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M Zhang

Publications and source records attributed to M Zhang.

At least 901 records · Page 50Linked to original sources
PubMed1994

Crystallization and preliminary X-ray analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

Two crystal forms of bovine heart phosphotyrosyl protein phosphatase (BHPTP) have been examined by X-ray analysis. One crystal form grows as long rods with triclinic crystal symmetry and diffracts to 3 A resolution. The diffraction pattern of this form of the crystal shows twinning about a major axis. A second crystal form of BHPTP grows as flat trapezoidal prisms with monoclinic symmetry C2, and unit cell parameters a = 95.3 A, b = 43.3 A, c = 41.2 A and beta = 113.5 degrees. The unit cell dimensions indicate that there is one 18 kDa molecule per asymmetric unit. These crystals diffract to at least 2.2 A resolution and are resistant to decay in the X-ray beam.

Animals↗
PubMed1994

Characterization of trimethyllysine 115 in calmodulin by 14N and 13C NMR spectroscopy.

In this paper, we describe three approaches to study the single trimethyllysine 115 in calmodulin. First, 14N NMR spectroscopy has been used as a novel spectroscopic tool. Because of the unique symmetrical tetrahedral substitution of its side chain, the trimethyllysine residue gives rise to a sharp 14N NMR resonance; hence, this has allowed the detection and quantitation of the level of trimethylation. Trimethyllysine side chains of bovine testis calmodulin and yeast cytochrome c were shown to have a high mobility in aqueous solution as determined by 14N NMR relaxation measurements. Second, we have purified mammalian calmodulin from an overproducing Escherichia coli strain. By comparison of the 1H-13C heteronuclear multiple quantum coherence spectra of 13C-dimethylated calmodulin samples from bovine testis and E. coli, the resonance for Lys-115 in bacterially expressed calmodulin could be identified. pH titration experiments showed that epsilon-NH2 group of Lys-115 has a normal pKa value both in the apo and Ca2+ forms of the protein and in a complex of calmodulin with a 22-residue calmodulin-binding peptide derived from myosin light chain kinase. Third, we have shown that mutation of Lys-115 to the uncharged Gln residue does not alter the ability of the protein to stimulate the enzymes cyclic nucleotide phosphodiesterase and myosin light chain kinase. These results show that the trimethylation of Lys-115 is not caused by an unusual pKa and reactivity of its epsilon-NH2 group and that its side chain remains flexible. Moreover, our data suggest that the introduction of a permanent positive charge on Lys-115 by trimethylation is also not the major reason for this specific post-translational modification.

Amino Acid Sequence↗
PubMed1994

The calmodulin-binding domain of caldesmon binds to calmodulin in an alpha-helical conformation.

The binding of calcium-calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. It has been reported that a 17-residue synthetic peptide encompassing the residues Gly651-Ser667 of smooth muscle CaD constitutes its CaM-binding domain [Zhan, Q., Wong, S. S., & Wang, C.L.A. (1991) J. Biol. Chem. 266, 21810-21814]. This peptide does not share sequence homology with the CaM-binding domains of other proteins, and in addition, the binding of CaM to CaD is known to be relatively weak. Here we have investigated the properties of this atypical CaM-binding domain by NMR and circular dichroism (CD) spectroscopy. Two dimensional NMR studies performed in aqueous TFE mixture (75%/25%) showed that the peptide has the capacity to adopt an amphiphilic alpha-helical conformation. TRNOESY experiments and CD spectroscopy were used to determine that the CaD peptide binds in an alpha-helical conformation to CaM. The addition of TFE or the binding of the CaD peptide to CaM induces an alpha-helical structure only for the central 10 amino acid residues of the peptide. Titrations of CaM with the CaD peptide were followed by proton NMR and show the formation of a 1:1 complex and that the binding is calcium-dependent. The chemical shifts of 13C-methyl groups of specifically labeled Met residues and of the 15N backbone amide groups of CaM undergo changes upon addition of the CaD peptide; these data suggest that both domains and the central helix of CaM are involved in the binding of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Characterization of the calmodulin-binding domain of rat cerebellar nitric oxide synthase.

Nitric oxide (NO) has recently been identified as an intercellular messenger which is involved in the regulation of neurotransmission, vasorelaxation, and cytotoxicity. In cerebellum and endothelium this compound is synthesized by "constitutive" nitric oxide synthases (NOS); these are Ca(2+)-calmodulin (CaM)-dependent enzymes. A potential CaM-binding domain for the CaM-dependent NOS has previously been identified in the gene sequence. In this work, a synthetic 23 residue peptide encompassing the putative CaM-binding domain of rat cerebellar NOS was studied. The constitutive NOS peptide binds to CaM in a calcium-dependent manner with 1:1 stoichiometry as determined by polyacrylamide gel electrophoresis of the peptide-CaM complex in 4 M urea. Circular dichroism studies showed that the peptide binds to CaM in an alpha-helical conformation. Binding of the constitutive NOS peptide inhibits the stimulatory effect of CaM on cyclic nucleotide phosphodiesterase. From competition experiments between the peptide and phosphodiesterase we have determined a Kd of 2.2 nM for the peptide-CaM complex. Two-dimensional NMR and circular dichroism studies were used to determine the structure of the peptide in aqueous solution. In addition, the effect of increasing amounts of trifluoroethanol on the peptide structure was investigated. It was found that the peptide can adopt an alpha-helical structure which bears close resemblance to the structure of the CaM-bound form of the CaM-binding domains of myosin light chain kinases.

Amino Acid Oxidoreductases↗
PubMed1994

The malignancy of dementia. Predictors of mortality in clinically diagnosed dementia in a population survey of Shanghai, China.

OBJECTIVE: To evaluate the effect of dementing illnesses on the risk of dying, taking into account other conditions that would shorten survival. DESIGN: Five-year follow-up of community survey of dementia. SETTING: Five-year data were obtained for the 3531 subjects, aged 65 years and older, who participated in the 1987 population survey of dementia in Shanghai, China. MAIN OUTCOME MEASURE: Time to death. Relative risks of dying were calculated for demographic variables, dementia diagnoses based on findings of clinical evaluations, and 15 reported prevalent medical conditions using the proportional hazards model. RESULTS: In those subjects aged 65 to 74 years, the mortality risk ratio was 5.4 (95% confidence interval, 2.0 to 14.6) for Alzheimer's disease and 7.2 (95% confidence interval, 3.6 to 14.4) for vascular dementia. The risk ratio for Alzheimer's disease was similar to the mortality risk ratio for cancer (5.6 [range, 2.9 to 10.9]). In this age group, dementing illnesses were uncommon, and few deaths were therefore attributable to the dementing illnesses. In those subjects aged 75 years and older, the mortality risk ratios were 2.8 (95% confidence interval, 2.1 to 3.6) for Alzheimer's disease, 3.5 (95% confidence interval, 2.4 to 5.1) for vascular dementia, and 3.6 (95% confidence interval, 2.0 to 6.7) for "other dementias." Because these dementing disorders were common in those subjects aged 75 years and older, 23.7% of the risk of death could be attributed to these disorders. CONCLUSIONS: Both Alzheimer's disease and vascular dementias are truly malignant and constitute major risk factors for death in persons older than 75 years.

Aged↗
PubMed1994

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes.

The involvement of cytochrome b5 in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b5. Anti-b5 antiserum inhibited the reduction of cytochrome b5 by NADH-cytochrome b5 reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b5 is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b5. The results suggest that cytochrome b5 involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved.

Animals↗
PubMed1994

Solution conformation of a cyclic neurokinin antagonist: a NMR and molecular dynamics study.

The solution structure of a hexapeptide, cyclo (Gln-Trp-Phe-Gly-Leu-Met), which is a selective NK-2 antagonist, has been studied by a combination of two-dimensional nmr and molecular dynamics (MD) techniques. The simulation based on nmr and MD data resulted in the convergence to a family of structures. Free molecular dynamics for 50 ps in the presence of DMSO solvent molecules shows that the structure is energetically stable. One intramolecular hydrogen bond between the amide proton of Gln and the carbonyl oxygen of Gly was revealed. This result is consistent with the results from the measurement of the temperature coefficient of the amide protons. The extent of intermolecular hydrogen bonding between the amide protons of the peptide and DMSO was also revealed by the free MD simulation. The resulting structure of the cyclic peptide contains a variation type I' beta-turn in the Gly-Leu-Met-Gln segment. Comparison of the structure of this peptide with that of other NK-2 antagonist cyclic hexapeptides was made, and the activity of cyclic antagonists appears to be inversely related to the conformational rigidity of the cyclic peptides.

Amino Acid Sequence↗
PubMed1994

Use of antibodies to identify satellite cells with a light microscope.

Satellite cells are essential for the growth and regeneration of skeletal muscles but the study of them has been difficult due to the need to use an electron microscope. This article describes a method which enables satellite cells to be identified with a light microscope. Satellite cells were distinguished from: (1) myonuclei on the basis of whether the nuclei were inside or outside the plasmalemma of a fiber (stained with antidystrophin); and (2) interstitial cells depending on whether they were inside or outside of a fiber's basal lamina (stained with anticollagen IV). Some satellite cells were stained by the antidystrophin which further aided their identification. The question of whether all satellite cells are myogenic has been investigated and discussed. This method will facilitate the study of satellite cells in biopsies of human muscles and the estimation of satellite cell number in laboratory animals.

Animals↗
PubMed1994

Overexpression and characterization of recombinant human fusion protein IL-6/IL-2 (CH925).

An expression vector encoding the human recombinant fusion protein interleukin 6/interleukin 2 (IL-6/IL-2) was constructed. When a flexible linker had been synthesized and ligated with the IL-2 gene fragment by polymerase chain reaction (PCR) amplification, the IL-6 gene fragment was unidirectionally inserted into the upstream of the linker-IL-2 sequence. The molecule of the IL-6-linker-IL-2 fusion gene named E. coli DH5 alpha/pfIL-6/2 was cloned and identified by DNA sequencing. The expressed protein named as CH925 showed a strong band on SDS-PAGE and amounted to 32% of total cell protein, and its estimated molecular weight was about 37 kDa. The fusion protein purified by gel filtration and reversed-phase HPLC showed as almost homogeneous. CH925 possesses both IL-2 and IL-6 activities when assayed by CTLL2- and 7TD1-dependent cell lines, respectively. The specific activity of IL-2 was 2.1 x 10(6) U/mg while that of IL-6 was 2.3 x 10(8) U/mg. Our studies exhibited that CH925 exerted a significant augmentative effect on the growth of erythroid colony forming units (CFU-E), and synergized with erythropoietin (EPO) and/or IL-3 in a dose-dependent way. Our experimental results also showed CH925 at a low dose causing active lymphokine-activated killer (LAK) cell proliferation more vigorous than IL-2 and/or IL-6 (p < 0.001). CH925 is a novel fusion protein, being neither IL-6 nor IL-2, more potent than IL-2 and/or IL-6 and causing non-IL-2 and non-IL-6 functions of strong EPO-like and mild IL-3-like effects on erythroid progenitor cell growth. There is a potential for efficacious clinical application of CH925.

Base Sequence↗
PubMed1994

Modulation of connexins during differentiation of oval cells into hepatocytes.

The connexins are a family of related gap-junction proteins, implicated in embryonic development, cell growth control, and cellular differentiation. To identify connexins involved in liver cell differentiation, both in vivo and in vitro systems were employed to study expression of connexins 26, 32, and 43. Northern blot analysis and in situ hybridization were used to measure the levels of connexin expression and cellular localization of the transcripts, respectively. Normal liver expressed high connexin 32, low connexin 26, and barely detectable connexin 43. In vivo proliferation and differentiation of oval cells was at first accompanied by increased connexin 43 and decreased connexin 32 expression; later as the oval cells differentiated into hepatocytes, connexin 43 disappeared and connexin 32 increased to control levels. In situ hybridization showed that both oval cells and bile duct epithelial cells, but not hepatocytes, expressed connexin 43. A switch from connexin 43 to connexin 32 expression was observed following in vitro transformation and differentiation of rat liver epithelial cells toward the hepatocytic lineage. These results suggest that early progenitor cells in the liver express connexin 43 and a switch from connexin 43 to connexin 32 may signal commitment to hepatocytic differentiation.

Animals↗
PubMed1994

Autoradiographic localization of beta-endorphin binding in the pancreas.

Autoradiographic localization of 125I-labeled beta-endorphin binding in the rabbit pancreas demonstrated specific binding in the pancreatic islet cells. Binding was inhibited by (1) nonradioactive beta-endorphin, (2) the opioid antagonist naloxone, (3) the mu receptor agonists morphine and [D-Ala2, (Me)Phe4, Gly(ol)5]enkephalin, (4) the delta receptor agonist [D-penicillamine2, D-penicillamine5]-enkephalin, (5) the mu and delta agonist met-enkephalin and (6) the delta and kappa agonist dynorphin. Specific binding was not clearly demonstrable in the acinar portion of the rabbit pancreas. The binding characteristics of 125I-beta-endorphin in the pancreatic islets were comparable with those of mu and delta opioid receptors in the rabbit brain. In the pancreas, beta-endorphin binding appeared to be concentrated in discrete areas in the islets. Combined immunohistochemistry and autoradiography demonstrated that beta-endorphin binding was primarily concentrated in the glucagon-containing alpha and somatostatin-containing delta cells, but was also found in the insulin-containing beta cells to a lesser extent. Given the intraislet location of the opioid binding sites, and our previous finding of immunoreactive beta-endorphin in the pancreatic beta cells and the inhibitory effect of beta-endorphin on insulin secretion, it appears that beta-endorphin may serve a paracrine or autocrine function in the regulation of pancreatic hormone secretion.

Animals↗
PubMed1994

Genomic structure and chromosomal localization of processed pseudogenes for human RBP-Jk.

The functional gene for human recombination signal sequence-binding protein (RBP-Jk) and corresponding processed psudogenes have been isolated from various species, such as Drosophila, Xenopus, mouse, and human. Here we report the isolation of another two genomic pseudogenes of human RBP-Jk, named K2 and K7, from a cosmid library of Hela cells. The nucleotide sequences of both genes exhibited more than 95% homology to the functional human gene for RBP-Jk. Moreover, they did not contain any intron sequences and were interrupted by several stop codons in all frames. In situ hybridization demonstrated that the pseudogenes, K2 and K7, were localized at chromosomes 9p13 and 9q13, respectively. Their physical maps differed from those of the true functional gene and of the pseudogenes reported previously by Amakawa et al. (1993).

Base Sequence↗
PubMed1994

Reductive methylation and pKa determination of the lysine side chains in calbindin D9k.

The Lys residues in the 75-residue Ca(2+)-binding protein calbindin D9k were reductively methylated with 13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca(2+)- and apo-forms of the protein, the 13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. The pKa values of the individual Lys residues in Ca(2+)-calbindin D9k and apo-calbindin D9k were obtained by combining pH titration experiments and (1H, 13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca(2+)- and apo-forms of calbindin D9k has a unique pKa value. The Lys pKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca(2+)-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of the pKa values of the Lys residues.

Calbindins↗
PubMed1994

ELISA for the neuropeptide degrading endopeptidase 3.4.24.11 in human serum and leukocytes.

We describe the development of a new ELISA for the detection of neural endopeptidase 3.4.24.11 (NEP). Neutral endopeptidase 3.4.24.11 was determined in preparations of human granulocytes, mononuclear cells (MNC), and in serum. Human recombinant NEP was used as reference. Specificity of the mAbs was tested using APAAP, FACS analysis, and Western blot analysis. Lysis of the blood cells was performed by incubating the cells with 0.4% Tween-20 and repeated freezing cycles. The minimal detectable dose for recombinant NEP was 15 pg/ml. The recovery was 94 +/- 9%. The NEP was detectable in 15 out of 20 serum samples of 20 volunteers (mean +/- SEM, 245 +/- 88 pg/ml, n = 20)) and in all granulocyte preparations (1176 +/- 138 pg/10(7) cells, n = 20)). The results were reproducible among replicates (CV = 3 +/- 1%, n = 40), dilutions (CV = 8 +/- 2%, n = 5), and assays (CV = 12 +/- 4%, n = 5). With this new ELISA, a simple and reproducible method for the measurement of NEP 3.4.24.11 is described.

Blotting, Western↗
PubMed1994

Suppression of the allogeneic response by human IL-10: a critical role for suppression of a synergy between IL-2 and TNF-alpha.

The effect of IL-10 on T-cell activation by alloantigens in primary mixed lymphocyte reaction (MLR) was examined. IL-10 strongly suppressed proliferation and cytokine synthesis observed in this reaction. To determine the cytokine synthesis inhibition that was critical for the IL-10 induced suppression of proliferation in MLR, the effect of exogenous cytokines (IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IFN-gamma, or TNF-alpha) on this suppression was examined. None of these cytokines, when used at high concentration, was able to completely restore proliferation in the MLR to the levels observed in the absence of IL-10. However, IL-2 and TNF-alpha, when added alone at high concentration, could partially overcome the IL-10 induced suppression of proliferation in MLR. Moreover, when a combination of IL-2 and TNF-alpha was added at suboptimal doses to IL-10-suppressed MLR, complete restoration of the proliferative response was obtained. The ability of IL-10 to suppress proliferation in MLR was dependent on the type of cells used as stimulators. Thus, IL-10 suppressed proliferation in MLR when allogeneic normal peripheral blood mononuclear cells (PBMC), highly purified monocytes or B cells, were used, but not when B-cell lines were used as stimulators. Investigation of the effect of IL-10 on cytokine synthesis revealed that when B-cell lines were used as stimulators of MLR, IL-10 suppressed IFN-gamma and IL-2 synthesis but was unable to suppress TNF-alpha production. In contrast, CSA, which inhibited proliferation in MLR induced by B-cell lines, also inhibited TNF-alpha. IL-2 and IFN-gamma synthesis. Together these data suggest that the suppression of MLR by IL-10 requires the effective inhibition of both IL-2 and TNF-alpha production to suppress a synergy between these two cytokines.

Antibodies, Monoclonal↗
PubMed1994

Distribution of NADPH-diaphorase-positive neurons in the enteric nervous system of the human colon.

Recent investigations have demonstrated the colonization of NOS-immunoreactivity and NADPH-diaphorase staining in central and peripheral neurons. The current study investigates the presence and distribution of NADPH-diaphorase-stained nerve cells and fibres in whole-mount preparations of the enteric nervous system of the human colon. Numerous NADPH-diaphorase-stained nerve cell bodies were found in the plexus myentericus and in the plexus submucosus externus. In contrast, we found but very few NADPH-diaphorase-stained nerve cells in the plexus submucosus internus. The majority of the NADPH-diaphorase-stained nerve cells had morphological characteristics similar to those of the Dogiel type I neuron, i.e. possessing broad flat dendrites and one major axonal projection. NADPH-diaphorase-stained nerve fibres can be observed in all three ganglionic networks and in the aganglionic plexus of the circular muscle layer.

Colon↗