Biomedical subjects

Z Hu

Publications and source records attributed to Z Hu.

At least 163 records · Page 9Linked to original sources

PubMed1999

Identification, sequence analysis and phylogeny of the lef-2 gene of Helicoverpa armigera single-nucleocapsid baculovirus.

The baculovirus late expression factor 2 (LEF-2) is involved in DNA replication, and most likely function as a primase processivity factor. Lef-2 genes have been found in multinucleocapsid nucleopolyhedroviruses (MNPVs) and in granuloviruses (GVs), but not yet in single-nucleocapsid NPV (SNPV). Here, a lef-2 gene homolog was identified from SNPV of Helicoverpa armigera (HearNPV). The open reading frame of the HearNPV lef-2 gene is 696 nucleotides long, encoding a putative protein of 232 amino acids with an M(r) of about 26 kDa. The 5'-noncoding region contains two early (CAGT) consensus motifs for transcription initiation and three TATA boxes. Lef-2 transcripts started at a C, 29 nucleotides upstream of a putative translational start. A putative polyA signal, AATAAA, was found 76 nucleotides downstream of the translation stop codon. The HearNPV lef-2 gene has a low but significant degree of amino acid sequence identity (30%) to the lef-2 genes of 15 other baculoviruses of which nine were newly determined. The N-terminal half of the LEF-2 proteins contains one (I) and the C-terminal half two (II and III) conserved domains. Sixteen amino acids are absolutely conserved in those LEF-2 investigated and are probably critical for LEF-2 function. A phylogenetic tree of 16 baculovirus LEF-2 proteins was constructed by using maximum parsimony analysis and appeared to be comparable to a tree for ecdysteroid UDP-glucosyl transferases (Chen et al., 1997a). The genomic location of the lef-2 genes relative to polyhedrin/granulin and the clade structure of the gene trees suggest that genome organization and gene phylogeny are useful parameters to study the evolutionary history of baculoviruses. These two independent approaches also give a more complete picture of the ancestral relationship among baculovirus.

Amino Acid Sequence↗

PubMed1999

Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography (MECC) method for the separation and determination of five components, glycyrrhizic acid (GA), glycyrrhetinic acid (GTA) and 3,4'-dimethoxy-5-hydroxychalone, fermononetin and isoliquiritigenin, in extracts of Glycyrrhiza uralensis Fisch root was developed. Migration behavior of these analytes was studied by the systematic examination of the borate and sodium dodecyl sulfate (SDS) concentrations in the run buffer. The optimum separation for these analytes was achieved using 10 mmol l(-1) of tetraborate and 25 mmol l(-1) of SDS as the running buffer, with 17 kV of applied voltage. All experiments were performed using a 50.0 cm (42.4 cm effective length)x75 microm I.D. of fused-silica capillary. The apparent pKa values of GA and GTA and the binding constants for the association between the above five analytes and SDS were calculated in this study. A comparison of the extraction efficiency for GA and GTA from Glycyrrhiza uralensis Fisch root was made using ethanol, distilled water and chloroform as extraction reagents, respectively. These results provided very useful information to select the proper solvent to extract the desired components in Glycyrrhiza uralensis Fisch. The MECC method established in this paper was employed to analysis the above five active components.

Acids↗

PubMed1999

Analysis of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid and homoarginine in Lathyrus sativus by capillary zone electrophoresis.

A simple capillary zone electrophoresis (CZE) method has been developed for the simultaneous quantitative determination of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP) and homoarginine in Lathyrus sativus (LS; grass pea). A new Na2B4O7-Na2SO4 run buffer was used and the pH was 9.20, contents of beta-ODAP and homoarginine in crude extracts of LS plant material were determined with this method, the RSDs of peak areas of beta-ODAP and homoarginine were 2.62% and 3.61%, respectively. It was found that the equilibrium concentration ratio of alpha- and beta-ODAP decreased from 34.5/65.5 to 28.6/71.4 when the pH of the solution increased from pH 3.0 to pH 11.0.

Amino Acids, Diamino↗

PubMed1999

Axotomy-induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus.

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high-affinity PACAP receptors, PAC(1) and VPAC(2), in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time-dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20-fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10-fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC(1) and VPAC(2) receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC(2) receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC(1) receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses.

Animals↗

PubMed1999

Flavopiridol inhibits smooth muscle cell proliferation in vitro and neointimal formation In vivo after carotid injury in the rat.

BACKGROUND: Smooth muscle cell (SMC) proliferation is a critical component of neointimal formation in many models of vascular injury and in human lesions as well. Cell-cycle inhibition by gene transfer techniques can block SMC proliferation and lesion formation in animal models, although these methods are not yet applicable to the treatment of human disease. Flavopiridol is a recently identified, potent, orally available cyclin-dependent kinase inhibitor. METHODS AND RESULTS: Using human aortic SMCs, we found that flavopiridol in concentrations as low as 75 nmol/L resulted in nearly complete inhibition of basic fibroblast growth factor-induced and thrombin-induced proliferation. At this dose, flavopiridol inhibited cyclin-dependent kinase activity, as measured by histone H1 phosphorylation, but had no effect on mitogen-activated protein kinase activation. Induction of the cell cycle-related proteins cyclin D1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein was also blocked by flavopiridol. Flavopiridol had no effect on cellular viability. To test whether flavopiridol had a similar activity in vivo when administered orally, we examined neointimal formation in rat carotid arteries after balloon injury. Flavopiridol 5 mg/kg reduced neointimal area by 35% and 39% at 7 and 14 days, respectively, after injury. CONCLUSIONS: Flavopiridol inhibits SMC growth in vitro and in vivo. Its oral availability and selectivity for cyclin-dependent kinases make it a potential therapeutic tool in the treatment of SMC-rich vascular lesions.

Animals↗

PubMed1999

Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo.

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.

Animals↗

PubMed1999

Targeting tumor vasculature endothelial cells and tumor cells for immunotherapy of human melanoma in a mouse xenograft model.

An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

Adenoviridae↗

PubMed1999

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression.

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.

Animals↗

PubMed1999

Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1.

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The Kd values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.

Animals↗

PubMed1999

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

Amino Acid Sequence↗

PubMed1999

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase.

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.

Amino Acid Substitution↗

PubMed1999

Value of low dose dobutamine Doppler tissue imaging for detecting hibernating myocardium.

In order to assess the value of Doppler tissue imaging (DTI) in detecting viable hibernating myocardium, 20 patients with coronary artery disease and chronic left ventricular dysfunction underwent low dose dobutamine stress echocardiography and low dose dobutamine stress DTI. The results showed that among the 100 asynergic segments, 35 segments showed improvement after dobutamine infusion (group H) and no changes were observed in the remaining 65 segments (group N). The left ventricular echocardiographic score index decreased from 1.60 +/- 0.35 to 1.44 +/- 0.36 (n = 20, P < 0.01). During low dose dobutamine stress DTI, there was no difference in the values of velocity of S wave (V.) before dobutamine infusion between two groups. However, after dobutamine infusion, the values of Vs and VR in group H were significantly higher than those in group N (Vs: 10.1 +/- 3.0 cm/s vs 7.3 +/- 2.2 cm/s, P < 0.01; VR: 60% +/- 41% vs 25% +/- 32%, P < 0.001), 95.7% asynergic myocardial segments with VR < or = 0 had no viability while 86% asynergic segments with VR > 80% were viable myocardium. It is concluded that the different reactions to dobutamine stress between hibernating and necrosis myocardium could be showed by DTI and it is more clinically significant when VR < or = 0 and VR > 80%.

Coronary Disease↗

PubMed1999

The effects of lower intensity anticoagulation therapy on coagulation system in patients with mechanical prosthetic valves.

To evaluate the effect of lower intensity anticoagulation therapy in patients with mechanical prosthetic valves, laboratory-based hematological assays including prothrombin time (PT), activity of factor X, antithrombin III (AT III), D-dimer, fibrinogen (Fg) and platel et al pha-granular membrane protein (GMP-140) were performed in 65 patients who had been on warfarin treatment for over one month. The patients were assigned to 3 groups on the basis of their International Normalized Ratios (INR), ranging from 2.00 to 2.50; 2.51 to 3.00; 3.01 to 4.50, respectively. The results showed that the D-dimer, Fg, GMP-140 levels were higher after mechanical valve replacement than those before operation, indicating the activation of coagulation and fibrinolysis system and the damage of platelets. Lower intensity anticoagulation therapy (INR 2.00 to 2.50) could effectively inhibit the activity of factor X and increase the level of AT III. There were no appreciable differences among D-dimer, Fg, GMP-140 and AT III in the 3 anticoagulation intensity groups. These results suggest that in patients with new generation mechanical prosthetic valves, target anticoagulation level (INR 2.00 to 2.50) may result in good protection from thrombo-embolism.

Adolescent↗

PubMed1999

Functional domain analysis of the Saccharomyces MAL-activator.

MAL63 of the MAL6 locus and its homologues at the other MAL loci encode transcription activators required for the maltose-inducible expression of the MAL structural genes. We carried out a deletion analysis of LexA-MAL63 gene fusions to localize the functional domains of the Mal63 MAL-activator protein. Our results indicate that the sequence-specific DNA-binding domain of Mal63p is contained in residues 1-100; that residues 60-283 constitute a functional core region including the transactivation domain; that residues 251-299 are required to inhibit the activation function of Mal63p; and that the rest of the C-terminal region of the protein contains a maltose-responsive domain that acts to relieve the inhibitory effect on the activation function. Abundant overproduction of Mal63p does not overcome the negative regulation of MAL gene expression in the absence of maltose, suggesting that a titratable MAL-specific repressor similar to Gal80p is not involved in the negative regulation of the MAL-activator. A model for maltose-inducible autoregulation of the MAL-activator is presented.

Amino Acid Sequence↗

PubMed1999

Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R.

Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300-1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library.

Base Sequence↗

PubMed1999

Long-term lack of endogenous glucocorticoids down-regulates glucocorticoid receptor levels in the rat forebrain.

To understand the effect of a chronic lack of endogenous glucocorticoids on glucocorticoid receptor levels, the changes of glucocorticoid receptor content in the rat forebrain five months after adrenalectomy were investigated. In the long-term adrenalectomized rats that showed a hormone deficiency and loss of glucocorticoid receptor immunoreactivity in the forebrain, an intraperitoneal injection of corticosterone was used to elevate the serum hormone levels and recover glucocorticoid receptor immunoreactivity in the forebrain. One hour later, when the blood corticosterone returned to the normal level, the recovery of glucocorticoid receptor immunoreactivity in the forebrain was examined by immunohistochemistry. Since the complete restoration of glucocorticoid receptor immunoreactivity was shown to depend on the presence of normal levels of both serum hormone and intracellular glucocorticoid receptors, the weak reappearance of glucocorticoid receptor immunoreactivity in any forebrain area of the long-term adrenalectomized rats that had normal serum corticosterone might reflect the low intracellular glucocorticoid receptor levels there. Our results revealed a weak reappearance of glucocorticoid receptor immunoreactivity in some forebrain areas of the long-term adrenalectomized rats after corticosterone treatment; the hippocampal granule cell layer and cerebral cortex in particular showed very weak recovery of glucocorticoid receptor immunoreactivity. Conversely, neurons in the CA1/CA2 subfields of the hippocampal pyramidal cell layer, immediately adjacent to the granule cell layer on the same brain section, exhibited a strong reappearance of glucocorticoid receptor immunoreactivity, to near normal levels. These results suggest that, five months after adrenalectomy, the intracellular glucocorticoid receptor content decreased in the rat granule cell layer and cerebral cortex. Therefore, the long-term lack of endogenous glucocorticoids after adrenalectomy might down-regulate but not up-regulate the intracellular glucocorticoid receptor level, and the presence of glucocorticoids is important for the continued synthesis of glucocorticoid receptors.

Adrenalectomy↗

PubMed1999

Topological regulation of cell division in Escherichia coli involves rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD and MinE.

Placement of the Z ring at midcell in Escherichia coli is assured by the action of the min system, which blocks usage of potential division sites that exist at the cell poles. This activity of min is achieved through the action of an inhibitor of division, MinC, that is activated by MinD and topologically regulated by MinE. In this study, we have used a functional GFP-MinC fusion to monitor the location of MinC. We find that GFP-MinC is a cytoplasmic protein in the absence of the other Min proteins. The addition of MinD, a peripheral membrane protein that interacts with MinC, results in GFP-MinC appearing on the membrane. In the presence of both MinD and MinE, GFP-MinC oscillates rapidly between the halves of the cell. Thus, MinC is positioned by the other Min products, but in a dynamic manner so that it is in position to inhibit Z ring assembly away from midcell.

Adenosine Triphosphatases↗

PubMed1999

Development of tissue-simulating optical phantoms: poly-N-isopropylacrylamide solution entrapped inside a hydrogel.

The average turbid optical properties of the N-isopropylacrylamide (NIPA) polymer solution entrapped inside a polyacrylamide hydrogel (called an NIPA/PAAM gel system) were studied using a multiwavelength oblique-incidence reflectometer. The turbidity of such a system can be drastically changed by simply switching the temperature from below the low critical solution temperature of the NIPA, around 33 degrees C, to above. The absorption coefficient and the reduced scattering coefficient were obtained as a function of wavelength for samples with selected NIPA and blue dextran concentrations. It is found that the scattering of the optical phantom comes from the NIPA polymer chains and the absorption from the blue dextran. The turbid optical properties of an NIPA/PAAM gel system can be tuned to simulate biological tissues at a specific wavelength by varying compositions of NIPA and blue dextran and further modified by controlling the temperature.

Acrylic Resins↗