Biomedical subjects

H Zhu

Publications and source records attributed to H Zhu.

At least 307 records · Page 17Linked to original sources

PubMed1998

Co-detection by two imidazoline receptor protein antisera of a novel 85 kilodalton protein.

Imidazoline receptors (I-receptors) are considered as potential therapeutic targets for a spectrum of stress-induced illnesses. Yet, I-receptors remain poorly defined at the molecular level. In this study, candidate imidazoline receptor proteins were compared using two imidazoline receptor-selective antisera of diverse origins. One antiserum was derived from affinity-purified imidazoline-binding protein. The second antiserum was produced as an anti-idiotypic antiserum, from purified IgG selective for imidazolines. Despite such diverse origins, both antisera co-identified an 85 kDa band on western blots from a variety of tissues. The integrity of the 85 kDa band was dependent on protection by eight different protease inhibitors. Other proteolytic breakdown products (obtained after homogenization with only one protease inhibitor) were comparable in size to previously reported smaller immunoreactive bands. The full-size 85 kDa band was also enriched in plasma membrane fractions and abundant in rat PC12 cells and brain regions known to be abundant in I1 binding sites. Furthermore, the immunodensity of the 85 kDa band, against anti-idiotypic antiserum, was linearly correlated with reported I1 site radioligand Bmax values (r2 = 0.8736, P = 0.0002) across nine rat tissues. Therefore, a possible candidate for the full-length imidazoline receptor(s) appears to be an 85 kDa protein.

Animals↗

PubMed1998

Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE.

We report here the inactivation of a member of the Ice/Ced-3 (caspase) family of cell death genes, casp-11, by gene targeting. Like Ice-deficient mice, casp-11 mutant mice are resistant to endotoxic shock induced by lipopolysaccharide. Production of both IL-1alpha and IL-1beta after lipopolysaccharide stimulation, a crucial event during septic shock and an indication of ICE activation, is blocked in casp-11 mutant mice. casp-11 mutant embryonic fibroblast cells are resistant to apoptosis induced by overexpression of ICE. Furthermore, we found that pro-caspase-11 physically interacts with pro-ICE in cells, and the expression of casp-11 is essential for activation of ICE. Our data suggest that caspase-11 is a component of ICE complex and is required for the activation of ICE.

Animals↗

PubMed1998

Intron-exon structure of the MET gene and cloning of an alternatively-spliced Met isoform reveals frequent exon-skipping of a single large internal exon.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor that stimulates epithelial cell mitogenesis, motility, invasion, and morphogenesis. Its receptor is encoded by the MET proto-oncogene, a transmembrane receptor tyrosine kinase. Several studies have suggested a role for MET as a dominant oncogene in tumor development and progression. Conversely, MET is located at a region on chromosome 7q31 frequently deleted in carcinomas, suggesting that recessive mutations in MET may exist in certain cancers. To facilitate a search for mutations in MET, we have obtained the intron-exon structure of the human MET gene. We present the genomic structure of the first member of the Met receptor family to be characterized. Interestingly, MET contains a large second exon of 1214 nucleotides. We show that this exon, containing the AUG for the Met receptor, is frequently skipped in normal human tissues and cell lines, and corresponds to a ubiquitously expressed 7 kb Met transcript. This transcript yields no detectable protein product in vivo. Thus, unlike other genes, in which alternative splicing often gives rise to proteins with distinct activities, exon-skipping of MET exon 2 is predicted to decrease the abundance of a Met mRNA encoding a functional Met receptor.

Alternative Splicing↗

PubMed1998

Transcriptional activation of the p34cdc2 gene by cdc2 promoter binding factor/nuclear factor-Y in fetal rat ventricular myocytes.

To determine how myocardial terminal differentiation is regulated by cell cycle control genes, we studied cdc2 expression in rat cardiac muscle and found that cdc2 mRNA and protein levels were reduced in neonatal compared with fetal ventricles and became undetectable in juvenile and adult ventricles. To further determine whether cdc2 downregulation is attributed to a decrease in transcription, transient expression assay was performed using the progressively truncated 6.2-, 1.8-, 1.1-, 0.7-, and 0.1-kb human cdc2 5' flanking regions. All five fragments activated reporter expression in fetal myocytes and were significantly less active in neonatal myocytes. The 0.1-kb fragment showed 65% of the activity of the 6.2-kb fragment. A protein binding site that contains an inverted CCAAT box was identified within the 0.1-kb fragment by DNase I footprint assay and named the cdc2 promoter binding factor (CPBF) site. Point mutations within the CPBF site that abolish CPBF binding significantly decreased both 0.1- and 6.2-kb promoter activities. Competition and antibody supershift assays suggested that CPBF was identical or related to the transcription factor, nuclear factor Y (NF-Y). The 0.1-kb promoter activity was suppressed by a dominant-negative NF-Y mutant in fetal myocytes. Taken together, our results demonstrate that cardiac cdc2 expression is downregulated after birth and turned off when the juvenile stage is attained. A 0.1-kb promoter fragment of cdc2 contains major information for both cdc2 transcriptional activation and suppression in fetal and neonatal myocytes, respectively. NF-Y or its related factor plays a critical role in activating the 0.1-kb cdc2 promoter.

Animals↗

PubMed1998

Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2-.) production by biological systems. However, its validity as a O2-.-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2-.. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2-. has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2-. production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2-. in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 microM in all of the O2-.-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/ NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2-. production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/ xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2-.-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2-.. Thus, LDCL still appears to be a valid probe for detecting O2-. production by enzymatic and cellular sources.

Acridines↗

PubMed1998

Human magnetic resonance imaging at 8 T.

In this work, we present the first human magnetic resonance image (MRI) obtained at ultrahigh field strengths (8 T). We demonstrate that clinical imaging will be possible at 8 T and that reasonable quality head images can be obtained at this field strength. Most importantly, we emphasize that the power required to excite the spins at 8 T is much lower than had previously been predicted by the nuclear magnetic resonance theory. A 90 degree pulse in the head at 8 T requires only approximately 0.085 J of energy (90 W for a 2-lobe 4 ms sinc pulse). Based on measurements at 4 T, 1-2 J of energy should have been utilized to achieve a 90 degree excitation at 8 T. The fact that the energy required for spin excitation at 8 T is much lower than predicted by the NMR theory, will be extremely important to the viability of ultrahigh field imaging, since concerns related to power absorption and specific absorption rate (SAR) violations at ultrahigh field are alleviated. As such, it will be possible to utilize RF intensive pulse sequences and adiabatic spin excitation at 8 T without significant risk to the subject.

Brain↗

PubMed1998

Subtyping of transferrin by isoelectric focusing in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients ranging from pH 5.05 to 5.60 was used to study the distribution of transferrin (Tf) subtypes and their gene frequencies from 188 unrelated healthy donors of the Han population in Beijing. Six phenotypes (TfC1, TfC2, TfC1C2, TfC1C3, TfC1Dchi and TfC2Dchi) were detected and Tf*C3 has never before been reported in the Han population. Gene frequencies were as follows: Tf*C1 = 0.7420, Tf*C2 = 0.2420, Tf*C3 =0.0027, Tf*Dchi = 0.0133. There is good agreement between the observed and expected values corresponding to the Hardy-Weinberg equilibrium (sum (chi2) = 0.9183, df = 2, 0.5 < P < 0.75). The allele frequencies for Tf*C1, Tf*C2 and Tf*Dchi agreed with those previously reported for the Chinese Han population.

Humans↗

PubMed1998

The role of glutamate in the locus coeruleus during opioid withdrawal and effects of H-7, a protein kinase inhibitor, on the action of glutamate in rats.

To investigate the role of glutamate in the locus coeruleus (LC) during opioid withdrawal, rats were continuously infused with morphine (a mu-opioid receptor agonist, 26 nmol/microl/h) or butorphanol (a mu/delta/kappa-mixed opioid receptor agonist, 26 nmol/microl/h) intracerebroventricularly (i.c.v.) via osmotic minipumps for 3 days. A direct LC injection of glutamate (1 or 10 nmol/5 microl) or naloxone (an opioid receptor antagonist, 24 nmol/5 microl) induced withdrawal signs in morphine- or butorphanol-dependent animals. However, these agents failed to precipitate any withdrawal signs in saline-treated control animals. On the other hand, the expression of withdrawal signs precipitated by the administration of glutamate or naloxone in opioid-dependent animals was completely blocked by concomitant infusion with 1 or 10 nmol/microl/h of an inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase and protein kinase C, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine]. In animals that had been infused with opioids in the same manner, i.c.v. injection of naloxone (48 nmol/5 microl) precipitated withdrawal signs and increased extracellular fluid levels of glutamate in the LC of morphine- or butorphanol-dependent rats measured by in vivo microdialysis method. However, concomitant infusion with H-7 inhibited the increases of glutamate levels in the LC. These results strongly suggest that an expeditious release of glutamate in the LC region plays an important role in the expression of physical dependence on opioids. Furthermore, the action on glutamate release might be increased by the enhancement of cAMP-dependent protein kinase and/or protein kinase C activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Expression of anti-CD4 human/murine chimeric antibody and their killer tumor activity.

From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup II (A) and kappa chain subgroup III, respectively. The VH and VL genes were subcloned into p gamma 1-Expr and p kappa-Expr respectively, then transfected into XL2-Blue. The VH- p gamma 1 and VL- p kappa were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specifically in vitro.

Animals↗

PubMed1998

Differential expression of Met/hepatocyte growth factor receptor in subtypes of non-small cell lung cancers.

Hepatocyte growth factor (HGF)/scatter factor (SF) is a multifunctional factor that stimulates epithelial cell motility, invasion and morphogenesis. Its receptor is a transmembrane tyrosine kinase encoded by the Met proto-oncogene. Several studies have suggested a possible role for HGF/Met in tumor development and progression. To investigate the potential roles of Met in human lung cancer biology, we have studied the mRNA and protein expression of Met in normal lung tissue, primary non-small cell lung carcinoma (NSCLC), and NSCLC cell lines. The results indicated a differential pattern of Met expression among various subtypes of NSCLC. The majority of squamous cell carcinoma (SQCC), either in vivo or in vitro, expressed Met mRNA and its protein product at levels much lower than or similar to normal lung tissue or bronchial epithelium. Moreover, SQCC characteristically over-expressed a variant Met mRNA which corresponds to a 5' partially deleted transcript produced by alternative splicing. In contrast, the expression of Met mRNA and its protein product in adenocarcinoma (ADC) and large cell undifferentiated carcinoma were more heterogeneous. Overexpression was demonstrated in approximately 35% and 20% of these subtypes of NSCLC, respectively. Among ADC, intermediate to high levels of Met immunoreactivity correlated with greater degree of tumor differentiation. Furthermore, an accentuation of Met immunoreactivity was often noted in cancer cells at the advancing edge of tumors. These findings support a role for Met in lung cancer cell invasion and differentiation in vivo, but its expression and functions may be modified by the differentiation phenotype of the tumor cells.

Adenocarcinoma↗

PubMed1998

Analysis of the accuracy of determining average molecular weights of narrow polydispersity polymers by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be used to determine number- and weight-average molecular weights of narrow polydispersity polymers. In this work, several possible sources of error in determining molecular weights of polymers with narrow polydispersity by MALDI-TOFMS are rigorously examined. These include the change in polymer distribution function, broadening or narrowing of the overall distribution, and the truncation of selected oligomer peaks within a distribution (i.e., the oligomer peaks at the high- and low-mass tails expected to be observed are not detected). These variations could be brought about by a limited detection sensitivity, background interference, and/or mass discrimination of oligomer analysis in MALDI-TOFMS. For narrow polydispersity polystyrenes, it is shown that by using an appropriate MALDI matrix and sample preparation protocol and a sensitive ion detection instrument, no systematic errors from these possible variations were detected within the experimental precision (0.5% relative standard deviation) of the MALDI method. It is concluded that MALDI mass spectrometry can provide accurate molecular weight and molecular weight distribution information for narrow polydispersity polymers, at least for polystyrenes examined in this work. The implications of this finding for polymer analysis are discussed.

Indicators and Reagents↗

PubMed1998

Somatic mutations of the mitochondrial genome in human colorectal tumours.

Alterations of oxidative phosphorylation in tumour cells were originally believed to have a causative role in cancerous growth. More recently, mitochondria have again received attention with regards to neoplasia, largely because of their role in apoptosis and other aspects of tumour biology. The mitochondrial genome is particularly susceptible to mutations because of the high level of reactive oxygen species (ROS) generation in this organelle, coupled with a low level of DNA repair. However, no detailed analysis of mitochondrial DNA in human tumours has yet been reported. In this study, we analysed the complete mtDNA genome of ten human colorectal cancer cell lines by sequencing and found mutations in seven (70%). The majority of mutations were transitions at purines, consistent with an ROS-related derivation. The mutations were somatic, and those evaluated occurred in the primary tumour from which the cell line was derived. Most of the mutations were homoplasmic, indicating that the mutant genome was dominant at the intracellular and intercellular levels. We showed that mitochondria can rapidly become homogeneous in colorectal cancer cells using cell fusions. These findings provide the first examples of homoplasmic mutations in the mtDNA of tumour cells and have potential implications for the abnormal metabolic and apoptotic processes in cancer.

Base Sequence↗

PubMed1998

Development of heritable melanoma in transgenic mice.

Transfer of genetic material into recipient cells by transfection has been used successfully to isolate genes responsible for particular phenotypic traits. By using this strategy, DNA fragments were isolated that when transfected into appropriate uncommitted cells will commit the recipient cells to undergo adipocyte differentiation. Transgenic mice were generated with one of the active DNA clones, Clone B. The transgenic mice were expected to display an adipocyte-related phenotype; however, the animals developed melanin containing tumors at a young age. Insertion of Clone B into the mouse DNA probably interrupted a gene(s) that is involved in the regulation of cell growth, specifically regulation of cell growth in melanin-producing cells. Histopathologic analysis of these mice showed dark spots on the ear lobes of the animals as early as 10-12 d of age. By 3 mo, in addition to the ear lobes, pigmented tumors could be observed in other organs. A significant number of these transgenic mice died within 1 y of age. The melanomas developed spontaneously in these animals in the absence of any known chemical carcinogen or ultraviolet radiation. This line of mice provides a way of identifying genes involved in regulation of cell growth control and differentiation. These mice also serve as a model system to investigate the molecular, genetic, and phenotypic characterization and development of melanomas.

Animals↗

PubMed1998

cDNA sequence analysis of monoclonal antibodies against the human placental acidic isoferritin.

By using human placental acidic isoferritin (PAF) as antigen to immunize BALB/c mice and conventional cell fusion, we have established three mouse hybridoma cell lines that secrete IgG monoclonal antibodies (MAbs) to PAF, termed as Z-2-3, Z-2-5, and Z-3-6, respectively. In ELISA, the MAbs were shown to react specifically with human PAF. We then applied the polymerase chain reaction (PCR) technique to clone variable region genes of the heavy (V(H)) and light (V(L)) chains of these MAbs, and appropriate full-length cDNA clones were obtained and characterized by nucleotide sequence analysis. V(H) and V(L) segments of anti-human PAF MAbs belong to the J558 and Vkappa19 family, respectively. The nucleotide sequence of Z-3-6 in the V(H) segment is highly homologous to that of MAb 18.1.16 except for their diversity minigenes. The light chain sequences of these MAbs show high homology with that of MAb cc92. It is implied that the D segment and the nucleotides inserted at the V(H)-D and D-J splice junctions are mostly responsible for the specificity of Z-3-6, and that the differences between the V(H) and V(L) sequences of these MAbs may determine their different affinity or recognition of different antigenic determinants.

Animals↗

PubMed1998

Identification of two new hydrophobic residues on basic fibroblast growth factor important for fibroblast growth factor receptor binding.

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several types of vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitopes required for receptor binding of the growth factor. Using structure-based site-directed mutagenesis, two critical areas on the bFGF surface for the high affinity receptor binding have already been identified [Springer, B.A., Pantoliano, M.W., Barberal, F.A., Gunyuzlu, P.L., Thompson, L.D., Herblin, W.F., Rosenfeld, S.A. and Book, G.W. (1994) J. Biol. Chem., 269, 26879-26884; Zhu, H.Y., Ramnarayan, K., Anchin, J., Miao, Y., Sereno, A., Millman, L., Zheng, J., Balaji, V.N. and Wolff, M.E. (1995) J. Biol. Chem., 270, 21869-21874; Zhu, H.Y., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M.E. (1997) Protein Engng, 10, 417-421]. According to these studies, one receptor binding site includes two polar residues Glu96 and Asn104 on bFGF whereas the other includes four hydrophobic residues Tyr24, Tyr103, Leu140 and Met142. Using a protein modelling technique, we report here the identification of a new hydrophobic patch on bFGF which includes residues Tyr73, Val88 and Phe93. The role of this area on receptor binding affinity was evaluated by mutating each of these residues individually and determining the mutated protein's (mutein's) receptor binding affinity. In addition, we examined the role of two other hydrophobic residues, Phe30 and Leu138, on bFGF for high-affinity receptor binding. These two residues are the neighbors of the hydrophobic residues Tyr24 and Tyr103, respectively. Replacement of Val88 and Phe93 with alanine reduced the receptor binding affinity about 10- and 80-fold, respectively, compared with wild-type bFGF. In contrast, substitution of Phe30 and Leu138 with alanine has no effect on the receptor binding affinities. We conclude that the newly identified hydrophobic residues, Val88 and Phe93, are crucial for the receptor binding. The present data, together with the previous identification of four hydrophobic residues (Tyr24, Tyr103, Leu140 and Met142), suggests that there are two hydrophobic receptor binding sites on the bFGF surface. Our findings can be employed in the discovery and design of potent bFGF antagonists using computational methods.

Amino Acid Substitution↗

PubMed1998

Organically-bound 3H concentration in rice around atomic energy facilities.

Rice samples collected around atomic energy facilities in the Tokai area were analyzed for organically-bound 3H (OBT). Measurement results were compared with those for samples from control areas in Japan and the People's Republic of China. OBT concentrations of rice in the Tokai area were found to be 4 times higher than the control area as maximum. Although this relatively high 3H concentration shows the effect of 3H release from the facilities into the environment, the concentration level is comparable with those in the rice sample from a northern interior region of China. The OBT concentration of rice sample from the general environment in interior China was 4.5 times higher than the control area in Japan. The radiation dose from the increasing 3H level is negligible in comparison with that from natural radiation.

Air Pollutants, Radioactive↗

PubMed1998

Identification of a full-length cDNA for an endogenous retrovirus of miniature swine.

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attachment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.

Amino Acid Sequence↗

PubMed1998

Identification, characterization, and mapping of Ecm1, a locus affecting extracellular matrix production and lesion size in Cochliobolus heterostrophus.

A mutant of Cochliobolus heterostrophus lacking the outer layer of extracellular matrix around its germ tubes and hyphae was obtained by mutagenizing protoplasts. The mutant not only lacks the outer matrix, but also produces much smaller lesions on corn leaves than nonmutant strains; the area of mutant lesions averages 0.6 mm2 compared with 5.8 mm2 for nonmutant lesions. Genetic analysis demonstrated that the failure to produce the outer matrix cosegregates with the reduced lesion size, indicating that the two traits are controlled by the same locus, designated Ecm1 (Extracellular Matrix Deficient). The mutant retains normal growth on media and normal abilities to germinate, form appressoria, and penetrate corn leaves. This indicates that the outer matrix is not necessary for infection prior to entrance of the fungus into the leaf. It also indicates that the pathogenicity defect in this mutant is manifested after penetration. To facilitate future tests of whether the pathogenicity defect is caused by the lack of the outer matrix, Ecm1 was mapped. Seven markers linked to Ecm1 were found by analysis of amplified fragment length polymorphisms. Ecm1 maps to chromosome 4; the closest markers to Ecm1 are 5 cM distant, which is estimated to represent about 115 kb.

Ascomycota↗