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Biomedical subjects

M Zhang

Publications and source records attributed to M Zhang.

At least 775 records · Page 43Linked to original sources
PubMed1996

Antisense blockade of inducible nitric oxide synthase in glial cells derived from adult SJL mice.

Increasing evidence suggests a correlation between cytokine-induced nitric oxide synthase (iNOS) and demyelination in Multiple sclerosis (MS). Inhibition of iNOS may therefore be a novel therapeutic approach in MS. To test an antisense oligodeoxynucleotide (ODN) knockdown strategy for inhibiting iNOS, we used lipopolysaccharide (LPS) together with gamma-interferon (IFN-gamma) to induce iNOS in adult mouse mixed glial cell cultures. We administered an iNOS-derived antisense phosphorothiorate oligodeoxynucleotide (S-ODN) to block the induction. The antisense ODN treatment resulted in significant inhibition of LPS and IFN-gamma induced iNOS mRNA and protein expression. It also inhibited nitric oxide (NO) and cyclic GMP (cGMP) production in a dose dependent fashion. Sense and random S-oligo had no effect in any of these studies. These data indicate the efficacy and specificity of the antisense oligodeoxynucleotide approach in inhibiting iNOS in glial cells.

Animals↗
PubMed1996

Site-directed mutagenesis, kinetic, and spectroscopic studies of the P-loop residues in a low molecular weight protein tyrosine phosphatase.

The structure of the specific phosphate binding loop (P-loop) of bovine protein tyrosine phosphatase (BPTP) is very similar to that present in high M(r) PTPases. Site-directed mutagenesis was used to explore the role of several conserved residues involved in forming the P-loop of BPTP. Thus, Ser-19 and Ser-43 were individually mutated to alanines, and Asn-15 was mutated to alanine and glutamine. The 1H NMR spectra of the mutants showed good conservation of global secondary structure when compared to wild-type enzyme. Kinetic measurements revealed that only S19A and N15A had substantially altered catalytic activities toward p-nitrophenyl phosphate at pH 5.0, with both mutants exhibiting Vmax values that were 0.25-0.33% of wild-type enzyme. Further kinetic analyses of the N15A and S19A mutants were performed using phosphomonoester substrates with varied phenolic leaving groups. For S19A, the slope of the correlation between Vmax and the substrate leaving group pKa was significantly altered, consistent with a change of the rate-determining step from dephosphorylation to phosphorylation. This was confirmed by partitioning experiments employing methanol as an alternative nucleophile in the dephosphorylation step. Thus, mutating Ser-19 to alanine reduced the efficiency of nucleophilic attack by Cys-12. It is concluded that Ser-19 acts to facilitate the ionization and orientation of Cys-12 for optimal reaction as a nucleophile and as a leaving group. It also appears that Asn-15, Ser-19, His-72, and to a lesser extent Ser-43 serve structural functions that allow the active site to adopt an optimal geometry for phosphate binding. The Asn-15 to Ala mutation appears to disrupt the hydrogen-bonding network, with an accompanying alteration of the geometry of the P-loop. These conclusions are also consistent with changes in the stability of the respective proteins, as measured by urea denaturation.

Amino Acid Sequence↗
PubMed1996

Cell cycle-dependent expression of TAP1, TAP2, and HLA-B27 messenger RNAs in a human breast cancer cell line.

Tumor cells are generally poorly responsive to immunotherapy. The results presented here suggest that antigen presentation of somatic tumor cells may be diminished greatly in quiescence and may be determined in part by growth regulation. Peptides produced by proteasomes are transported into the endoplasmic reticulum by transporter proteins TAP-1 and TAP-2, where they bind and stabilize MHC class I molecules required for antigenic presentation on the cell surface. TAP-1 and TAP-2 mRNAs were undetectable in quiescent, serum-deprived human breast cancer cells (21PT). They appeared 10 h after serum induction, near the G(1)-S boundary. In contrast, HLA-B27 mRNA was biphasically up-regulated. These mRNAs were significantly down-regulated in most tissues that contain mainly terminally differentiated, nonproliferating cells. All of the investigated breast cancer cell lines showed lower expression levels of these mRNAs than did the corresponding normal cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1996

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: thermal- and trifluoroethanol-induced denaturation at neutral pH.

The unfolding and refolding of alpha-helical proteins has been extensively studied, demonstrating formation of intermediate structures which retain the native-like alpha-helix but lack the tertiary structure. Studies on the folding of proteins consisting primarily of beta-sheet are interesting since, unlike the alpha-helix, the beta-sheet requires the formation of peptide hydrogen bonds between two or more polypeptide segments which may be far apart in the linear sequence. Here we have studied the unfolding of the beta-sheet-containing protein tumor necrosis factor-alpha (TNF-alpha). This protein exists as a symmetric trimer in solution. Murine TNF-alpha begins to melt at 60 degrees C and unfolds to a soluble structure with a transition midpoint of 66 degrees C. This reaction is irreversible. This unfolded form contains a considerable amount of (approximately 30%) alpha-helix, as determined by circular dichroism. Human TNF-alpha begins to melt at 60 degrees C and precipitates concurrently with unfolding, such that there is no soluble protein present by 70 degrees C. The secondary and tertiary structures of murine TNF-alpha unfold simultaneously, suggesting that unfolding from the native to the unfolded state occurs cooperatively. The thermal-induced denaturation is very insensitive to protein concentration, indicating that trimer to monomer conversion, if it occurs, is not rate-limiting. Trifluoroethanol induces alpha-helix in both human and murine TNF-alpha, further demonstrating the propensity of TNF-alpha to form alpha-helix. The different behavior of human versus murine TNF-alpha upon thermal unfolding is due to differences in the solubility of the unfolded protein, the murine form being more soluble. These results indicate that TNF-alpha can form alpha-helix when the long range interactions conferred by the native structure are removed during unfolding.

Animals↗
PubMed1996

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation.

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.

Animals↗
PubMed1996

Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth.

The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.

Amino Acid Sequence↗
PubMed1996

Topological folding and proteolysis profile of P-glycoprotein in membranes of multidrug-resistant cells: implications for the drug-transport mechanism.

P-glycoprotein (Pgp)1 is a polytopic membrane protein and functions as an energy-dependent drug efflux pump. It is responsible for multidrug resistance (MDR) in cancer cell lines. Recently, the topological structure of Pgp has been investigated. However, the results are in dispute. A major question concerning the Pgp topology is the membrane orientation of the loop linking TM4 and TM5 (loop 4) and the loop linking TM8 and TM9 (loop 8). In this study, we generated polyclonal antibodies specific to these two loops. In combination with a panel of other well-characterized site-specific polyclonal- and monoclonal antibodies of Pgp, we tested the membrane orientation of these two loops of Pgp in multidrug-resistant cells using immunocytochemistry and proteolysis/membrane protection assay. Our results showed that (1) both loops 4 and 8 are located extracellularly whereas other domains, such as the ATP-binding sites, are in the cytoplasm and (2) proteolysis of Pgp is not a random event and the trypsin-sensitive sites are cleaved in orders. Since the Pgp was not genetically manipulated in this study, in contrast to previous studies, we believe that naturally expressed Pgp molecules have an unconventional topology. We speculate that this alternate topology of Pgp may represent a different functional state of the protein. Further detailed analysis of Pgp topology will help to understand the fundamental mechanism of drug transport by Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The cervicothalamic tract terminates in Cat301-sparse regions of the cat VPL.

The termination pattern of the cervicothalamic tract (CTI), labelled with anterogradely transported WGA-HRP, was compared with the immunolabelling pattern obtained with the monoclonal antibody Cat301 in adjacent sections through the ventral posterolateral nucleus (VPL). CTT terminations are located in peripheral parts of the medial and lateral parts of the VPL (VPLm and VPL1), being more extensive in the caudal than in the rostral parts of the subnuclei, and in the dorsal part of VPL1 and dorsolateral part of VPLm, regions that are all sparse in CAt301 immunoreactivity. Central regions of the VPL with dense Cat301 immunolabelling contain only very sparse CTT termination. Thus, our findings show that the CTT innervates a compartment of the VPL that is characterized by sparse Cat301 immunoreactivity.

Animals↗
PubMed1996

FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex.

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection.

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C18 column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5-4000 ng/ml. The recovery of internal standard "8212" at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.

Acetic Acid↗
PubMed1996

Rheumatoid factor idiotypic and antigenic specificity is strongly influenced by the light chain VJ junction.

The aim of this study was to define the structural basis for rheumatoid factor (RF) specificity and for the expression of the RF light chain-associated Ids, 4C9 and 6B6.6, by determining the reactivity of recombined heavy and light chains of Ig derived from monoclonal B cell lines of patients with rheumatoid arthritis and of light chains with site-directed mutations. We found that expression of the 4C9 and 6B6.6 Ids resulted from use of the VkIIIa genes Humkv 328 and Vg, but only in the presence of a permissive VJ junction. Expression of the Ids was independent of heavy chain use for the Humkv328-encoded light chains, but was highly dependent on the associated heavy chain for the Vg-encoded light chains. The RF specificity of the Abs was primarily heavy chain dependent, but the light chain VJ junction was critical in determining the relative avidity of the Abs for Fc. Our study points to the critical contribution of the somatically generated VJ junction to RF autoantibody specificity and to the expression of the two RF-associated Ids studied.

Amino Acid Sequence↗
PubMed1996

Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule.

We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.

Amino Acid Sequence↗
PubMed1996

Cost-effectiveness of clozapine monitoring after the first 6 months.

BACKGROUND: Clozapine is effective in treating patients with schizophrenia who do not respond to conventional neuroleptic drugs. The drug is unique in that it is available only with a US Food and Drug Administration-mandated system for weekly monitoring of patients' white blood cell counts. No study has been conducted to evaluate the cost-effectiveness of this mandatory monitoring system. METHODS: A benchmark case was established by utilizing cumulative incidence rates of agranulocytosis from a recent study with a large sample of clozapine-treated patients. We assumed a 20% mortality among patients with agranulocytosis, $30.61 in monitoring costs each week, and 14.4 years of remaining life expectancy after detection of agranulocytosis. Based on these bench-mark assumptions, cost-effectiveness ratios in dollars per quality-adjusted life-year were calculated for the first, second, and third 6-month periods during which a patient was receiving clozapine. Sensitivity analyses were performed with more conservative assumptions in 5 alternative scenarios. RESULTS: In the benchmark case, costs per quality-adjusted life-year gained were $61,694, $925,418, and $420,644 for the first, second, and third 6-month periods of clozapine treatment, respectively. In the alternative scenarios, these costs ranged from $7923 to $46,056 for the first 6-month period and from $54,025 to $690,850 for the second and third 6-month periods. CONCLUSIONS: While the costs of monitoring patients with schizophrenia in the first 6-month period of clozapine treatment seem to be justifiable, monitoring thereafter may not be cost-effective because of the very low incidence of agranulocytosis in the later periods.

Adult↗
PubMed1996

Intravenous vs. intraprostatic administration of N-methyl-N-nitrosourea to induce prostate cancer in rats.

To develop an improved model of human prostate cancer, 16-wk-old Wistar rats were treated orally for 18 days with the antiandrogen, flutamide (50 mg/kg body weight [BW]/day), followed by 3 days of s.c. testosterone (100 mg/kg BW). There were the only treatments the control animals received (Group 1, n = 10). On the day after the third testosterone injection, N-methyl-N-nitorsourea (MNU) was administered via the tail vein at a dose of 50 mg/kg BW (Groups 2, n = 40 and 3, n = 20); in some rats, a second dose was delivered by the same route 22 wk later (Group 3). A smaller dose of MNU (15 mg/kg BW) was administered intraprostatically (Group 4, n = 20) to a fourth group. In Groups 2, 3, and 4, silastic capsules containing testosterone were implanted s.c. approximately every 6 wk beginning 1 wk post-MNU. Accessory sex gland tumors arose in MNU-treated rats in Group 2 (12/40, 30%). Group 3 (8/20, 40%), and Group 4 (8/20, 40%); 90% were macroscopic (25/28). There were no neoplasms in these organs in the control rats (Group 1, 0/10). These accessory sex gland neoplasms were adenocarcinomas or undifferentiated carcinomas which appeared to be derived from the prostate based on location and histological characteristics, although the size and spread of some of the tumors precluded definitive localization of the tissue of origin. The incidence of these neoplasms was similar in rats given a single dose of MNU intraprostatically or two doses of MNU i.v., but the animals treated intraprostatically maintained higher body weights and developed fewer extraneous tumors. The average (+/- SD) latent period for clinical or postmortem detection of prostate neoplasia after MNU was shortest in the rats given two i.v. doses (39 +/- 3 wk) compared with the single i.v. dose (45 +/- 6 wk) or an intraprostatic dose (56 +/- 7 wk). In 57% of the cases (16/28), the prostate tumors metastasized to distant sites. An activating point mutation was detected in codon 12 of the Ki-ras oncogene in the MNU-induced primary prostate tumors (8/10 examined), and metastases arising from these prostate tumors (2/3) but was absent in normal prostate tissue (0/6). This study demonstrates that two systemic doses of MNU increase the incidence and decrease the latency of prostate neoplasms compared with a single dose, and that a single dose of MNU injected intraprostatically induces prostate adenocarcinoma without many of the other tumors and weight loss typically found after i.v. administration.

Adenocarcinoma↗