides in bivalve's samples. Presuming that the antimicrobial compounds were proteins or peptides. In SDS-PAGE on 12% gel, the crude proteins M. meretrix and M. casta showed 5-6 bands ranging from 45-223 kDa. They represent potential pharmacological leads perhaps possessing novel and uncharacterized mechanisms of action that might ultimately benefit the ongoing global search for clinically useful antimicrobial agents.
To investigate the antimicrobial activity of the tissue extracts of Babylonia spirata (B. spirata) against nine bacterial and three fungal pathogens.
Crude extract of gastropod was tested for inhibition of bacterial and fungal growth. Antibacterial assay was carried out by disc diffusion method and in vitro antifungal activity was determined against Czapex Dox agar. The antimicrobial activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract. Molecular size of muscle protein was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). And fourier transform infrared spectroscopy (FTIR) spectro photometry analysis was also studied.
The maximum inhibition zone (12 mm) was observed against Pseudomonas aeruginosa in the crude ethanol extract of B. spirata and the minimum inhibition zone (2 mm) was noticed against Staphylococcus aureus in the crude methanol extract of B. spirata. Water extract of B. spirata showed the highest activity against Vibrio parahaemolyticus, Staphylococcus aureus and Candida albicans. Ethanol, acetone, methanol, chloroform and water extracts showed antimicrobial activity against almost all the bacteria and fungus. Compared with water extracts, ethanol and methanol extracts showed higher activity against all pathogens. The molecular weight of protein of the gastropod sample ranged from 2-110 kDa on SDS-PAGE. FTIR analysis revealed the presence of bioactive compounds signals at different ranges.
The research shows that the great medicinal value of the gastropod muscle of B. spirata may be due to high quality of antimicrobial compounds.
The vibrational spectrum of a molecule is considered to be a unique physical property and is characteristic of the molecule. As such, the infrared spectrum can be used as a fingerprint for identification by the comparison of the spectrum from an “unknown” with previously recorded reference spectra. This is the basis of computer-based spectral searching. In the absence of a suitable reference database, it is possible to effect a basic interpretation of the spectrum from first principles, leading to characterization, and possibly even identification of an unknown sample. This first principles approach is based on the fact that structural features of the molecule, whether they are the backbone of the molecule or the functional groups attached to the molecule, produce characteristic and reproducible absorptions in the spectrum. This information can indicate whether there is backbone to the structure and, if so, whether the backbone consists of linear or branched chains. Next it is possible to determine if there is unsaturation and/or aromatic rings in the structure. Finally, it is possible to deduce whether specific functional groups are present. If detected, one is also able to determine local orientation of the group and its local environment and/or location in the structure. The origins of the sample, its prehistory, and the manner in which the sample is handled all have impact on the final result. Basic rules of interpretation exist and, if followed, a simple, first-pass interpretation leading to material characterization is possible. This article addresses these issues in a simple, logical fashion. Practical examples are included to help guide the reader through the basic concepts of infrared spectral interpretation.
Persistent infections involving slow-growing or non-growing bacteria are hard to treat with antibiotics that target biosynthetic processes in growing cells. Consequently, there is a need for antimicrobials that can treat infections containing dormant bacteria. In this Review, we discuss the emerging concept that disrupting the bacterial membrane bilayer or proteins that are integral to membrane function (including membrane potential and energy metabolism) in dormant bacteria is a strategy for treating persistent infections. The clinical applicability of these approaches is exemplified by the efficacy of lipoglycopeptides that damage bacterial membranes and of the diarylquinoline TMC207, which inhibits membrane-bound ATP synthase. Despite some drawbacks, membrane-active agents form an important new means of eradicating recalcitrant, non-growing bacteria.
This review focuses on defensins and cystein rich peptides, which are the most abundant natural antimicrobial peptides (AMPs) described in molluscs. These are compact peptides, 3-5 kDa in molecular mass, cationic and amphipatic; the presence of at least six cysteine residues forming three or four disulfide bridges is their prime structural characteristic. A 3-D structural characterization of these molecules has been included in recent investigations, using currently-available techniques. AMPs have been purified from hemocytes, epithelial tissue and plasma as well as cloned and chemically synthesized. Their antibacterial activity against Gram-positive and Gram-negative bacteria and fungi has been shown; only a synthetic mytilin fragment has displayed activity against viruses.
Antimicrobial peptides are small-sized, cationic and amphipathic molecules able to neutralize pathogenic microorganisms. Their antimicrobial effects tie them to mechanisms of immune defense, which is why they have been normally purified from immune cells. We describe an apparently new group of antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis . 20 specimens yielded 40 g of gills which produced 16 mg of an enriched fraction with antimicrobial activity as low as 0.045 μg/μl over reference strains. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. The resulting post Sep-pak C-18® 20% acetonitrile (ACN) eluate was fractionated by reverse phase HPLC and all resulting fractions were the source for in vitro antimicrobial activity evaluation. Active fractions were characterized by SDS-containing protein gel electrophoresis. All fractions were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria and 10 times more efficient over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules.
We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.
Rheum emodi is a Himalayan herb of family Polygonaceace, known for its medicinal properties owing to the presence of chrysophanol, emodin, physcion, aloe emodi anthraquinones as major constituents. The present investigation aims to synthesize TiO2 nanoparticles (TiO2 NPs) using R. emodi root extract. The synthesized nanoparticles were characterized using UV–Vis, FTIR, FESEM, EDX, AFM, TEM and XRD techniques. UV–visible spectra indicated the surface plasmon resonance (SPR) at 336 nm. The size distribution profiles were analyzed through XRD which revealed the crystalline nature of nanoparticles with average size of 45 nm. TEM and SEM micrographs showed that the particles are spherical and uneven. FTIR spectra reflected the evidence of the presence of anthraquinones, possible bio-molecules responsible for stabilization, reduction and capping of TiO2 NPs. The cytotoxic potential of TiO2 NPs was tested against liver cancer cells (HepG2) lines which was
found very effective and the formulation can be used in nano-drug to treat cancer cells in future.
Antimicrobial peptides are small-sized, cationic and amphipathic molecules able to neutralize pathogenic microorganisms. Their antimicrobial effects tie them to mechanisms of immune defense, which is why they have been normally purified from immune cells. We describe an apparently new group of antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis . 20 specimens yielded 40 g of gills which produced 16 mg of an enriched fraction with antimicrobial activity as low as 0.045 µg/µl over reference strains. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. The resulting post Sep-pak C-18® 20% acetonitrile (ACN) eluate was fractionated by reverse phase HPLC and all resulting fractions were the source for in vitro antimicrobial activity evaluation. Active fractions were characterized by SDS-containing protein gel electrophoresis. All fractions were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria and 10 times more efficient over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules.
Anti-bacterial activity was evaluated in different crude extracts of commercially important, edible freshwater bivalves, namely Lamellidens marginalis, collected from the Godavari river of Andhra Pradesh (India). Three different procedures, viz Aqueous, Chloroform and Acetone, were followed to prepare the extracts. The efficacy of the extraction procedure was assessed on the antimicrobial activity. Antimicrobial assay was carried out against 8 bacterial strains (3 gram positive and 5 gram negative bacteria) and 1 species of fungi. The acetone extracts showed significant activity against the tested organisms as compared to aqueous and chloroform extracts. The findings of the present study confirmed that the antimicrobial activity in freshwater bivalves appeared to be dependent on the extraction.
Anti-bacterial activity was evaluated in different crude extracts of five commercially important edible marine bivalves, namely Meretrix casta (Chemnitz), Polymesoda (Geloina) P. erosa (Solander), Perna viridis (Linnaeus), Crassostrea gryphoides (Schlothim) and Villorita cyprinoides (Grey), collected from the coast of Goa (India). Three different procedures, viz. methanol (MeOH), PBS and acid-enzyme hydrolysis (AEH), were used to prepare the extracts. The efficacy of the extraction procedure was assessed on the antimicrobial activity. Antimicrobial assay was carried out against 8 bacterial strains (3 Gram positive and 5 Gram negative bacteria) and 1 species of fungi. The AEH extracts showed higher activity against the tested organisms as compared to MeOH and PBS extracts. The findings of the present study confirmed that the antimicrobial activity in bivalves appeared to be dependent on the extraction process. Considerable interspecies variation was also observed.
Structural differences between α-chitin from shrimp (Crangon crangon) and β-chitin from squid (Loligo), as well as between their precipitated products from N,N-dimethylacetamide-LiCl solutions, are indicated by the CP-MAS 13C-NMR, FT-IR, FT-Raman spectra and the X-ray diffractograms.The 13C-NMR spectra in the solid state of α-chitin consisting of eight major resonances suggest a high degree of structural homogeneity. In the spectrum of β-chitin, the C-3 and C-5 signals merged in a single resonance.The bands contour of deconvoluted and curve-fit FT-IR and FT-Raman spectra shows a more detailed structure of α-chitin in the region of OH, NH and CO stretching regions; in particular the Amide I band is split, whereas a single broad band dominates in the corresponding β-form.Precipitation treatments induce a general disorder in α-chitin, while the metastable structure of β-chitin tends toward a more ordered architecture.All the present spectroscopic data of chitin samples are consistent with the corresponding X-ray diffraction patterns.
MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.
Plasma from the mussel Mytilus galloprovincialis previously immunized by injecting them with bacteria contains several bactericidal proteins. One protein, MGD-1, was purified by reverse-phase HPLC of supernatant from acidified cell-free hemolymph. Its biological activity is directed against both gram-positive and gram-negative bacteria but it is not cytotoxic towards human erythrocytes nor protozoa. As determined by mass spectrometry, the molecular mass of MGD-1 is 4418 Da. Primary-structure analysis revealed 38 amino acids including 8 cysteines and a modified amino acid residue in position 28. Computer searches unambiguously recognized the signature of an arthropod defensin, but the presence of two extra cysteines and of one modified amino acid suggest that it is a previously unknown member of that family.
Previous research has shown that mytilins and MGDs are two types of 4-kDa, cysteine-rich, cationic antimicrobial peptides, which are abundant in hemocytes of the mussels, Mytilus galloprovincialis and M. edulis. The expression of the genes encoding these peptides has been analyzed in the hemocytes of animals subjected to various stress factors, as well as during larval development. Variations in gene expression in adult mussels have been tested under conditions of physical stress, bacterial challenge and heat shock. The results suggest that in adult mussels, the MGD2 gene may be over-expressed with physical and temperature stress, but that reduced expression occurs with bacterial challenge. Gene expression during development has been analyzed using different larval and post-larval stages, ranging from 4-day-old veliger larvae to 32-day-old post-larvae. The results show that the expression of both mytilin B and MGD2 is developmentally regulated, but neither gene is expressed in mussels until after larval settlement and metamorphosis. Finally, the genes encoding two isoforms of these peptides have been cloned and sequenced, revealing that both genes contain four exons and three introns.
The aim of this study was to identify the presence of antimicrobial activities in the hemolymph of commercially important mollusc species. Acidic extracts were prepared from whole Cerastoderma edule (Cardiidae), Ruditapes philippinarum (Veneridae), Ostrea edulis (Ostreidae), Crepidula fornicata (Calyptraeidae) and Buccinum undatum (Buccinidae) and fractionated by Solid Phase Extraction. Fractions were first assayed for antibacterial activity against two indicator strains by an antagonism well diffusion method. Minimal Inhibitory Concentrations (MICs) were then determined against a panel of target bacteria including three Gram-positive and seven Gram-negative bacteria, some of which are aquaculture-pathogenic marine strains. In parallel, antiviral activities were assayed in vitro against Herpes simplex virus type 1 and Vero cells by cell viability. The broadest antibacterial activity was found in fractions from C. edule, but the highest activity was found in O. edulis (gills + mantle). At the antiviral level, the most active fraction was also attributed to C. edule acidic extract. Furthermore, SPE-fractions from other molluscs (40% SPE-fraction from O. edulis (gills + mantle), and 80% SPE-fractions from B. undatum, C. edule and R. philippinarum extracts) displayed lower EC50 values than the 40% SPE from C. edule but at in vitro concentrations causing more cytotoxicity. These promising results have led us to consider purification of the active compound(s) which may be related to mollusc defense mechanisms.
Gene-encoded antimicrobial peptides (AMPs) serve a major role in host defense systems against pathogens. In this study, cDNA of a new mollusk defensin was identified from a normalized cDNA library constructed from whole tissues of disk abalone. Abalone defensin peptide (pro-defensin) has a 198-bp coding sequence comprised of a putative 66 amino acids with a mature defensin consisting of 48 amino acid residues. The presence of an invertebrate defensin family domain, an arrangement of six cysteine residues and their disulfide linkage in C(1)-C(4), C(2)-C(5) and C(3)-C(6) form, an alpha helix in three-dimensional structure and a phylogenetic relationship suggests that abalone defensin could be a new member of the invertebrate defensin family, and related to arthropod defensins. In non-stimulated abalone, defensin transcripts were constitutively expressed in all examined tissues including hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas. It was observed that abalone defensin transcripts were significantly induced in hemocytes, gills and digestive tract at different time intervals after infection by pathogenic bacteria mixture containing Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes. Our overall results suggest that disk abalone defensin could be involved in the immune response reactions as a host defense against pathogenic bacteria.
Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. We previously characterized the first AMP from an oyster, a defensin, that was shown to be continuously expressed in the mantle of Crassostrea gigas. In this study, we report the cDNA cloning of two new isoforms of the defensin AMP family (Cg-defh1 and Cg-defh2) from the hemocytes of the oyster. The deduced amino acid sequences reveal two peptides of 73 amino acid residues with a mature portion consisting of 43 amino acid residues. Cy-Defh1 and CyDefh2 share 86% amino acid identity and belong to the "arthropod-molluscs defensin family". qRT-PCR analyses indicate that Cg-defh2 is continuously expressed in the hemocytes of C. gigas. In addition, after a bacterial challenge, the level of Cg-defh2 transcripts decreases dramatically in the circulating hemocyte population and this decrease can be correlated with an increase of Cg-defh2 transcripts in the gill and the mantle tissue, suggesting a possible migration of the hemocytes expressing Cg-defh2 towards the tissues implicated in the first defense barrier of the oyster. These results would suggest an important role of Cg-Defh2 in the oyster response to a microbial challenge. (c) 2006 Elsevier Ltd. All rights reserved.
Antimicrobial peptides (AMPs) are widely expressed and rapidly induced at epithelial surfaces to repel assault from diverse infectious agents including bacteria, viruses, fungi and parasites. Much information suggests that AMPs act by mechanisms that extend beyond their capacity to serve as gene-encoded antibiotics. For example, some AMPs alter the properties of the mammalian membrane or interact with its receptors to influence diverse cellular processes including cytokine release, chemotaxis, antigen presentation, angiogenesis and wound healing. These functions complement their antimicrobial action and favor resolution of infection and repair of damaged epithelia. Opposing this, some microbes have evolved mechanisms to inactivate or avoid AMPs and subsequently become pathogens. Thus, AMPs are multifunctional molecules that have a central role in infection and inflammation.
Diversity of mRNAs from mytilin B, one of the five mytilins identified in the Mediterranean mussel, Mytilus galloprovincialis, has been investigated from circulating hemocytes. One mussel expressed simultaneously two to ten different mytilin B mRNAs as observed in denaturing gradient gel electrophoresis (DGGE), defining 10 individual DGGE patterns (named A to J) within the mussels from Messina, Sicily (Italy). Three patterns accounted for 79% of the individuals whereas other patterns were found in only 2-7% of the 57 analyzed mussels. Base mutations were observed at specific locations, mainly within COOH-terminus and 3'UTR, leading to 36 nucleotide sequence variants and 21 different coding sequences (cds) segregating in two different clusters. Most of the base mutations were silent, and the number of pro-peptide variants was restricted to four. Finally, as the two amino acid replacements occurred within COOH-terminus, mature peptide from mytilin B appeared unique. Multiple sequencing of partial mytilin B gene from one mussel revealed that one to four randomly distributed mutation points occurred within intron-3. Only one sequence out of the 91 analyzed contained 16 mutation points. In addition, this sequence was the only one containing four out of the six mutation points occurring within exon-4, that code for most of the COOH-terminus domain, including the unique amino acid replacement. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure for COOH-terminus domain.
Antimicrobial peptides (AMPs) are promising novel antibiotics, because they exhibit broad antimicrobial spectra and do not easily induce resistance. For clinical applications, it is important to develop potent AMPs with less toxicity against host cells. This review article summarizes the molecular basis for the cell selectivity (bacteria versus host cells) of AMPs and various attempts to control it, including the optimization of physicochemical parameters of peptides, the introduction of D-, fluorinated, and unusual amino acids into peptides, the constraining of peptide conformations, and the modification of peptides by polymers. Pros and cons of these approaches are discussed.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
A rapid and sensitive colorimetric assay has been developed to evaluate the sensitivity of herpes simplex viruses (HSV) to antiviral agents. The chessboard titration of viruses and antiviral drugs and the automatic reading and analysis of the data allows objective and accurate results to be rapidly obtained. Virus sensitivity was expressed as an ED50 value which was the concentration of drug (micrograms/ml) reducing viral cpe by 50%. The ED50 values of antiviral drugs [acetylguanosine (ACV), idoxuridine (IDU), deoxycytidine (IDC) and bromovinyl deoxyuridine] for several HSV reference strains were determined and the method was then applied to clinical specimens. The selection of ACV and IDU resistant mutants was performed on a cloned sensitive HSV 1 ocular strain. We observed cross-resistance between ACV and IDU for the mutants isolated. The resistance to thymidine-kinase-dependent antiviral agents varied in inverse ratio to the thymidine kinase activity induced by HSV strains.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.
Extracts of the granular haemocytes of Carcinus maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein of approximately 11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Gram-positive bacteria. In addition its activity is stable after heating to 100 degrees C and is retained at concentrations as low as 10 microgram.mL-1. It has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the four-disulphide core proteins. Partial cDNA sequence data has been obtained.