Improvement of allograft viability with organs procured from non-heart-beating donors in porcine liver transplantation.
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Animals↗
Biomedical subjects
M Gu
Publications and source records attributed to M Gu.
Cytokeratin immunoreactivity in Ewing's sarcoma: prevalence in 50 cases confirmed by molecular diagnostic studies.
Ewing's sarcoma (ES) and primitive neuroectodermal tumor (PNET) are characterized by the presence of the specific t(11;22)(q24;q12) or variants thereof, producing diagnostic EWS fusion transcripts. Cytokeratin has been reported sporadically to be expressed in some cases of ES/PNET. However, its prevalence has not been assessed systematically in a series of cases with confirmatory molecular or cytogenetic evidence of a diagnostic translocation. We present in detail three index patients in whom strong cytokeratin immunoreactivity was a confounding factor in the diagnosis. To establish further the prevalence of cytokeratin immunoreactivity in a series of well-characterized ES/PNET, we then performed immunohistochemical studies with antibodies CAM5.2 and AE1/AE3 on 50 cases of ES/PNET diagnosed at Memorial Sloan-Kettering Cancer Center in which molecular evidence of a specific ES/PNET-associated translocation were available. Immunoreactivity to cytokeratin was present in 10 cases (20%), in five diffusely and five focally. There was no significant association between cytokeratin expression and the following parameters: patient age, sex, skeletal and extraskeletal primary site, and the type of EWS fusion transcript. Cytokeratin expression, a manifestation of epithelial differentiation, is present in as many as 20% of ES/PNET in either a diffuse or focal pattern.
Adult↗
Nitric oxide-induced increase in p21(Sdi1/Cip1/Waf1) expression during the cell cycle in aortic adventitial fibroblasts.
This study was performed to investigate whether the expression of p21(Sdi1/Cip1/Waf1), one of the cyclin-dependent kinase inhibitor proteins, could be regulated by nitric oxide (NO) and might account for the antiproliferative effect of NO. Quiescent adventitial fibroblasts were stimulated to proliferate by serum addition and by NO donors added during different phases of the cell cycle. [(3)H]Thymidine incorporation was markedly reduced by S-nitroso-N-acetyl-penicillamine (SNAP) added either with serum at quiescence or at later time point in the cell cycle. Northern and Western blot analyses showed that addition of SNAP either at quiescence or 15 hours after serum addition induced a rapid induction of p21 mRNA and protein. Immunoprecipitation studies and electrophoretic mobility shift analysis indicate that the treatment of cells with SNAP induced the phosphorylation of p53 (a tumor suppressor protein) and enhanced the ability of p53 to bind DNA when SNAP was added during the cell cycle. The increased expression of p21 mRNA or p53 activation during late G(1) or S phase was also caused by addition of 8-bromo-cGMP and effectively blocked by a specific inhibitor of the soluble guanylate cyclase. Furthermore, this response to SNAP was blocked by an inhibitor of protein kinase G. These studies implicate NO as a potential regulator of the cell cycle in aortic adventitial fibroblasts through a cGMP-mediated transcriptional mechanism involving the induction of p21.
Animals↗
Laminin and alpha7beta1 integrin regulate agrin-induced clustering of acetylcholine receptors.
The clustering of acetylcholine receptors (AChRs) in the post-synaptic membrane of skeletal muscle is an early developmental event in the formation of the neuromuscular junction. Several studies show that laminin, as well as neural agrin, can induce AChR clustering in C2C12 myofibers. We recently showed that specific isoforms of the alpha7beta1 integrin (a receptor normally found at neuromuscular junctions) colocalize and physically interact with AChR clusters in a laminin-dependent fashion. In contrast, induction with agrin alone fails to promote localization of the integrin with AChR clusters. Together both agrin and laminin enhance the interaction of the integrin with AChRs and their aggregation into clusters. To further understand this mechanism we investigated cluster formation and the association of the alpha7beta1 integrin and AChR over time following induction with laminin and/or agrin. Our results show that the alpha7beta1 integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable association of the integrin and AChRs and this association is independent of clustering. In addition to laminin-1, merosin (laminin-2/4) is present both before and after formation of neuromuscular junctions and also promotes AChR clustering and colocalization with the integrin as well as synergism with agrin. Using site directed mutagenesis we demonstrate that a tyrosine residue in the cytoplasmic domain of both (&agr;)7A and (&agr;)7B chains regulates the localization of the integrin with AChR clusters. We also provide evidence that laminin, through its association with the alpha7beta1 integrin, reduces by 20-fold the concentration of agrin required to promote AChR clustering and accelerates the formation of clusters. Thus laminin, agrin and the alpha7beta1 integrin act in a concerted manner early in the development of the post-synaptic membrane, with laminin priming newly formed myofibers to rapidly and vigorously respond to low concentrations of neural agrin produced by innervating motor neurons.
Agrin↗
[Information behavior of microsatellite loci in genome scanning].
OBJECTIVE: To determine the heterozygosity and polymorphism information content of 139 microsatellite loci in Han population. METHODS: Multiplex approach was used to analyze the 139 loci. The amplified fragments were subjected to electrophoresis in PAGE gel and analyzed with Genescan( TM) and Genotyper(TM). RESULTS: The heterozygosities are between 0.35 to 0.89, with the heterozygosities of 88% loci >0.60. PICs are in the range of 0.32 and 0.88, with PICs of 95% loci >0.50. CONCLUSION: The polymorphism information content of microsatellite loci is very high and their distribution varies in different races and populations.
Aged↗
[Proliferation of attenuated infections bursal disease virus(IBDV) on Vero cell on large scale].
Vero cell was selected as productive carrier in production of Attenuated IBDV. The optimal conditions in culturing IBDV in agitating bottle were figured out. After proliferation, supernatant was harvested in batch process under the condition of five-liter-agitated fermenter. The result shows it was successful to culture IBDV by this methodology.
Animals↗
Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.
We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.
14-3-3 Proteins↗
The cytoplasmic domain of the platelet glycoprotein Ibalpha is phosphorylated at serine 609.
The alpha chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIbalpha is phosphorylated at Ser(609); this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIbalpha C-terminal sequence S(606)GHSL(610) only when Ser(609) is phosphorylated. Immunoabsorption with anti-pS609 removed almost all of the GPIbalpha from platelet lysates, indicating a high proportion of GPIbalpha phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3zeta. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3zeta binding. A synthetic phosphopeptide corresponding to the GPIbalpha C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3zeta. Thus, phosphorylation at Ser(609) of GPIbalpha is important for 14-3-3zeta binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIbalpha staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIbalpha molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser(609) of GPIbalpha regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading.
14-3-3 Proteins↗
Early but not delayed continuous arteriovenous hemofiltration improves cardiovascular function in sepsis in dogs.
OBJECTIVE: Continuous arteriovenous hemofiltration (CAVH) has been advocated as treatment to remove inflammatory mediators and thereby to improve hemodynamic parameters in sepsis. However, the results obtained with CAVH have been inconsistent. In a canine model of bacteremic Pseudomonas aeruginosa pneumonia, we tested the hypothesis that the time course of the institution of CAVH may be important in obtaining a beneficial treatment effect. METHODS: Two protocols were performed in phenobarbital-anesthetized dogs. In the early hemofiltration study (EHS), CAVH for 3 h was initiated 2 h post-pneumonia before mean arterial pressure (MAP) fell. In the late hemofiltration study (LHS), CAVH for 3 h was initiated at 5 h post-pneumonia when a decrease in MAP had already occurred. Hemodynamic measurements included cardiac output (CO), stroke volume (SV), and stroke work (SW). Myocardial depressant activity [filterable cardiodepressant substance (FCS)] found in plasma was assessed by bioassay at each measurement interval. RESULTS: In EHS, after 5 h of sepsis, SW, CO, and SV in the hemofiltered pneumonia group were higher as compared with the nonhemofiltered pneumonia group. In contrast, in LHS, no differences in hemodynamic parameters were found between the two pneumonia groups. In both EHS and LHS, plasma FCS activity was decreased to similar extents by CAVH. CONCLUSION: These results suggest the time course of institution of CAVH may be important in obtaining a beneficial treatment effect in sepsis.
Animals↗
Determination of power and sample size in the design of clinical trials with failure-time endpoints and interim analyses.
An important but difficult task in the design of a clinical trial to compare time to failure between two treatment groups is determination of the number of patients required to achieve a specified power of the test. Because patients typically enter the trial serially and are followed until they fail or withdraw from the study or until the study is terminated, the power of the test depends on the accrual pattern, the noncompliance rate, and the withdrawal rate in addition to the actual survival distributions of the two groups. Incorporating interim analyses and the possibility of early stopping into the trial increases its complexity, and although normal approximations have been developed for computing the significance level of the test when the log-rank or other rank statistics are used, there are no reliable analytic approximations for evaluating the power of the test. This article presents methods, based on Monte Carlo simulations and recent advances in group sequential testing with time-to-event responses, to choose appropriate test statistics, compute power and sample size at specified alternatives, check the adequacy of commonly used normal approximations of the type I error probability, and assess the performance of different interim analysis strategies. It also presents two computer programs implementing these methods.
Adrenergic beta-Antagonists↗
Ceramide second messengers and ceramide assays.
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Ceramides↗
On the complexities of ceramide changes in cells undergoing apoptosis: lack of evidence for a second messenger function in apoptotic induction.
The generation of cellular ceramides as a second messenger has been implicated as a regulatory and required step for the induction of apoptosis. In this study, we have applied a recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cells and the chemical agents anisomycin or geranylgeraniol in HL-60 cells. The mass spectrometric method has significant advantages over traditional methods for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously. We quantitiated ceramides ranging from C14 to C26, finding that their basal levels and relative distribution varied significantly, both within and between different cell types. However, we were not able to detect any significant changes in either total ceramide content or species distribution until 1 h or more post-stimulation with any of these treatments, by which time the cells were in an advanced stage of apoptosis. Differences were also seen between all three treatments in the ceramide species distribution observed in these late stages of apoptosis. These data indicate that in vivo ceramide generation occurs as a consequence of apoptosis rather than as an essential second messenger involved in its induction. They also pose new questions about the potential roles that certain ceramide species may play in the late stages of apoptosis, and demonstrate a clear need to utilize the resolving power of mass spectrometry-based assays in any future investigations into the biological function of ceramides.
Anisomycin↗
[The expression of the receptors of estrogen, progesterone, epidermal growth factor and growth hormone in uterine adenomyosis and a comparative study after treatment with androgen].
OBJECTIVES: To investigate the pathogenesis of adenomyosis and its immunopathological study after treatment with androgen. METHODS: Estrogen receptor (ER), progestoren receptor (PR), epidermal growth factor receptor (EGFR) and growth hormone (GH) were determined by immunohistochemical SABC method in 30 cases of adenomyosis (group I) and another 24 cases of adenomyosis accompanying leiomyoma (group II); ER, PR were determined with the same methods in 24 cases of adenomyosis who had been treated with methyltestosterone before operation (group III). RESULTS: (1) Both the eutopic and ectopic endometrium in group I showed ER, PR, EGFR, GH positive. The positive expression rate had no significant differences (P > 0.05). The strong positive rate of ER, PR in the eutopic endometrium were significantly higher than that in the ectopic (P < 0.05). (2) ER, PR, EGFR, GH in the eutopic and ectopic endometrium in group I and group II all showed positive but no significant differences (P > 0.05). (3) Both ER and PR showed positive in group I, II and group III. There were no significant differences (P > 0.05). (4) Among the 24 cases treated with androgen, the positive and strong positive rate of ER and PR in both eutopic and ectopic endometrium of 11 cases proved effective were higher than that of 13 ineffective cases and there were significant and striking differences (P < 0.05, < 0.01). CONCLUSIONS: ER, PR, EGFR, GH all expressed in the eutopic and ectopic endometrium. They played important roles in promoting the development of adenomyosis.
Adult↗
[Effects of integrins on the implantation of embryo, fetal development and obstetric and gynecologic diseases].
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Embryo Implantation↗
A novel ligand-binding site in the zeta-form 14-3-3 protein recognizing the platelet glycoprotein Ibalpha and distinct from the c-Raf-binding site.
We reported previously that the zeta-form 14-3-3 protein (14-3-3zeta) binds to a platelet adhesion receptor, glycoprotein (GP) Ib-IX, and this binding is dependent on the SGHSL sequence at the C terminus of GPIbalpha. In this study, we have identified a binding site in the helix I region of 14-3-3zeta (residues 202-231) required for binding to GPIb-IX complex and to the cytoplasmic domain of GPIbalpha. We also show that phosphorylation-dependent binding of c-Raf to 14-3-3zeta requires helix G (residues 163-187) but not helix I. Thus, the GPIbalpha-binding site is distinct from the binding sites for RSXpSXP motif-dependent ligands. Furthermore, we show that wild type 14-3-3zeta has a higher affinity for GPIb-IX complex than recombinant GPIbalpha cytoplasmic domain. Deletion of helices A and B (residues 1-32) disrupts 14-3-3zeta dimerization and decreases its affinity for GPIb-IX. Disruption of 14-3-3zeta dimerization, however, does not reduce 14-3-3zeta binding to recombinant GPIbalpha cytoplasmic domain. This suggests a dual site recognition mechanism in which a 14-3-3zeta dimer interacts with both GPIbalpha and GPIbbeta (known to contain a phosphorylation-dependent binding site), resulting in high affinity binding.
14-3-3 Proteins↗
A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering.
The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.
Agrin↗
An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene.
Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and/or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinase-based system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob/ob (leptin-deficient) mice. In contrast to evidence of liver toxicity, inflammation, and cellular infiltration observed with Ad-leptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications.
Adenoviridae↗
Plasmid DNA encoding transforming growth factor-beta1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model.
Transforming growth factor beta is a potent immunomodulator with both pro- and antiinflammatory activities. Based on its immunosuppressive actions, exogenous TGF-beta has been shown to inhibit autoimmune and chronic inflammatory diseases. To further explore the potential therapeutic role of TGF-beta, we administered a plasmid DNA encoding human TGF-beta1 intramuscularly to rats with streptococcal cell wall-induced arthritis. A single dose of 300 microg plasmid DNA encoding TGF-beta1, but not vector DNA, administered at the peak of the acute phase profoundly suppressed the subsequent evolution of chronic erosive disease typified by disabling joint swelling and deformity (articular index = 8.17+/-0. 17 vs. 1.25+/-0.76, n = 6, day 26, P < 0.01). Moreover, delivery of the TGF-beta1 DNA even as the chronic phase commenced virtually eliminated subsequent inflammation and arthritis. Both radiologic and histopathologic as well as molecular evidence supported the marked inhibitory effect of TGF-beta1 DNA on synovial pathology, with decreases in the inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and the expression of proinflammatory cytokines that characterize this model. Increases in TGF-beta1 protein were detected in the circulation of TGF-beta1 DNA-treated animals, consistent with the observed therapeutic effects being TGF-beta1 dependent. These observations provide the first evidence that gene transfer of plasmid DNA encoding TGF-beta1 provides a mechanism to deliver this potent cytokine that effectively suppresses ongoing inflammatory pathology in arthritis.
Animals↗