Biomedical subjects
Publications and source records attributed to J Tu.
A series of compounds that utilize the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold was synthesized and shown to be highly effective inhibitors of recombinant human skin chymase.
Glucokinase (GK) is the rate-limiting enzyme in the glycolytic pathway of the beta-cell and, even in the rat fetus at 22-days gestation, immediately before birth, acts as a sensor of glucose influencing the rate of glucose utilization. However, when GK first appears in islets during beta-cell development is unknown. Whether GK is expressed in fetal glucagon-producing cells is also unknown. To determine this information, fetal rat islets were examined at 16-, 18-, and 22-days gestation. GK was identified immunocytochemically in both beta- and alpha-cells at all these ages, with the number of GK immunoreactive cells positively correlated to the fetal age from 16-22 days. Western blot analysis of islet protein extracts demonstrated the presence of GK, at 52 kDa, at 16 days and thereafter. To determine whether glucose had any effect on regulation of GK biosynthesis, fetal islets were cultured in medium containing a wide range of concentrations of glucose for 7 days. The amount of GK protein was significantly decreased in low concentrations of glucose and augmented at high concentrations. In conclusion, GK was expressed in both beta- and alpha-cells in fetal rat islets during development. GK is an integral part of the function of both of these cells at all stages in the development of the fetal islet.
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RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate a variety of methods to induce chronic pancreatitis and its radiologic expression by experimentally inducing this condition in cats. MATERIALS AND METHODS: Chronic inflammatory and fibrosing pancreatitis was produced in cats by intraductal injection of 1.5 mL of 94% ethanol in one group or by a combination of intraductal and intraparenchymal injection of ethanol together with partial obstruction of the main pancreatic duct to 70% of its original lumen by fixation of a small catheter in the papilla. For comparison, other cats underwent total obstruction of the main pancreatic duct. All groups, as well as untreated control cats (n = 3), underwent repeat laparotomy to obtain biopsy specimens. RESULTS: Cats with total obstruction showed progressing fibrosis with dilatation of ductules occasionally infiltrated with granulocytes. From 26 weeks on, acini and islets of Lnagerhans became atrophic. Radiographs showed progressive but diffuse dilatation of ducts, ductules, and side branches. Cats from the other two groups had interlobular inflammation and fibrosis with flattened and irregular ductular epithelium. Later, ductular proliferation occurred, interstitial inflammation subsided, and fibrosis increased. Radiographs showed very irregular ducts and ductules with stenosis and dilatation. From 26 weeks on, no substantial differences were observed between the cats who received only intraductal injection of ethanol and the cats who underwent the combination of procedures. CONCLUSION: The histopathologic and radiographic alterations that evolved from damage to the ductal epithelium in the cat resembled the features of chronic pancreatitis in humans and differed from those caused by total obstruction of the main pancreatic duct in cats.
BACKGROUND: Conservative site-specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposon-encoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites. RESULTS: The resolvase encoded by transposable element ISXc5 from the gram-negative phytopathogen Xanthomonas campestris shows two features which distinguish it from other known resolvases. First, it is more closely phylogenetically related to invertases than other resolvases. In particular, two functionally important regions seem highly conserved between this resolvase and members of the invertase subfamily. Second, the enzyme exhibits a large extension of its carboxy-terminal domain with unknown function. We purified ISXc5 resolvase and analysed its resolution reaction in vitro. Our biochemical and DNA topological analysis reveals that critical features of resolution are similar, if not identical, to that carried out by gammadelta resolvase. However, despite its apparent similarity to invertases, we were unable to detect recombination on standard substrates for DNA inversion, in either the presence or absence of Fis. CONCLUSIONS: ISXc5 resolvase employs a reaction mechanism which is common to members of the resolvase family. Its position near the evolutionary borderline to invertases and its high degree of identity within two functionally important regions with members of the DNA invertase subfamily suggest that only a few replacements of critical residues may suffice to convert this resolvase into a functional, possibly Fis-dependent invertase.
Two peptide libraries, Ac-MXXXXXBBRM and Ac-VXXXXXBBRM, were constructed on TentaGel solid support to search for ligands that bind tightly with the H9724 Lyme antibody. By using an on-bead ELISA, approximately 120 ligands were selected as candidates for further study. Matrix-assisted laser desorption ionization mass spectrometry analysis of the candidate ligands indicated a high rate of occurrence of certain amino acids at the randomized positions. On the basis of the initial screening results, a small library was designed and iteratively synthesized. Subsequent library screenings led to the identification of four peptides, Ac-PQEEGX-NH2 (X = R, K, A, D), that showed specific affinity to the antibody. This combination of solid-phase screening and iterative synthesis is an effective strategy for rapid identification of ligands that bind tightly with disease-specific antibodies and should be applicable, at least in principle, to other ligand-receptor systems. This combinatorial library approach can also be a useful tool for the discovery of novel diagnostic agents.
The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 45 kDa membrane protein with a higher apparent molecular weight of 50-85 kDa due to glycosylation. Previously, we had demonstrated that N-glycosylation plays a critical role in the GM-CSF receptor-ligand interaction. To assess the activity of the alpha subunit of the human GM-CSF receptor (GMRalpha) in a lower eukaryote, we expressed GMRalpha in the yeast S. cerevisiae and found that the protein has a lower apparent molecular weight compared with that expressed in mammalian cells. Using indirect immunofluorescence microscopy, we showed that GMRalpha protein expressed in yeast localizes to the plasma membrane. Although the yeast-expressed GMRalpha is able to interact with anti-GMRalpha antibody, the heterologously expressed receptor does not bind GM-CSF. Our results indicate that specific sites and/or forms of glycosylation of the GM-CSF receptor are crucial for ligand binding.
A library of Ac-XXXXXPAPRM decapeptides was prepared on a TentaGel solid support using the approach of split synthesis and the strategy of partial chain termination. Epitope screening of the library (17(5) approximately 1.4 x 10(6) decapeptides) with a Lyme monoclonal antibody (H9724) and subsequent MALDI-MS analysis of candidate peptides from colored beads revealed a consensus structure of Xi-DLSXj (Xi = V, L, Y; Xj = G, R). These identified sequences presented no homology to the natural epitope from Borrelia burgdorferi flagellin. However, they were found to resemble a fragment at the N-terminus of human heat shock protein (Hsp60). Our results confirmed that H9724 cross-reacts between bacterial and human proteins and provided support for an autoimmunity mechanism of Lyme disease.
In order to design a feasible somatic cell gene delivery system for the treatment of type I diabetes, a suitable cell type needs to be determined. We have previously shown that the stable transfection of the full-length insulin cDNA into the human liver cell line, (HEP G2ins) resulted in synthesis, storage and acute regulated release of insulin to analogues of cAMP, but not to the physiological stimulus glucose. In attempting to explain the lack of glucose responsiveness of the HEP G2ins cells we have stably transfected these cells with the human islet glucose transporter GLUT 2 (HEP, G2ins/g cells). The HEP G2ins/g cell clones exhibit glucose-stimulated insulin secretion and glucose potentiation of the secretory response to nonglucose secretagogues. While glucose responsiveness commenced at a lower concentration than normal islets, a secretion curve approaching normal physiological conditions was generated. Immunoelectron microscopy revealed the presence of insulin-containing granules, similar in size and appearance to those of the normal beta cell. These results demonstrate that while it is most likely that the HEP G2ins/g cell line predominantly secretes insulin via the constitutive pathway, significant acute regulated release was seen in response to glucose, and thus represents significant progress in the creation of a genetically engineered 'artificial beta cell' from a human hepatocyte cell line.
Glucokinase (GK) is the glucose sensor in the adult beta-cell, resulting in fuel for insulin synthesis and secretion. Defects in this enzyme in the beta-cell are responsible for the genetic disorder maturity-onset diabetes of the young, with the beta-cell being unable to secrete insulin appropriately when challenged with glucose. The human fetal beta-cell is also unable to secrete insulin when exposed to glucose, but whether GK is present and functional in this developing cell is unknown. To determine the expression of GK in human fetal pancreatic tissue, cytosolic protein was extracted from human fetal islet-like cell clusters (ICCs) at 17-19 weeks gestation and examined for protein content and enzyme activity. On Western blots, a single band corresponding to GK was seen at 52 kDa, and this was similar to that obtained from human adult islets. The maximal velocity (Vmax) of GK was less in fetal ICCs than that in adult islets (8.7 vs. 20.7 nmol/mg protein x h); similar K(m) values were found in both ICCs and islets. No attempt was made to determine which cells in an ICC contained GK. Glucose utilization was determined radiometrically; the Vmax of the high K(m) component was less in ICCs than in islets (31.3 pmol/ICC x h vs. 101.4 pmol/islet.h). Culture of ICCs for 3-7 days in medium containing 11.2 mmol/L glucose resulted in a 3.7-fold increase in the Vmax of GK and a 1.8-fold increase in glucose utilization. These enhanced activities of glucose phosphorylation and glycolysis, however, did not lead to the beta-cell being able to secrete insulin when exposed to glucose. In conclusion, glucokinase is present and functional in human fetal ICCs, but the inability of the human fetal beta-cell to secrete insulin in response to an acute glucose challenge is not due to immaturity of this enzyme.
Three metalic salts Ferrous, Mgnesium and Calcium were used to induce and to prevent Vi antigen of S. typhi loss. All of the 96 Vi-II phage typing standard strains belonged to V type (a recovery rate of 100%). Out of 1320 local strains, 1292 were type V (a recovery rate of 97.7%) and 28 remained type W with a Vi loss rate of 2.1%. The results showed that induction and prevention of Vi antigen of S. typhi loss by the three metalic salts was higher than that of other reports which showed a loss rate of 5% Vi antigen of S. typhi.
Phosphorylation of the three Xp12-specific phosphoproteins was drastically reduced by rifampicin, an antibiotic that specifically inhibits the host-cell RNA polymerase. However, this inhibitory effect could not be found in spontaneous mutants of Xanthomonas oryzae pv. oryzae whose RNA polymerase are resistant to the drug. The inhibitory effect of rifampicin treatment also resulted suppression of the Xp12 multiplication cycle. This implies the physiological significance of this effect and supports our previous prediction that phosphorylation plays an important role in the life cycle of Xp12. The acid- and alkali-labile character of the Xp12-specific phosphoproteins and the chemical stability of the phosphoryl linkages show that the corresponding protein kinase catalyzes the formation of an acyl phosphorylation. Subsequent fractionation of cell lysate revealed that the phosphoproteins were located in the periplasm. Actinomycin D, which affects transcription through DNA condensation rather than its binding to RNA polymerase, was not able to cause the inhibition effect. On the other hand, cerulenin was found to reduce the acyl phosphorylation which hints at a possible role of cell membrane in the phosphorylation. Here we present the evidence for the functional involvement of the rifampicin treatment on protein phosphorylation. A possible mechanism of rifampicin on the alternation of acyl phosphorylation is proposed.
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Protein phosphatase type I (PP1) is involved in diverse cellular processes, and its activity toward specific substrates is thought to be controlled by different regulatory or targeting subunits. To identify regulatory subunits and substrates of the Saccharomyces cerevisiae PP1, encoded by GLC7, we used the two-hybrid system to detect interacting proteins. Among the many proteins identified were Gac1, a known glycogen regulatory subunit, and a protein with homology to Gac1. We also characterized a new gene designated GIP1, for Glc7-interacting protein. We show that a Gip1 fusion protein coimmunoprecipitates with PP1 from cell extracts. Molecular and genetic analyses indicate that GIP1 is expressed specifically during meiosis, affects transcription of late meiotic genes, and is essential for sporulation. Thus, the Gip1 protein is a candidate for a meiosis-specific substrate or regulator of PP1. Finally, we recovered two genes, RED1 and SCD5, with roles in meiosis and the vesicular secretory pathway, respectively. These results provide strong evidence implicating PP1 function in meiosis. In addition, this study indicates that the two-hybrid system offers a promising approach to understanding the multiple roles and interactions of PP1 in cellular regulation.
The cause of the poor secretion of insulin in response to glucose by the beta-cell in the fetal rat pancreas is thought to be immaturity of the metabolism of glucose. Glucokinase (GK), a key enzyme in glycolysis, is the glucose sensor that maintains glucose homeostasis in the adult beta-cell; its role in the fetal beta-cell has not been determined. The aim of this study was to examine whether GK was functional in phosphorylation of glucose in the fetal islet, and if so, to determine what factors regulated this activity. Similar Km values were found in both fetal and adult islets: 7.4 vs. 7.7 mmol/l. The maximal GK velocity (Vmax) of the fetal islet and the contribution of GK to total glucose phosphorylation were also not significantly different from their adult counterparts. Western blot analysis of protein extracts from fetal and adult islets confirmed the presence of GK at 52 kDa. To determine if glucose had any effect on the Vmax of GK, islets were cultured for 7 days in medium containing low (1.4 or 2.8 mmol/l), normal (5.6 mmol/l), or high (11.2 or 16.8 mmol/l) concentrations of glucose. The maximal GK velocity increased linearly with increasing concentrations of glucose (r = 0.93; P < 0.01). To determine whether it was possible to up- and down-regulate Vmax of GK, islets were cultured in either a low (1.4 mmol/l) or high (30 mmol/l) concentration of glucose for 7 days and then switched to the opposite concentration for a further 3 days. The Vmax of GK in the fetal islet was upregulated 3.8-fold when the glucose concentration was raised. Conversely, the Vmax was downregulated 3.6-fold when the glucose concentration was lowered. The same phenomenon was also observed in the adult islet. These data indicate that GK is the glucose sensor for the fetal rat islet, just as it is for the adult islet. Since glucose did not cause insulin secretion from the fetal islet, it was important to examine whether this substrate had any effect on its own metabolism. Glucose utilization was estimated, and its Vmax was found to increase linearly with increasing concentrations of glucose (r = 0.96; P < 0.01). We conclude that the inability of the fetal rat beta-cell to secrete insulin in response to glucose cannot be explained by immaturity of GK or the glycolytic pathway.
Attention to an architectural framework in the development of clinical applications can promote reusability of both legacy systems as well as newly designed software. We describe one approach to an architecture for a clinical workstation application which is based on a critical middle tier of distributed object-oriented services. This tier of network-based services provides flexibility in the creation of both the user interface and the database tiers. We developed a clinical workstation for ambulatory care using this architecture, defining a number of core services including those for vocabulary, patient index, documents, charting, security, and encounter management. These services can be implemented through proprietary or more standard distributed object interfaces such as CORBA and OLE. Services are accessed over the network by a collection of user interface components which can be mixed and matched to form a variety of interface styles. These services have also been reused with several applications based on World Wide Web browser interfaces.
The efficacy of Kexue Ning (stopping hemoptysis) in the treatment of acute hemoptysis, and its comparison with that of the control group (treated with western medicine) are reported in this paper. The results showed that both the time for cessation of hemoptysis and the cure rate were better in the therapeutic group than in the control group. In addition, marked improvement in blood rheology after treatment was also found in the therapeutic group, but not in the control group.
Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway.