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Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos
... Recently, transgenic cloned animals were used to produce valuable proteins for the development of new medicines and therapies [8][9][10][11]; however, the efficiency of animal production by SCNT is extremely low. This inefficiency is due to various reasons, including the low number of cells and aberrant gene expression in cloned embryos and the poor efficiency with which pregnancies develop to term and fetuses survive [12,13]. To overcome the low efficiency of cloning, multiple approaches have been investigated such as using different types of donor cell lines, a variety of culture systems, different fusion methods, and chemicals [2,3,[14][15][16][17]. ...
... In the present study, total cell number in the blastocyst was lower in the cloned embryos than in either cloned aggregated or fertilized embryos. The total cell number per embryo is an important criterion that determine the viability of the embryo after transfer into a surrogate mother [12,13]. The low number of cell in the cloned embryo was associated with a low percentage of survivability after embryo transfer in several species [18,19,[29][30][31][32]. Chesne et al. [30] have reported around 40% reduction in cell number of the cloned rabbit embryos compared to control counterparts. ...
... Early embryos vary in their responses to different environments, and fetuses with defects may suffer abortion more easily. Actually, in cat, a certain number of proteins are abnormally expressed in 21-day-old placentas of fetuses derived from cloned embryo transfer, and this phenomenon is suggested to be associated with fetal loss during pregnancy [21]. Recent studies have suggested that most X-linked genes may function not only in sex-related transcriptional differences, but also in the regulation of autosomal gene expression in fetuses. ...
Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.
Ethnopharmacological relevance
Shoutai Wan (STW) is a classic herbal formula for the treatment of recurrent spontaneous abortion (RSA), and clinical studies have shown the effectiveness of STW on RSA. However, the molecular mechanism of STW treatment of RSA is still unclear.
Methods
(1) Animal experiments: The normal pregnancy model was established with CBA/J*BALB/C, and the RSA model was established by CBA/J*DBA/2. The RSA model CBA/J*DBA/2 pregnant mice were randomly divided into four groups (RSA model group, STW low, medium and high dose groups) according to the order of pregnancy, respectively. The drug administration starts from the first day of pregnancy to the 14th day of pregnancy. The embryo loss rate (ELR) of each group was calculated. (2) Proteomic analysis of decidual tissue: The total protein of decidual tissue of each group was isolated by solid phase pH gradient 2-DE technique. The differentially expressed protein spots were analyzed and identified by PDQuest images; the peptide quality fingerprinting (PMF) was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then, the proteins were identified by Mascot software searching, their functions were identified by bioinformatics strategy. (3) The expression of HSP27, α-enolase and Transferrin was detected by Western blotting and the expression of Annexin A2 and Transferrin was detected by immunohistochemistry. (4) The differential proteins and potential targets were analyzed by systematic biological strategy.
Results
(1) Compared with the normal group, the ELR in the RSA model group was significantly higher (P < 0.01). Compared with the model group, the ELR in the STW high, medium dose groups was lower (P < 0.01). (2) A 2-DE map of the decidual tissue of the RSA model group, normal pregnancy group, STW low, medium and high dose groups was established. Thirty proteins were identified. (3) The results of western blot showed that the expression of HSP27 and a-enolase in the RSA model group was higher than that in the normal group, and the expression of transferrin was lower (P <0.01). Compared with the model group, the expression of HSP27 and a-enolase in STW high, medium dose groups was decreased (P <0.01); Compared with the model group, the expression of Transferrin in the STW high dose group was increased (P < 0.01). (5) A lot of RSA treatment-related targets, biological processes and pathways were found after the systematic biological analysis.
Conclusion
(1) STW may reduce the ELR of the RSA mice. (2) The results of proteomics suggest that RSA is a complex process involving multiple proteins. STW can regulate the expression of various proteins in the decidual tissue of RSA mice, suggesting that it can act on multiple targets. (3) The results of western blotting of HSP27, a-enolase, transferrin were consistent with the results of proteomic analysis. (4) STW may achieve therapeutic effects by interfering with the targets, biological processes and signaling pathways discovered in this study.
The peptidyl-prolyl cis/trans isomerase (PPIase) activity and the expression of cyclophilins were studied in 6-day-old rabbit preimplantation embryos cultured under physiological and increased oxygen concentrations of 5% and 20% O(2), respectively. The PPIase activity was completely inhibited by cyclosporin A (CsA). The inhibitor of FK506-binding proteins, rapamycin, had no effect on the PPIase activity, indicating that the PPIase activity in rabbit blastocysts originates from cyclophilins. Using CsA affinity chromatography, only one cyclophilin with a molecular mass of about 17.8 kDa was separated. The cDNA of rabbit cyclophilin was cloned and sequenced. Analysis of the 682-base pair cDNA revealed an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular weight of 17.83 kDa. Homologies of 90% and 96% for the cDNA and amino acid sequence, respectively, to the human CyP18 were found, suggesting that the novel rabbit cyclophilin is a member of the CyP18 family (rabCyP18). The transcription level of rabCyP18 mRNA was 8.3 +/- 0.6 pg in 100 ng total RNA in noncultured blastocysts. In vitro culture with moderate oxygen stress (20% O(2)) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O(2). Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.
Because relevant biochemical changes are known to begin at the choriodecidual interface some weeks before actual clinical onset of labor, we hypothesized that the preterm choriodecidua may display gene and protein expression patterns specific to preterm labor. Transcriptomic (microarray) and proteomic (2-dimensional gel electrophoresis [2DGE]) profiling methodologies were used to compare changes in choriodecidual tissue collected from women who delivered before 35 weeks of gestation following spontaneous preterm labor (n = 12) and gestation-matched nonlaboring controls (n = 7). Additionally, 2DGE was used to compare differences in protein expression during term and preterm labor and to construct a choriodecidual proteome map. Overall, expressed transcripts and proteins indicated active tissue remodeling independent of labor status and an association with inflammatory processes during labor. Spontaneous, infection-induced and abruption-associated preterm deliveries were each defined by distinct transcriptional profiles. Proteins osteoglycin and progesterone receptor component 2 (PGRMC2) were upregulated during term and preterm labor while galectin 1, annexin 3, annexin 5, and protein disulfide isomerase (PDI) were upregulated only during preterm labor, suggesting a probable association with the underlying pathology. Together, these results represent novel data that warrant further investigations to elucidate plausible causal relationships of these molecules with spontaneous preterm delivery.
POU5F1 and NANOG play important roles in the maintenance of embryonic stem cell pluripotency. Recently, we isolated cat embryonic stem (ES)-like cells from cat blastocysts generated in vivo. In an effort to identify genetic markers for the characterization of cat ES-like cells, we have determined the coding sequences (CDSs) of cat POU5F1 (cPOU5F1) and NANOG (cNANOG). The sequence identities of cPOU5F1 with orthologous genes of the human and mouse were 92 and 82%, respectively, at the nucleotide level and 94 and 83%, respectively, at the amino acid level. We identified POU-specific and POU homeodomain sequences in the CDS of cPOU5F1. The sequence identities of cNANOG with its human and mouse orthologs were 69 and 68%, respectively, at the nucleotide level and 69 and 58%, respectively, at the amino acid level. We identified a homeodomain, SMAD4 domain and tryptophan repeat domain (W/QXXXX) in the CDS of cNANOG. We examined the expression of cPOU5F1 and cNANOG mRNA in ES-like cells and fibroblast feeder cells by RT-PCR. Transcripts of cPOU5F1 and cNANOG were detected at a high level in ES-like cells. However, these two genes were undetectable in cat fibroblast feeder cells and 6 adult tissues. We also examined ES-like cells by immunocytochemistry and demonstrated that cPOU5F1 and cNANOG are present at high levels in cat ES-like cells and are undetectable in cat fibroblast feeder cells. These results confirmed that cat ES-like cells can be successfully isolated from in vivo-produced blastocysts and that the expression of cPOU5F1 and cNANOG can be used as a biomarker for characterization of cat ES-like cells.
Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.
Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.
Cytochrome c (Cyt c) is located within the mitochondrial intermembrane space, and it is an essential constituent of the respiratory chain. The translocation of Cyt c from mitochondria to the cytosol is an important step in the apoptotic signaling pathway, linking mitochondrial changes to the activation of execution caspases (Liu et al, 1996). Once translocated into the cytosol, Cyt c together with Apaf-1 and procaspase-9 form a multiprotein complex, the apoptosome, which initiates the activation of caspase-3 (Li P et al, 1997). Studies in cell-free sytems have shown that addition of Cyt c to cytosols directly activates caspase-3, whereas immunodepletion of Cyt c from cell homogenates prevents caspase-3 activation. Furthermore, microinjection of Cyt c into intact cells has been shown to induce apoptosis (Li F et al, 1997). Although Cyt c release has been observed in many experimental models of apoptosis, the mechanism of translocation across the outer mitochondrial membrane is unclear. One question is whether Cyt c release occurs by a selective transport mechanism into the cytosol, or by a nonselective change of permeability in the outer mitochondrial membrane. To distinguish between these two possibilities, we monitored the translocation of Cyt c and of the mitochondrial intermembrane space marker adenylate kinase (ADK).
Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.
The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8-/- embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8-/- lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8-/- embryos. Finally, we show that K8-/- concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8-/- embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.
This work was undertaken in order to study the developmental competence of nuclear transfer (NT) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.
Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly
and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated
that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined
annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected
macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block
was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339,
2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular
bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided
with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential
for the proper assembly of HIV in MDM.
The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.
Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy.
Most heat shock proteins (HSPs) have pro-survival functions. However, the role of HSP60, a mitochondrial matrix protein, is somewhat controversial with both pro-survival and pro-apoptotic functions reported. Here we show that in numerous apoptotic systems HSP60 protein accumulates in the cytosol. In BMD188-induced cell death, HSP60 accumulates in the cytosol with significant mitochondrial release. In contrast, in apoptosis induced by multiple other inducers, the cytosolic HSP60 accumulates without an apparent mitochondrial release. The short interfering RNA-mediated knockdown experiments revealed that in BMD188-induced apoptosis, HSP60 has a pro-death function and that the pro-death role of HSP60 seems to involve caspase-3 maturation and activation in the cytosol. In contrast, HSP60 appears to play a pro-survival role in other apoptotic systems where there is no apparent mitochondrial release as its knockdown promotes cell death. In these latter apoptotic systems HSP60 does not associate with active caspase-3. In both cases, HSP60 does not appreciably interact with Bax. Taken together, our results suggest the following: 1) cytosolic accumulation of HSP60 represents a common phenomenon during apoptosis induction; 2) cytosolic HSP60 accumulation during apoptosis occurs either with or without apparent mitochondrial release; and 3) the cytosolically accumulated HSP60 possesses either pro-survival or pro-death functions, which involves differential interactions with caspase-3.
Prohibitin 1 (PHB1) is a highly conserved protein that is mainly localized to the inner mitochondrial membrane and has been implicated in regulating mitochondrial function in yeast. Because mitochondria are emerging as an important regulator of vascular homeostasis, we examined PHB1 function in endothelial cells. PHB1 is highly expressed in the vascular system and knockdown of PHB1 in endothelial cells increases mitochondrial production of reactive oxygen species via inhibition of complex I, which results in cellular senescence. As a direct consequence, both Akt and Rac1 are hyperactivated, leading to cytoskeletal rearrangements and decreased endothelial cell motility, e.g., migration and tube formation. This is also reflected in an in vivo angiogenesis assay, where silencing of PHB1 blocks the formation of functional blood vessels. Collectively, our results provide evidence that PHB1 is important for mitochondrial function and prevents reactive oxygen species-induced senescence and thereby maintains the angiogenic capacity of endothelial cells.
Pregnancy loss can be defined as an abortion which happens before 20 weeks of gestation. We will call those abortions occurring within the first 12 weeks early pregnancy loss (EPL). In 1956 Hertig et al. [1] described the morphologic features of abnormal eggs for the first time. In their study, they addressed the problem of pregnancy loss at the previllous stage and considered that it amounted to more or less 32% before the establishment of gestational amenorrhea. It is supposed that roughly 50% of all conceptions abort [2]. Moreover, the frequency of recurrent abortion (repeated pregnancy loss) increases after each incident: it has been evaluated at 12% after one loss, 25% after two, and 39% after three. The rate may increase even further with subsequent abortions [3]. By definition, habitual (recurrent) abortion needs three consecutive losses. Some authors consider that two consecutive incidents might be sufficient since the likelihood of pregnancy loss has increased twofold. Conversely, after a normal pregnancy, with a healthy child, the likelihood of subsequent abortion decreases.
Objective
To improve the precision and reliability of estimates of the association between preoperative serum albumin concentration and surgical outcomes.
Design
Prospective observational study. Patients were followed up for 30 days postoperatively. Multiple logistic regression models were developed to evaluate serum albumin level as a predictor of operative mortality and morbidity in relation to 61 other preoperative patient risk variables.
Setting
Forty-four tertiary care Veterans Affairs (VA) medical centers.
Patients
A total of 54,215 major noncardiac surgery cases from the National VA Surgical Risk Study.
Main Outcome Measures
Thirty-day operative mortality and morbidity.
Results
A decrease in serum albumin from concentrations greater than 46 g/L to less than 21 g/L was associated with an exponential increase in mortality rates from less than 1% to 29% and in morbidity rates from 10% to 65%. In the regression models, albumin level was the strongest predictor of mortality and morbidity for surgery as a whole and within several subspecialties selected for further analysis. Albumin level was a better predictor of some types of morbidity, particularly sepsis and major infections, than other types.
Conclusions
Serum albumin concentration is a better predictor of surgical outcomes than many other preoperative patient characteristics. It is a relatively low-cost test that should be used more frequently as a prognostic tool to detect malnutrition and risk of adverse surgical outcomes, particularly in populations in whom comorbid conditions are relatively frequent.
Objective To investigate levels of messenger RNA (mRNA) encoding thiol protein disulphide isomerase, in human amnion, chorion and placenta during pregnancy and in relation to term and preterm labour.
Design Amnion, chorion and placenta from 33 women delivered between 24 and 41 weeks of gestation were used in the study.
Setting Reproductive Molecular Research Group, Department of Obstetrics and Gynaecology, University of Cambridge Clinical School, Rosie Maternity Hospital, Cambridge.
Results Women who were delivered spontaneously before 30 weeks of gestation had higher levels of mRNA encoding thiol protein disulphide isomerase in placenta and chorion than those who were delivered spontaneously after this time (placenta P < 0.01, chorion P < 0.01) and compared with those who were delivered by elective caesarean section before 30 weeks of gestation (placenta P < 0.001, chorion P < 0.05). In the group in whom spontaneous labour occurred, at all gestations studied, there were increased levels of mRNA encoding thiol protein disulphide isomerase in the placenta (P < 0.001) and chorion (P < 0.001) compared with the amnion.
Conclusion Changes in the steady state level of mRNA encoding thiol protein disulphide isomerase may play a role in the onset of preterm labour before 30 weeks of gestation.
The finestructural changes of the interareolar porcine placenta during pregnancy are described. After perfusion fixation of the placenta the change in the thickness of the placental barrier from day 30 to day 110 of gestation is much more evident than after immerson fixation as has been used by all former authors. The alterations are due to the indentation of both the trophoblast and uterine epithelium by their corresponding capillary-network. This indentation is limited to the lateral wall and the summit of the chorionic ridges, while at the base the trophoblast as well as the uterine epithelium remains high columnar.This indicates that in the interareolar porcine placenta, which is represented by the chroionic ridges and the corresponding endometrial folds, at least two different areas with different structure and function may be discerned.1)
The lateral side and the top of the chorionic ridges seem to be predestinated for gaseous exchange. The placental barrier in this area is often less than 2 m.
2)
The transport of blood-borne nutrients takes place at the base of the chorionic ridges. This transport seems to be facilitated by an intercellular channel system between the uterine epithelial cells.
The ultrastructure of the areolae in the porcine placenta is described. The areolae occur on day 30 of pregnancy as a dome-shaped formation over the openings of the uterine glands. The lumen of the areolae is filled with the secretions of the uterine glands, the so-called histiotroph. The areolae lining epithelium is high collumnar, possesing long microvilli, a well-developed apical tubular system and numerous coated vesicles. This indicates that the epithelium has a high absorptive capacity. Our histochemical investigations reveal a high content of glycoproteins within the areolar lumen. The importance of one of the glycoprotein components of the histiotroph, uteroferrin, is discussed in connection with iron transfer from mother to the fetus.
The ultrastructure of feline placental labyrinth has been studied from the 45th to the 63rd day of gestation. The endotheliochorial structure is composed of: a thick hypertrophied maternal endothelium with giant decidual cells; an “interstitial inert substance”; a continuous syncytial layer (syncytiotrophoblast) and a discontinuous cellular layer (cytotrophoblast) of trophoblast; and a thin foetal endothelium with its surrounding mesenchyme. Foetal capillaries increasingly invade the syncytiotrophoblast. Maternal endothelial hypertrophy is reduced in the last days of gestation. Thus the interval between maternal and foetal bloods may in some areas become 2 μ, in late pregnancy.
Foetal endothelium, syncytiotrophoblast, and maternal endothelium demonstrate ultrastructural features of transfer function. The “interstitial inert substance”, which separates maternal from foetal tissue was always evident though with variable thickness. The giant decidual cells which are reduced in late pregnancy, show one or several “glycogen bodies”. The syncytiotrophoblast, where some steroïd biosynthesis has been demonstrated, has an extensive rough endoplasmic reticulum suggestive of protein hormone synthesis activity.
Endothelial cell migration and proliferation, central steps in both physiologic and pathologic angiogenesis, require cytoskeletal-dependent remodeling, which is, in large part, achieved by the dynamic regulation of the β-actin network. Specifically, the β-actin network has previously been shown to be (i) enriched in regions of highly motile cytoplasm, and (ii) modulated by its isoactin-specific barbed-end capping protein, βcap73. We hypothesize that regulated over-expression of βcap73 could disrupt angiogenesis by capping β-actin-filament assembly thus inhibiting the incipient cellular migration and microvascular morphogenesis that ensues. Indeed, upon infection of capillary endothelial cells (cEC) with an adenovirus encoding the full-length βcap73 (Ad-βcap73), there is a robust cellular rounding response that occurs concomitantly with cytoskeletal disruption, as visualized with immunofluorescence microscopy. Further, we demonstrate that over-expression of Ad-βcap73 inhibits cEC migration in wound healing studies. Quantitative in vitro angiogenesis assays reveal that Ad-βcap73 not only prevents endothelial cells from forming capillary-like networks, but also induces the collapse of preformed endothelial tubes. In testing whether Ad-βcap73 impairs angiogenic events by inducing anoikis/apoptosis, we demonstrate that βcap73 infection activates a caspase-3-mediated cell death response as observed by quantitative Western blotting and immunofluorescence analyses. Altogether, these findings suggest that endothelial-specific targeting and βcap73 over-expression may represent an innovative therapeutic approach capable of abrogating pathologic angiogenesis.
Production of cloned mammals by somatic cell nuclear transfer is associated with functional and structural abnormalities of placentation and with abnormal fetal development. A proteomic analysis was performed in domestic cats (Felis catus) to compare cloned term placentas (CTP) obtained from cesarean section (CS) to control placentas obtained from CS or vaginal delivery. The expression of 20 proteins was altered in CTP (p<0.05) compared to control placentas. The two control groups showed that the method of delivery, vaginal delivery or CS, did not affect protein expression (p>0.05). A total of 13 proteins were up-regulated in CTP, including apoptosis-related cathepsin D (CD), annexin A1 and heat shock protein 27 (HSP 27), and seven proteins were down-regulated in CTP, including prohibitin (PHB). The expression of PHB and CD was confirmed by Western blotting and immunofluorescence staining. The abnormal expression of PHB and CD correlated with the generation of reactive oxygen species, leading to decreased mitochondrial membrane potential and telomeric DNA, which are associated with cellular senescence and apoptosis. In summary, a specific pattern of abnormal protein expression is associated with the impaired development and functions of cloned placentas and hence with decreased fetal viability. Strategies aimed at restoring normal placental protein expression may increase the efficiency of somatic cell nuclear transfer and transgenic cat production and help restore endangered species.
Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse.
The ultrastructure of feline placental labyrinth has been studied from the 45th to the 63rd day of gestation. The endotheliochorial structure is composed of: a thick hypertrophied maternal endothelium with giant decidual cells; an 'interstitial inert substance': a continuous syncytial layer (syncytiotrophoblast) and a discontinuous cellular layer (cytotrophoblast) of trophoblast; and a thin fetal endothelium with its surrounding mesenchyme. Fetal capillaries increasingly invade the syncytiotrophoblast. Maternal endothelial hypertrophy is reduced in the last days of gestation. Thus, the interval between maternal and fetal blood may in some areas become 2μ, in late pregnancy. Fetal endothelium, syncytiotrophoblast, and maternal endothelium demonstrate ultrastructural features of transfer function. The 'interstitial inert substance', which separates maternal from fetal tissue was always evident though with variable thickness. The giant decidual cells which are reduced in late pregnancy, show one or several 'glycogen bodies'. The syncytiotrophoblast, where some steroid biosynthesis has been demonstrated, has an extensive rough endoplasmic reticulum suggestive of protein hormone synthesis activity.
1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.
The finestructural changes of the interareolar porcine placenta during pregnancy are described. After perfusion fixation of the placenta the change in the thickness of the placental barrier from day 30 to day 110 of gestation is much more evident than after immerson fixation as has been used by all former authors. The alterations are due to the indentation of both the trophoblast and uterine epithelium by their corresponding capillary-network. This indentation is limited to the lateral wall and the summit of the chorionic ridges, while at the base the trophoblast as well as the uterine epithelium remains high columnar. This indicates that in the interareolar porcine placenta, which is represented by the chorionic ridges and the corresponding endometrial folds, at least two different areas with different structure and function may be discerned. 1) The lateral side and the top of the chorionic ridges seem to be predestinated for gaseous exchange. The placental barrier in this area is often less than 2 micrometers. 2) The transport of blood-borne nutrients takes place at the base of the chorionic ridges. This transport seems to be facilitated by an intercellular channel system between the uterine epithelial cells.
Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
The placenta has multiple functions, being the organ which provides oxygen and nutrients to the developing conceptus. In the placenta, the enzyme carbonic anhydrase (CA) may provide ions for exchange with Na+, K+, and Cl- in transepithelial movement of ions and fluid, as well as facilitating carbon dioxide diffusion. It can also be active in intermediary metabolism, such as gluconeogenesis, urea, and fatty acid synthesis. Placental material from pig, horse, cow, mink, rat, and human was therefore investigated, representing placenta types with variations in shape, internal architecture, and nature of the interhemal barrier. After glutaraldehyde fixation, sections were stained by a histochemical CA-method demonstrating all active isozymes. The most striking feature in common was a positive reaction in the maternal capillaries, when present, as in pig, horse, cow, and mink. In the maternal epithelium, the activation of CA was only observed in the pig, which also exhibited the strongest activity at the maternal interface, which reacted moderately in rat, weakly in horse, and was not visible in cow and human. The trophoblast was positive in pig and rat, whereas it was negative in horse, cow, human, and mink placentae except for few scattered trophoblast cells in pig, horse, and cow, which showed very intense activity. In the fetal capillaries, a positive reactivity was only observed in mink and human. The utilization of CA in placental transfer and metabolism is thus highest in the pig, rat, and mink, compared with horse, cow, and human. It can therefore be concluded that the activation and localization of CA in the placental interhemal barrier varies considerably among species.
To improve the precision and reliability of estimates of the association between preoperative serum albumin concentration and surgical outcomes.
Prospective observational study. Patients were followed up for 30 days postoperatively. Multiple logistic regression models were developed to evaluate serum albumin level as a predictor of operative mortality and morbidity in relation to 61 other preoperative patient risk variables.
Forty-four tertiary care Veterans Affairs (VA) medical centers.
A total of 54215 major noncardiac surgery cases from the National VA Surgical Risk Study.
Thirty-day operative mortality and morbidity.
A decrease in serum albumin from concentrations greater than 46 g/L to less than 21 g/L was associated with an exponential increase in mortality rates from less than 1% to 29% and in morbidity rates from 10% to 65%. In the regression models, albumin level was the strongest predictor of mortality and morbidity for surgery as a whole and within several subspecialties selected for further analysis. Albumin level was a better predictor of some types of morbidity, particularly sepsis and major infections, than other types.
Serum albumin concentration is a better predictor of surgical outcomes than many other preoperative patient characteristics. It is a relatively low-cost test that should be used more frequently as a prognostic tool to detect malnutrition and risk of adverse surgical outcomes, particularly in populations in whom comorbid conditions are relatively frequent.
We recently reported the identification of human calumenin, a novel Ca(2+) binding, transformation-sensitive and secreted protein [Vorum et al. (1998) Biochim. Biophys. Acta 1386, 121-131; Vorum et al. (1999) Exp. Cell Res. 248, 473-481] belonging to the family of multiple EF-hand proteins of the secretory pathway that include reticulocalbin, ERC-55, Cab45 and crocalbin. In order to further investigate the extracellular functions of calumenin we immobilized the recombinant protein to a column. After application of a placental tissue extract we were able to elute one protein that interacts with calumenin in the presence of Ca(2+). Amino acid sequencing identified this protein as serum amyloid P component (SAP). Furthermore, we verified and characterized the calumenin-SAP interaction by the surface plasmon resonance technique. The findings indicate that calumenin may participate in the immunological defense system and could be involved in the pathological process of amyloidosis that leads to formation of amyloid deposits seen in different types of tissues.
Matricellular proteins form a group of extracellular matrix (ECM) proteins that do not subserve a primary structural role, but rather function as modulators of cell-matrix interactions. Members of the group, including thrombospondin (TSP)-1,TSP-2, SPARC, tenascin (TN)-C, and osteopontin (OPN), have been shown to participate in a number of processes related to tissue repair. Specifically, studies in knockout mice have indicated that a deficiency in one or more of these proteins can alter the course of wound healing. More recently, TSP1, TSP2, and SPARC have also been implicated in the foreign body response, an unusual reaction to injury that occurs after the implantation of biomaterials. This review will focus on the roles of these proteins in the response to injury in mice and will show how studies of this pathophysiological process can elucidate some of the intrinsic properties of these matricellular proteins.
Here, we present the comparative analysis of the two keratin (K) gene clusters in the genomes of man, mouse and rat. Overall, there is a remarkable but not perfect synteny among the clusters of the three mammalian species. The human type I keratin gene cluster consists of 27 genes and 4 pseudogenes, all in the same orientation. It is interrupted by a domain of multiple genes encoding keratin-associated proteins (KAPs). Cytokeratin, hair and inner root sheath keratin genes are grouped together in small subclusters, indicating that evolution occurred by duplication events. At the end of the rodent type I gene cluster, a novel gene related to K14 and K17 was identified, which is converted to a pseudogene in humans. The human type II cluster consists of 27 genes and 5 pseudogenes, most of which are arranged in the same orientation. Of the 26 type II murine keratin genes now known, the expression of two new genes was identified by RT-PCR. Kb20, the first gene in the cluster, was detected in lung tissue. Kb39, a new ortholog of K1, is expressed in certain stratified epithelia. It represents a candidate gene for those hyperkeratotic skin syndromes in which no K1 mutations were identified so far. Most remarkably, the human K3 gene which causes Meesmann's corneal dystrophy when mutated, lacks a counterpart in the mouse genome. While the human genome has 138 pseudogenes related to K8 and K18, the mouse and rat genomes contain only 4 and 6 such pseudogenes. Our results also provide the basis for a unified keratin nomenclature and for future functional studies.
Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT.
In the endotheliochorial placenta of the cat, the maternal surface epithelium and parts of the connective tissue have to be removed to bring the fetal blood vessels in close contact to the maternal capillaries. The composition of the extracellular matrix (ECM) in the feline uterus is not known and it is still not clear if and which parts of the maternal ECM persist during gestation in the placental labyrinth. We demonstrated various extracellular matrix components (collagen types I, III, IV, and laminin) and matrix metalloproteinases (MMP-1, -2, -13) using immunohistochemistry and studied the distribution of intermediate filaments (vimentin, cytokeratin) and alpha-smooth muscle actin (SMA) in the placental girdle on specimens of different stages of gestation. Collagen types I and III were mainly present in the fetal chorionic lamellae whereas diminished in the maternal placental labyrinth part. Collagen IV and laminin were expressed in fetal basement membranes and mesenchyme. Maternal endothelial cells and stromal cells showed a positive immunoreaction for anti-collagen type IV and laminin. MMP-2 was identified in the maternal stroma, including decidual cells. Endothelia of maternal blood vessels within the labyrinth contained MMP-1, -2 and -13, probably associated with angiogenesis. In the trophoblast MMP-1 and -13 were demonstrated. Maternal stem vessels were accompanied by a thick layer of syncytiotrophoblast. Around these vessels, collagen type I and SMA were present in a periendothelial region between the endothelium and the trophoblast. These findings indicate that a strictly regulated balance between ECM deposition and ECM degradation in the feline placental labyrinth is necessary for proper placental development and function.
The heterogeneous nuclear ribonucleoproteins (hnRNP) are a family of proteins which share common structural domains, and extensive research has shown that they have central roles in DNA repair, telomere biogenesis, cell signaling and in regulating gene expression at both transcriptional and translational levels. Through these key cellular functions, individual hnRNPs have a variety of potential roles in tumour development and progression including the inhibition of apoptosis, angiogenesis and cell invasion. The aims of this review are to provide an overview of the multi functional roles of the hnRNPs, and how such roles implicate this family as regulators of tumour development. The different stages of tumour development that are potentially regulated by the hnRNPs along with their aberrant expression profiles in tumour tissues will also be discussed.
Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.
Fertilization of mammalian eggs is followed by successive cell divisions
and progressive differentiation, first into the early embryo and
subsequently into all of the cell types that make up the adult animal.
Transfer of a single nucleus at a specific stage of development, to an
enucleated unfertilized egg, provided an opportunity to investigate
whether cellular differentiation to that stage involved irreversible
genetic modification. The first offspring to develop from a
differentiated cell were born after nuclear transfer from an
embryo-derived cell line that had been induced to become
quiescent1. Using the same procedure, we now report the birth
of live lambs from three new cell populations established from adult
mammary gland, fetus and embryo. The fact that a lamb was derived from
an adult cell confirms that differentiation of that cell did not involve
the irreversible modification of genetic material required for
development to term. The birth of lambs from differentiated fetal and
adult cells also reinforces previous speculation1,2 that by
inducing donor cells to become quiescent it will be possible to obtain
normal development from a wide variety of differentiated cells.
The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.
A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.
Messenger RNA encoding thiol protein disulphide isomerase in amnion, chorion and placenta in human term and preterm labour
- J J Morrison
- D S Charnock-Jones
- S K Smith
Morrison JJ, Charnock-Jones DS, Smith SK. Messenger RNA encoding
thiol protein disulphide isomerase in amnion, chorion and placenta
in human term and preterm labour. Br J Obstet Gynaecol 1996;103:
873-8.
Viable offspring derived from fetal and adult mammalian cells
- Wilmut
Messenger RNA encoding thiol protein disulphide isomerase in amnion, chorion and placenta in human term and preterm labour
- Morrison