Biomedical subjects
Publications and source records attributed to M Gu.
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.
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.
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.
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.
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.
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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.
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.
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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.
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.
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.
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.
The cause of neuronal loss in patients with idiopathic Parkinson's disease is unknown. Oxidative stress and complex I deficiency have both been identified in the substantia nigra in Parkinson's disease but their place in the sequence of events resulting in dopaminergic cell death is uncertain. We have analysed respiratory chain activity, iron and reduced glutathione concentrations in Parkinson's disease substantia innominata and in the cingulate cortex of patients with Parkinson's disease, Alzheimer's disease and dementia with Lewy bodies to investigate their association with neuronal death and Lewy body formation. No abnormalities of mitochondrial function, iron or reduced glutathione levels were identified in Parkinson's disease substantia innominata or cingulate cortex. Mitochondrial function also appeared to be unchanged in cingulate cortex from patients with Alzheimer's disease and from patients with dementia with Lewy bodies, however, iron concentrations were mildly increased in both, and reduced glutathione decreased only in Alzheimer's disease. These results confirm the anatomic specificity of the complex I deficiency and decreased levels of reduced glutathione within the Parkinson's disease brain and suggest that these parameters are not associated with cholinergic cell loss in Parkinson's disease nor with Lewy body formation in this or other diseases. We propose that our data support a 'two-hit' hypothesis for the cause of neuronal death in Parkinson's disease.
Gene gun-mediated DNA vaccination stimulates an immune response characterized by the activation of IgG-secreting B cells and IFN-gamma-secreting T cells. To monitor the contribution of cells at the site of vaccination to this process, transfected skin was periodically removed and grafted onto naive recipients. Immediate removal of vaccinated skin abrogated the development of an immune response. Low-level IgG production was stimulated when the vaccination site was left in place for > or = 5 h, with the strength of this response increasing the longer the site remained intact (for up to 2 wk). Measurable primary T cell responses were observed in animals whose vaccination site remained in place for > or = 1 day. Skin grafts transferred 0 to 24 h postvaccination stimulated a primary immune response in naive recipients. Memory B and T cells were generated in animals whose site of vaccination remained intact for 5 to 12 h. Skin transferred within 12 h of vaccination triggered memory B and T cell development in graft recipients, while the removal of skin >12 h postvaccination did not reduce memory in vaccinated mice. These findings suggest that 1) primary immunity is induced by cells that migrate rapidly from the site of immunization, 2) nonmigratory cells influence the magnitude of this primary response, and 3) migratory cells alone are responsible for the induction of immunologic memory.
We identified and partially characterized another member of the uncoupling protein termed UCP3. Human and mouse UCP3 protein sequences are 86% identical to each other, and 73% and 59% identical to UCP2 and UCP1, respectively. Expression of human UCP3 in yeast resulted in a drastic decrease of mitochondria membrane potential. Northern analysis showed that UCP3 was highly expressed in skeletal muscle in human, rat, and mouse. Mapping of UCP3 placed it to the same chromosomal region of UCP2 in both human and mouse, a region that is linked to obesity and hyperinsulinemia. Furthermore, adenovirus-mediated leptin expression in obese ob/ob mice led to increased expression of UCP3 in skeletal muscle. The data indicate that UCP3 encodes a muscle-specific uncoupling protein that may play an important role in the regulation of energy expenditure and development of obesity.
Several groups have identified mitochondrial complex I deficiency in Parkinson's disease (PD) substantia nigra and in platelets. A search for any mitochondrial DNA (mtDNA) mutation underlying this defect has not yet produced any consistent result. We have made use of a mtDNA-less (p0) cell line to determine if the complex I deficiency follows the genomic transplantation of platelet mtDNA. From a preselected group of PD patients with low platelet complex I activity, 7 patients were used for detailed study. All 7 patients were used for mixed cybrid analysis and demonstrated a selective 25% deficiency of complex I activity. Individual clonal analysis of A549 p0/PD platelet fusion cybrids from 1 of the patients expressed combined complex I and IV deficiencies with 25% and 20% decreased activities in the PD clones, respectively. Histocytochemical, immunocytochemical, and cellular functional imaging studies of these clones showed the cells within the clones were heterogeneous with respect to cytochrome c oxidase (COX) function, COX I content, and mitochondrial respiratory chain activity. These results are in agreement with a previous study and support the proposition that an mtDNA abnormality may underlie the mitochondrial defect in at least a proportion of PD patients. This p0 technology may serve as a means to identify the subgroup of PD patients in whom an mtDNA defect may contribute to development of the disease.
Mitochondria play a critical role in cellular energy metabolism. The identification of a respiratory chain defect in Parkinson's disease (PD) provides not only a direct link with toxin models of parkinsonism but also insight into the mechanisms involved in etiology and pathogenesis. The presence of the complex I deficiency in PD substantia nigra and platelets suggests the involvement of a systemic cause. Genomic transplantation studies have been undertaken that involve the transfer to a novel nuclear background of mitochondrial DNA (mtDNA) from PD patients with a complex I defect, followed by both mixed and clonal expansion of the resulting cybrids. The mixed cybrids with the PD mtDNA expressed the complex I defect present in the original PD donor platelets. Clonal expansion of one such mixed cybrid culture produced a spectrum of clones with complex I and complex IV activities, ranging from severe deficiency to normal range, a pattern typical of a heteroplasmic mtDNA mutation. Histochemical, immunohistochemical, and functional assessments of delta psi(m) all showed a pattern in the PD clones typical of that produced by a mtDNA mutation. Patients with focal dystonia and a platelet complex I defect were used as disease controls for the cybrid studies. The mitochondrial abnormality was eradicated by transfer of dystonia mtDNA to a control nuclear background in both mixed and clonal cybrids, with no evidence of clonal heterogeneity. These results help to validate our findings in the PD patients and suggest that the complex I deficiency in dystonia is not due to an abnormality of mtDNA. We hypothesize that the mtDNA defect alone may be the cause of PD in a proportion of patients and may contribute to pathogenesis in others. Identification of the mtDNA genotype responsible for PD may allow the testing of neuroprotective strategies in appropriate patients.