Biomedical subjects
Publications and source records attributed to G Xu.
The stability of Jingu Tongxiao Pills was studied on the basis of accelerated destruction and sample-reserving observation at room temperature. According to the changes of tanshinone IIA in the sample, the time of efficacy determined by the two methods is 1.89 and 2.30 years respectively. Based on the analysis of other indices, the effective period is tentatively determined as two years.
Four compounds were isolated from the roots of Lycium chinense and identified as beta-sitosterol, betaine hydrochloride, scopoletin and E-ferulic acid octacosyl ester. The last one was isolated from the genus for the first time.
Commitment of mammalian cells to enter S phase enables the transcription factor E2F-1 to activate certain genes whose products mediate cell cycle advance. In S phase, E2F-1 forms stable complexes with cyclin A-kinase, which in turn eliminates E2F-1DNA binding function. Here, we show that suppression of E2F-1 DNA-binding activity by cyclin A-kinase is linked to orderly S phase progression. Disruption of this linkage resulted in S phase delay/arrest followed by regrowth or apoptosis, depending upon whether the DNA-bound E2F-1 could transactivate. Hence, the unscheduled presence of E2F-1 on specific DNA sequences during S phase can activate a specific S phase checkpoint, thereby linking transcription, DNA replication, and cell cycle control.
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To investigate the degree of conservation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans, and to help direct its functional analysis, we cloned a homolog (designated nim-1) from Neurospora crassa. Over the catalytic domain NIM-1 is 75% identical to NIMA, but overall the identity drops to 52%. nim-1 was able to functionally complement nimA5 in A. nidulans. Mutational analysis of potential activating phosphorylation sites found in NIMA, NIM-1, and related protein kinases was performed on NIMA. Mutation of threonine 199 (conserved in all NIMA-related kinases) inhibited NIMA beta-casein kinase activity and abolished its in vivo function. This site conforms to a minimal consensus phosphorylation site for NIMA (FXXT) and is analogous to the autophosphorylation site of cyclic-AMP-dependent protein kinases. However, mutation of a unique cysteine residue found only in the catalytic site of NIMA and NIM-1 had no effect on NIMA kinase activity or function. Three temperature-sensitive alleles of nimA that cause arrest in G2 were sequenced and shown to generate three different amino acid substitutions. None of the mutations prevented accumulation of NIMA protein during G2 arrest, but all prevented the p34cdc2/cyclin B-dependent phosphorylation of NIMA normally seen during mitotic initiation even though p34cdc2/cyclin B H1 kinase activity was fully activated.
A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.
Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.
The retinoblastoma gene product (pRB) is a known tumor suppressor, capable of arresting growth in mid-to-late G1. Part of its growth suppression action arises from interaction(s) with one or more members of the E2F family of transcription factors. These proteins most likely contribute to progression from G0 to S phase in mammalian cells, and pRB binding most likely inhibits aspects of their suspected growth-promoting function. Given their growth-stimulating potential, we asked whether one or more E2F alleles can function as oncogenes. Uncloned pools of NIH 3T3 cells producing the pRB binding target E2F-1, E2F-2, or E2F-3 grew in semisolid medium. In addition, they grew to much higher saturation density than controls. From the study of cells producing selected E2F-1 mutant species, it appears that E2F DNA-binding function contributes to, and pRB/E2F binding suppresses, soft-agar growth. Thus, three E2F family members can act as oncogene products, suggesting that part of the normal role of pRB is to down-modulate this potential activity.
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Cytosine methylation within DNA has been implicated in genetic imprinting, X-chromosome inactivation, regulation of tissue-specific gene expression, aging, and cancer. Unfortunately, DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) from various mammalian sources have been difficult to isolate and stabilize, precluding investigations of these critical enzymes. We describe a novel FPLC purification of the 190,000 Mr DNA methyltransferase from mouse Friend erythroleukemia cells. The homogeneous 190 kD Mr form of the enzyme is the only polypeptide detected at various stages of cell growth and has not undergone detectable N-terminal proteolysis.
Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)
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Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5' end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding region and established that it is composed of 59 exons. Furthermore, we have defined the 3'-untranslated region (3'-UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3'-UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template.
The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac alpha 2-3Gal, Neu5Gc alpha 2-3Gal and Neu5Ac alpha 2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac alpha 2-3Gal was higher than the activities with Neu5Gc alpha 2-3Gal and Neu5Ac alpha 2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac alpha 2-6Gal to Neu5Ac alpha 2-3Gal to be 0.13:0.2, and the ratio Neu5Gc alpha 2-3Gal/Neu5Ac alpha 2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac alpha 2-6Gal/Neu5Ac alpha 2-3Gal and 0.51:0.76 for Neu5Gc alpha 2-3Gal/Neu5Ac alpha 2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac alpha 2-6Gal and Neu5Gc alpha 2-3Gal linkages when compared with strains isolated in an earlier year, 1930.
BACKGROUND & AIMS: The Smith-Lemli-Opitz syndrome is a recessive inherited disorder characterized by neurological developmental defects and dysmorphic features with a defect in cholesterol synthesis at the conversion of 7-dehydrocholesterol to cholesterol. BM 15.766 inhibits 7-dehydrocholesterol-delta 7-reductase and reproduces the biochemical defect. The aim of this study was to investigate the effects of cholesterol, cholic acid, and lovastatin feeding on rats fed BM 15.766. METHODS: Plasma cholesterol and 7-dehydrocholesterol concentrations were related to the hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. RESULTS: With the inhibitor treatment, plasma cholesterol concentrations decreased 67%; 7-dehydrocholesterol concentrations increased from trace to 17 mg/dL; and hepatic HMG-CoA reductase activity and messenger RNA levels were stimulated 74% and two times, respectively. In inhibitor-treated rats, feeding cholesterol increased plasma cholesterol concentrations 3.7 times, decreased 7-dehydrocholesterol concentrations 88%, and reduced elevated HMG-CoA reductase activity and messenger RNA levels 74% and 49%. Feeding cholic acid increased plasma cholesterol without reducing 7-dehydrocholesterol concentrations. The combination of cholic acid and cholesterol enhanced plasma cholesterol 9.5 times without decreasing 7-dehydrocholesterol levels. Feeding lovastatin depressed plasma cholesterol further without reducing 7-dehydrocholesterol levels. CONCLUSIONS: Cholesterol is essential to correct abnormal cholesterol synthesis induced by BM 15.766 in rats by expanding the pool and inhibiting HMG-CoA reductase. Neither cholic acid nor lovastatin are effective separately, but cholic acid plus cholesterol may offer some additional benefit.
We examined beta-cell function and replication in spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY). Rats were subjected to 90% pancreatectomy (Px) or sham operation at the age of 8 weeks, and islet function and regeneration were examined 4 weeks after surgery. Plasma glucose levels were higher in SHR than in WKY (509 +/- 38 v 325 +/- 109 mg/dL, P < .0001) 1 week after Px and throughout the experimental period. Plasma glucose responses to intravenous injection of glucose (0.5 g/kg body weight) were not different in the sham-operated animals of the two strains, whereas plasma insulin responses were greater in SHR than in WKY. No insulin responses to glucose were observed in either strain of Px rats. The insulin content of the remnant equivalent (6.7 +/- 2.1 v 4.2 +/- 0.4 micrograms, P < .05) and whole pancreas (156.7 +/- 10.7 v 123.8 +/- 23.5 micrograms, P < .01) in sham-operated rats was greater in SHR than in WKY. However, insulin content was lower (P < .05) in Px-SHR (1.0 +/- 0.2 microgram) than in Px-WKY (3.9 +/- 1.7 micrograms). Histological examination showed that fibrotic degeneration of islets was much greater in Px-SHR than in Px-WKY. These data strongly suggest that the beta cells of SHR were more vulnerable to reduction of islet mass than those of WKY. Our data also suggest that hyperinsulinemia and/or insulin resistance in SHR has a deleterious effect on beta-cell replication.
The effects of vanadate on B-cell function and replication in rats after 90% partial pancreatectomy (Px) were compared with insulin therapy. At the age of 4 weeks, male Wistar rats were subjected to sham operation or Px. Vanadate (0.2 mg/ml) was given in drinking water for 3 weeks starting at 2 weeks after surgery. Regular insulin (2.4 units/day) was administered as a continuous subcutaneous infusion through an osmotic pump. Plasma glucose levels were significantly higher in the Px rats than in the sham rats from 1 week after surgery. Vanadate lowered plasma glucose levels to near normal values in the Px rats as early as 2 days. The effect was sustained throughout the experiment. The hypoglycemic effect of insulin was less than that of vanadate. During an i.p. glucose tolerance test, plasma glucose levels were decreased in the Px rats treated with vanadate or insulin, while plasma insulin levels were not affected. The insulin content in the Px rats treated with vanadate was significantly (P < 0.01) greater than in the insulin-treated Px rats. Histological examination showed fibrotic degeneration in the enlarged islets of Px rats, whereas the normal structure was retained in most islets of the Px rats treated with vanadate and insulin. In addition, B-cell areas within the islet were restored to normal levels not only in the insulin-treated Px rats but in the vanadate-treated Px rats. However, both vanadate and insulin failed to stimulate proliferative activity of the B-cells. These data suggest that vanadate is a new therapeutic option to ameliorate the diabetic state after Px.
We assessed the effect of CS-045, a new hypoglycemic agent, on B-cell function in partially pancreatectomized rats. At the age of 4 weeks, male Wistar rats were subjected to 90% pancreatectomy (Px). For 2 weeks starting at 6 weeks after surgery the Px rats were treated with CS-045 (CS rats) mixed with chow pellets in a proportion of 0.2% (w/w). To compare the efficacy of CS-045 with that of insulin therapy, an osmotic pump was implanted to release insulin (1.2 units/day) into the intraperitoneal cavity of the Px rats (Is rats). Plasma glucose levels in the CS and Is rats were significantly lower than in the control Px rats; however, no marked improvement in plasma glucose or insulin levels was observed in glucose tolerance test (2 g/kg, i.p.) in the CS rats. Insulin secretion by the isolated perfused pancreas in response to 16.7 mM glucose showed a biphasic pattern, but was slightly reduced in the Px and CS rats compared with the Is rats. Insulin secretion induced by 19 mM arginine was unaffected by the treatment. The insulin content of the CS rats was significantly greater than in the Px and Is rats. Histological observations suggested regranulation of the pancreatic islets of the CS rats. B-cell areas within the islet were restored to normal levels in the Cs and Is rats. These findings indicate that the hypoglycemic effect of CS-045, which is not mediated by insulin secretion from the residual pancreas, prevents destruction of the islet.