Thermal stable subtilisin with oxidation-resistant property.
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Hot Temperature↗
Biomedical subjects
L Zhu
Publications and source records attributed to L Zhu.
The human serotonin 5-HT2C receptor: complete cDNA, genomic structure, and alternatively spliced variant.
The complete 4775-nt cDNA encoding the human serotonin 5-HT2c receptor (5-HT2cR), a G-protein-coupled receptor, has been isolated. It contains a 1377-nt coding region flanked by a 728-nt 5'-untranslated region and a 2670-nt 3'-untranslated region. By using the cloned 5-HT2cR cDNA probe, the complete human gene for this receptor has been isolated and shown to contain six exons and five introns spanning at least 230 kb of DNA. The coding region of the human 5-HT2CR gene is interrupted by three introns, and the positions of the intron/exon junctions are conserved between the human and the rodent genes. In addition, an alternatively spliced 5-HT2CR RNA that contains a 95-nt deletion in the region coding for the second intracellular loop and the fourth transmembrane domain of the receptor has been identified. This deletion leads to a frameshift and premature termination so that the short isoform RNA encodes a putative protein of 248 amino acids. The ratio for the short isoform over the 5-HT2CR RNA was found to be higher in choroid plexus tumor than in normal brain tissue, suggesting the possibility of differential regulation of the 5-HT2CR gene in different neural tissues or during tumorigenesis. Transcription of the human 5-HT2CR gene was found to be initiated at multiple sites. No classical TATA-box sequence was found at the appropriate location, and the 5'-flanking sequence contains many potential transcription factor-binding sites. A 7.3-kb 5'-flanking 5-HT2CR DNA directed the efficient expression of a luciferase reporter gene in SK-N-SH and IMR32 neuro-blastoma cells, indicating that it contains a functional promoter.
Alternative Splicing↗
Decay Out of the Yrast and Excited Highly Deformed Bands in the Even-Even Nucleus 134Nd.
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Journal Article↗
On the relative ability of centromeric GNA triplets to form hairpins versus self-paired duplexes.
While tandem repeats of the human centromere DNA pentamer sequence TGRAA form stable "self-complementary" [TGRAATGRAA]2 duplexes (R = G or A) containing the GA-bracketed unpaired purine stack motif, their phase-shifted variants NAATGNAATG (N = A, G, C, T) were found to exist in solution as an equilibrium mixture of a duplex containing the GA-bracketed unpaired stack motif and a hairpin containing a single-residue loop closed by a sheared G x A pair. The stability of the hairpin form relative to duplex form of GNA triplets was found to be GCA>GAA/GTA>>GGA, with the CAATGCAATG sequence mostly in the hairpin form and the GAATGGAATG sequence mostly in the [GAATGGAATG]2 duplex form. The chemical shifts of the H1' and H4' protons of the central N residue in GNA triplets were found to differ markedly in the duplex and hairpin forms and are diagnostic indicators of which conformation the oligonucleotide adopts. Comparison between the structures of the G x A-closed C loop motif and the G x A-bracketed unpaired G-stack [GGA]2 motif reveals remarkably similar stacking by the loop C residue and the intercalated G residue on the adjacent sheared G x A pair. The anomalous upfield chemical shifts of the H1' and H4' protons in [GGA]2 motifs and the H4' proton in GCA loops, and the different sugar conformations in these two motifs, can be explained by interstrand versus intrastrand stacking of the central (G or C) deoxyribose with the adenine base. Based on these studies, a DNA sequence GTGGAATGGAATGGAAC was designed and shown to form a duplex containing three [GGA]2 motifs, while its 9G-->9C analog GTGGAATGCAATGGAAC was found to adopt a stable hairpin containing a (GGA)2 motif in the stem and a G x A-closed single C-loop.
Base Sequence↗
A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody.
Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity both in vitro and in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induce Xenopus oocyte maturation in vitro, as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation of Xenopus MAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3 (phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of the Xenopus IGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte-follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.
Androstadienes↗
Tetracycline-controlled gene expression system achieves high-level and quantitative control of gene expression.
The tetracycline-controlled gene expression system utilizes the control elements of the tetracycline resistance operon encoded in TnlO of Escherichia coli to control gene expression in eukaryotic cells. Here we demonstrate the quantitative control of the expression of the luciferase gene, dihydrofolate reductase gene, and bcl-2 gene in HeLa S3 or Chinese hamster ovary AA8 cells using the tetracycline-controlled gene expression system. Regardless of the host cell lines or the genes being expressed, there is a common range of tetracycline concentration within which the expression of genes is most sensitively regulated. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system is higher than that of the wild-type CMV promoter/enhancer-driven system. Nonetheless, careful selection of stably transfected clones is necessary to achieve the optimally regulated gene expression using this system.
Animals↗
G-protein-gated inward rectifier K+ channel proteins (GIRK1) are present in the soma and dendrites as well as in nerve terminals of specific neurons in the brain.
G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors.
Amino Acid Sequence↗
Adenocarcinoma of duodenum and ampulla of Vater: clinicopathology study and expression of p53, c-neu, TGF-alpha, CEA, and EMA.
Oncogenes, tumor suppressor genes, and growth factors are being explored as to their role in the initiation and progression of most neoplasms, but little information exists on the expression of oncoproteins or growth factors in adenocarcinoma of the duodenum or ampulla of Vater. This report covers expressions of p53, c-neu, TGF-alpha, CEA, and EMA in duodenal adenocarcinoma and ampullary adenocarcinoma, as well as correlations between expressions and tumor stage, histological grade and patient survival. The expression of p53, c-neu, TGF-alpha, CEA, and EMA has been studied in 15 duodenal adenocarcinomas and in eight ampullary adenocarcinomas by avidin-biotin-peroxidase complex indirect immunoperoxidase technique. The positive reaction for p53, c-neu, TGF-alpha, CEA, and EMA in duodenal adenocarcinoma was 20%, 60%, 60%, 73%, and 100%, respectively, and in ampullary adenocarcinoma, 13%, 100%, 50%, 63%, and 100%. Among the duodenal tumors, C-neu and p53 expression was noted more frequently in groups with high histological grades. Patients with c-neu positive duodenal adenocarcinoma had a shorter survival than the patients with c-neu negative duodenal adenocarcinoma (P < 0.01). C-neu product may serve as an unfavorable prognostic indicator in duodenal adenocarcinoma. No statistically significant correlation was found between the expressions of CEA, EMA, p53, and TGF-alpha and patient survival, tumor stage, or histological grade in either duodenal or ampullary adenocarcinomas.
Adenocarcinoma↗
The spin dynamics of heteronuclear multiple-spin systems formulated in the extended strong-narrowing limit.
Employing the perturbation approach directly in the multiply rotating frame, the master equation governing the spin dynamics of scalar-coupled heteronuclear multiple-spin systems has been derived. The resulting equation for the spin-density operator and the matrix form of the relaxation superoperator are relatively simple under the conditions of the "extended" strong-narrowing limit. The extended strong-narrowing limit, which requires that omega iI tau c << 1 and 2 pi Jij tau c << 1 [where I represents one of the spin types in the heteronuclear spin system, omega Ii is the chemical-shift range of spin type I in radians per second, Jij is the coupling constant J between spin i and spin j (like or unlike) in hertz, and tau c is the correlation time in seconds per radian], can be applied to heteronuclear spin systems of both small molecules and biopolymers in high-resolution liquid NMR. This newly developed formalism is used to investigate the effect of transverse cross relaxation on the apparent coupling constants in a heteronuclear 1H-1H-13C three-spin system. The calculation shows that, despite the strong dipolar interaction between directly bonded 1H and 13C, this perturbation on the apparent couplings is trivial. This result is in contrast to the homonuclear proton spin system, where the scalar coupling constants between two protons are significantly modulated if one proton is strongly dipole coupled to another proton. The underlying physical reasons for the different behavior of heteronuclear and homonuclear systems are explained.
Electron Spin Resonance Spectroscopy↗
Cell cycle control of T cell apoptosis induced by activation through the T cell antigen receptor.
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Animals↗
Metastatic pancreatic neuroendocrine carcinoma causing Cushing's syndrome. ACTH secretion by metastases 3 years after resection of nonfunctioning primary cancer.
CONCLUSION: Following resection of a nonfunctioning neuroendocrine carcinoma of the pancreas, subsequent metastases, in the absence of a primary cancer (resected), developed the capacity to secrete ACTH and create the Cushing syndrome. BACKGROUND: Although neuroendocrine carcinomas of the pancreas may produce one or more hormones and may switch secretion to a different hormone, no report is identified of a metastasis, in the absence of the primary tumor, developing de novo the capacity to secrete ACTH. METHODS: A nonfunctioning islet cell carcinoma was resected and immunochemically stained for multiple hormones. Three years later hepatic metastases were partially resected and stained as before. RESULTS: The primary cancer stained negative for ACTH and cortisol, positive for serotonin, and focally positive for gastrin. Three years later, after the development of a florid Cushing syndrome, the metastasis stained strongly for ACTH and negative for serotonin.
Adrenocorticotropic Hormone↗
Reaction of metallothionein with ethylenediamine-N,N,N',N'-tetraacetic acid.
The reactions of various rabbit MTs (Cd7Th, Zn7Th, Hg7Th, Bi7Th, Pb7Th) with EDTA were studied at different pH and EDTA concentrations, and were determined to be pseudo-first-order reactions. The reactions were all divided into two steps: one faster and EDTA-dependent, and the other EDTA-independent. The mechanism is also described in detail. Based on obtained results, the varied induction behaviors of MT formation for those metals are discussed.
Animals↗
Insulin sensitivity tested with a modified euglycemic technique in cats and rats.
A new insulin sensitivity test (IST) is described using a modified euglycemic clamp in cats and rats. The IST uses the amount of glucose required to be infused to maintain euglycemia over a 30-min period in rats and 60 min in cats following a bolus administration of insulin as the index of insulin sensitivity. Glucose levels are determined at short time intervals (2-5 min), and variable glucose infusion is used to hold glucose levels within a few percentage points of the basal pre-test glucose level. A new blood sampling procedure is described that allows each IST to be carried out using a total of only 0.5 mL of blood. The IST is sensitive and allows clear insulin dose effects to be demonstrated with 100 mU/kg requiring 355.0 +/- 14.3 mg/kg over 30 min and 50 mU/kg requiring 198.7 +/- 11.1 mg/kg. Five consecutive tests were reproducibly carried out (%CV = 3.0 +/- 0.5) over a 12-hr period in the cat with insulin, glucagon, and glucose levels remaining stable prior to each IST. Glucagon and norepinephrine plasma concentrations do not change significantly during the IST. The IST is sufficiently sensitive to allow demonstration of dose-response relationships for atropine-induced insulin resistance. The IST is thus sensitive, reproducible, and able to demonstrate acute insulin resistance in anesthetized cats and rats. The test is demonstrated in fed (rats) and fasted (cats) state.
Animals↗
PCBs do not induce DNA breakage in vitro in human lymphocytes.
Polychlorinated biphenyls (PCBs) are classified by IARC as non-mutagenic in vivo. However, despite almost 20 years of research, their mutagenicity in vitro is still debatable. In this work the in vitro cytochalasin B micronucleus test and the alkaline comet assay applied to human lymphocytes were used to study the genotoxicity of a PCB. PCB77, at concentrations ranging from 0.01 to 100 microg/ml, was used in whole blood or isolated lymphocyte cultures, with final dimethylsulfoxide percentages of 0.5-2%. In the micro-nucleus test lymphocytes were exposed for 48 h, and in the alkaline comet assay for 30 min, 1 h and 3 h. No increases of single strand breaks or micronucleus frequencies was found, in contrast to previously reported data. Our data indicate that PCB77 has no clastogenic properties in human lymphocytes.
Adult↗
Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay.
Polychlorinated biphenyls (PCBs) are stable pollutants, which can be found in almost every compartment of terrestrial and aquatic ecosystems. They are very lipophilic and therefore have the potency of accumulating in the fat stores of animals. The mechanisms by which PCBs exert their adverse effects are still unclear. It is known that PCBs induce some important biotransformation enzymes, but their mutagenic properties are still controversial. The DNA breakage and clastogenic potency of a planar PCB77 (3,3',4,4'-tetrachlorobiphenyl) was determined in vivo in fish, using the single cell gel electrophoresis or comet assay and the micronucleus test, on erythrocytes of the brown trout exposed for 3, 9 and 14 days to initial PCB concentrations of 780 and 918 pg/ml, dissolved in the water. Blood was taken by a caudal puncture and the erythrocytes were either deposited in an agarose gel (0.6%) for the comet assay or smeared directly on slides for the micronucleus test. Five fish were studied per treatment and 50 and 2000 erythrocytes per concentration and per animal were analysed for the comet assay and the micronucleus test respectively. Ethyl methanesulphonate (EMS) at a concentration of 25 mg/l water was used as a positive control. Although EMS induced a statistically significant increase of single strand breaks in the comet assay, in neither of the two tests used, were mutagenic effects due to PCB exposure observed.
Animals↗
Induction of autologous tumor-specific cytotoxic T-lymphocyte activity against a human renal carcinoma cell line by B7-1 (CD8O) costimulation.
Recently mouse models have shown that expression of costimulatory molecules such as B7-1 on tumor cells can induce tumor-specific immunity, suggesting that tumor cells modified to express costimulatory molecules can be a potential tumor vaccine. To investigate the importance of B7-1 co-stimulation in induction of autologous tumor immunity in humans, we established a renal carcinoma cell line, RCC-1, from a tumor resection and studied the patient's antitumor immune responses in vitro. The RCC-1 cell line constitutively expressed major histocompatibility complex (MHC) class I, intercellular adhesion molecule (ICAM)-1, and leukocyte function-associated antigen (LFA)-3 molecules, and MHC class II molecules were induced by interferon-gamma (IFN-gamma) treatment in vitro. However, neither RCC-1- nor IFN-gamma-treated RCC-1 cells expressed B7-1, and both failed to induce T-cell proliferative responses in mixed lymphocyte and tumor cell reaction (MLTR) assays, suggesting that the costimulatory signals provided by cell adhesion molecules such as ICAM-1 and LFA-3 were not sufficient to elicit an antitumor immune response. However, on transfection of the human B7-1 into RCC-1, these cells were able to induce a significant T-cell proliferation in MLTR assays. This T-cell response could be blocked by anti-B7 mAb treatment of the tumor cells. RCC-1B7 cells also induced the generation of tumor-specific cytolytic T lymphocytes to the parent RCC-1 cells in vitro, with little nonspecific cytolysis of an unrelated RCC line, A498, or autologous phytohemagglutinin (PHA) blasts. This specific cytotoxicity could be abrogated by anti-CD8 mAb and complement treatment. In summary, our study indicates that B7-1-CD28 interaction plays a critical role in induction of autologous tumor-specific cytotoxic T lymphocytes (CTLs) in humans, suggesting that the costimulatory molecule transfected tumor cells could be useful in expanding tumor-specific autologous CTL in vitro for adoptive tumor immunotherapy.
Antigens, Neoplasm↗
Direct observation of the double Auger decay of a K hole.
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Journal Article↗
The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter.
The Rb-related p107 protein has been implicated as an important control element in proper cell cycle progression. The p107 protein is thought to restrict cellular proliferation in part through its interaction with the E2F family of transcription factors and is, therefore, a specific target for regulation by several DNA viruses. Here, we demonstrate that p107 protein levels are induced in a biphasic manner in human fibroblasts during productive infection by the human cytomegalovirus (HCMV). Expression patterns of p107 protein levels during HCMV infection of human embryonic lung cells (HELs) demonstrate a sustained induction from early to late times of infection. We also demonstrate that the HCMV immediate-early protein IE1-72 complexes in vivo with the p107 protein and that this interaction can be reconstituted in an in vitro system by using reticulocyte-translated protein. Our data demonstrate that the interaction between p107 and the IE1-72 protein occurs at times of infection that temporally match the second tier of p107 protein induction and the phosphorylation pattern of the IE1-72 protein. Furthermore, we show here that the ability of p107 to transcriptionally repress E2F-responsive promoters can be overcome by expression of the IE1-72 protein. This effect appears to be specific, since the IE1-72 protein is not capable of relieving Rb-mediated repression of an E2F-responsive promoter. Finally, our data demonstrate that HCMV infection can induce cellular proliferation in quiescent cells and that IE1-72 expression alone can, to a degree, drive a similar progression through the cell cycle. These data suggest that IE1-72-mediated transactivation of E2F-responsive promoters through alleviation of p107 transcriptional repression may play a key role in the cell cycle progression stimulated by HCMV infection.
Carrier Proteins↗