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Biomedical subjects

X Zhang

Publications and source records attributed to X Zhang.

At least 19 recordsLinked to original sources
PubMed1992

An electron microscopical study of calcitonin gene-related peptide-like immunoreactive innervation of the anterior pituitary in the dog.

Our previous studies have demonstrated the presence of a considerable number of substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive (LI) nerve fibers in the anterior pituitary in several mammalian species. The present study investigated the ultrastructure of the CGRP-LI innervation of this gland in the dog. The CGRP-LI nerve fibers were unmyelinated, with a wealth of varicosities containing both small clear synaptic vesicles and large dense-cored vesicles. They were found to be in direct contact with every cell type of the anterior pituitary. However, only on corticotropes and somatotropes were CGRP-LI synaptic contacts identified. Most of them were asymmetrical in type. Occasional symmetrical synaptic contacts were also found. It is considered likely that direct neural factors may play a role in the regulation of the anterior pituitary.

Animals↗
PubMed1992

Chemical contrast in X-ray microscopy and spatially resolved XANES spectroscopy of organic specimens.

The scanning transmission x-ray microscope at the National Synchrotron Light Source has been used to record x-ray absorption near-edge structure (XANES) spectra from 0.01-square-micrometer regions of organic specimens. The spectral features observed reflect the molecular structure of the dominant absorbing atoms and provide the contrast mechanism for high-resolution imaging with chemical sensitivity. This technique was used with x-ray energies near the carbon K absorption edge to identify and map separate phases in various polymer blends and to map the DNA distribution in chromosomes with a spatial resolution of 55 nanometers.

Acrylic Resins↗
PubMed1992

Vasopressin receptors in the area postrema differentially modulate baroreceptor responses in rats.

This study examined the effects of microinjection of [Arg8]vasopressin (AVP) into the area postrema (AP) on baroreceptor reflex control of heart rate (HR) and sympathetic efferent discharge (SED) in anesthetized rats. Comparable increments in blood pressure evoked by systemic AVP, as opposed to phenylephrine, were associated with significantly greater reflex bradycardia. Similarly, AVP augmented the baroreflex-mediated sympathoinhibition; however, this effect was evident only with the lower increments in arterial pressure (< 45 mm Hg) i.e. following systemic administration of small doses of AVP. Beyond 45 mm Hg there was no further augmentation of baroreflex-mediated sympathoinhibition showing the non-linearity of the response compared to phenylephrine which was linear over a wide range of induced pressure increases. Microinjection of AVP into the AP produced a differential effect on HR and SED responses to baroreceptor activation by systemically administered phenylephrine, the baroreflex slope of HR response was attenuated whereas that of SED was enhanced. Microinjection of the V1 antagonist AVPX (d(CH2)5Tyr(Me)-AVP) into the AP abolished the inhibitory effect of AVP on the baroreceptor-HR response suggesting that V1 receptors are involved in this response. Further, AVPX inhibited the baroreceptor-SED response suggesting that V1 receptors in the AP are tonically involved in modulating the baroreceptor reflex control of SED. Qualitatively similar but smaller responses were obtained following microinjection of AVP into the nucleus tractus solitarii (NTS) suggesting involvement of neural input from the AP to the NTS in AVP-evoked responses in the AP. It is concluded that the AVP receptors in the AP differentially modulates the baroreceptor reflex control of HR and SED.

Animals↗
PubMed1992

Physiology and morphology of neurons in the dorsal motor nucleus of the vagus and the nucleus of the solitary tract that are sensitive to distension of the small intestine.

We have recently shown that distension-sensitive vagal afferents are part of a neural circuit affecting absorption of water in the rat small intestine. Our results indicated that vagal afferent activity directly or indirectly influences the activity of neurons in the dorsal motor nucleus of the vagus (DMNV). In the present study we pursued this interaction by examining the structure and function of neurons in the DMNV and nucleus of the solitary tract (NST) that responded to moderate distension of the small intestine. Distension-sensitive cells were filled by intracellular iontophoretic injection of horseradish peroxidase. A total of 43 distension-sensitive brainstem neurons were successfully characterized and labeled. Sixteen of the 17 NST neurons were excited by distension of the small intestine. Ten of the seventeen were restricted to the ipsilateral NST. Only two NST neurons possessed axons that terminated in the subjacent DMNV. In contrast to the response profile of the NST neurons, 24 of 26 DMNV neurons were inhibited by intestinal distension. Fourteen of the DMNV neurons appeared to contribute to the vagus nerve and 15 extended dendrites into the overlying NST. We propose that distension-induced inhibition of DMNV activity is accomplished by inhibitory NST neurons, which synapse on the dendrites of DMNV neurons in the NST.

Afferent Pathways↗
PubMed1992

Barbiturate tolerance: effects on GABA-operated chloride channel function.

Male ICR mice were fed powdered laboratory chow containing phenobarbital for 7 days to induce tolerance. Mice were sacrificed and brains assayed for changes in GABA-mediated chloride flux into brain membrane vesicles (microsacs). Concentration-dependent stimulation of chloride flux by GABA alone was not affected by the development of tolerance to phenobarbital. Phenobarbital potentiation of GABA-mediated chloride flux was significantly attenuated in the membranes prepared from phenobarbital-tolerant mice compared with those from pair-fed control mice. Similarly, stimulation of GABA-mediated flux by the benzodiazepine, flunitrazepam was also depressed in membranes from tolerant mice. However, the ability of ethanol and the benzodiazepine inverse agonist FG-7142 to modulate GABA-gated chloride flux was not affected by the development of phenobarbital tolerance. No significant changes in saturation [3H]diazepam binding parameters were observed. These findings suggest that there is a degree of cross-tolerance between phenobarbital and benzodiazepine agonist at the level of the GABA-operated chloride channel. Furthermore, although some reports have demonstrated behavioral cross-tolerance between ethanol and barbiturates, the present data suggest different mechanisms of tolerance development for these intoxicants at the level of the GABAA receptor chloride channel complex.

Animals↗
PubMed1992

Enantiomeric N-substituted N-normetazocines: a comparative study of affinities at sigma, PCP, and mu opioid receptors.

The optical antipodes of N-allyl-N-normetazocine (2; SKF 10047, NANM) were the original compounds used for the classification of the sigma receptor as distinct from other receptors such as the PCP (NMDA), opioid, and dopamine receptors. Later studies showed that (+)-N-(dimethylallyl)-N-normetazocine [(+)-4, (+)-pentazocine] was more potent and selective for the sigma receptor. In order to gain additional structure-activity relationship information, several N-substituted N-normetazocine analogs were prepared and evaluated for their sigma-1 ([3H]-(+)-3-PPP or [3H]-(+)-pentazocine), PCP ([3H]TCP), and mu opioid ([3H]DAMGO) receptor binding affinities. (+)-N-Benzyl-N-normetazocine [(+)-10)] possessed subnanomolar affinities for the sigma site, Ki = 0.67. The analog (+)-10 showed greater than 14,000- and 2400-fold selectivity, respectively, for the sigma receptor relative to the PCP and mu opioid receptors. The N-substituted N-normetazocines were enantioselective for the sigma site. The (+)-N-benzyl analog, (+)-10, showed a 55-fold selectivity relative to (-)-10. Analysis of the data also revealed that (+)-normetazocine [(+)-1] [Ki = 30 nM] possessed the highest affinity for the PCP receptor. However, (+)-metazocine [(+)-5] (Ki = 41 nM) was the most selective compound for the PCP receptor relative to the sigma (51-fold) and mu opioid (greater than 200-fold) sites.

Animals↗
PubMed1992

Hybrid system for protein secondary structure prediction.

We have developed a hybrid system to predict the secondary structures (alpha-helix, beta-sheet and coil) of proteins and achieved 66.4% accuracy, with correlation coefficients of C(coil) = 0.429, C alpha = 0.470 and C beta = 0.387. This system contains three subsystems ("experts"): a neural network module, a statistical module and a memory-based reasoning module. First, the three experts independently learn the mapping between amino acid sequences and secondary structures from the known protein structures, then a Combiner learns to combine automatically the outputs of the experts to make final predictions. The hybrid system was tested with 107 protein structures through k-way cross-validation. Its performance was better than each expert and all previously reported methods with greater than 0.99 statistical significance. It was observed that for 20% of the residues, all three experts produced the same but wrong predictions. This may suggest an upper bound on the accuracy of secondary structure predictions based on local information from the currently available protein structures, and indicate places where non-local interactions may play a dominant role in conformation. For 64% of the residues, at least two experts were the same and correct, which shows that the Combiner performed better than majority vote. For 77% of the residues, at least one expert was correct, thus there may still be room for improvement in this hybrid approach. Rigorous evaluation procedures were used in testing the hybrid system, and statistical significance measures were developed in analyzing the differences among different methods. When measured in terms of the number of secondary structures (rather than the number of residues) that were predicted correctly, the prediction produced by the hybrid system was also better than those of individual experts.

Amino Acid Sequence↗
PubMed1992

Catabolite gene activator protein (CAP) is not an "acidic activating region" transcription activator protein. Negatively charged amino acids of CAP that are solvent-accessible in the CAP-DNA complex play no role in transcription activation at the lac promoter.

It has been suggested that the catabolite gene activator protein (CAP) uses an "acidic activating region" transcription activation mechanism and that Glu171 of CAP is the critical amino acid of the "acidic activating region" of CAP (Irwin, N., and Ptashne, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8315-8319). In this paper, we show, contrary to the previously published report, that substitution of Glu171 of CAP fails to result in a specific defect in transcription activation at the lac promoter. Furthermore, in this paper, we show that substitution of each other negatively charged amino acid of CAP that is solvent-accessible in the CAP-DNA complex fails to result in a specific defect in transcription activation at the lac promoter. We conclude that CAP does not use an acidic activating region transcription activation mechanism in transcription activation at the lac promoter.

Amino Acids↗
PubMed1992

Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.

Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.

Alkylation↗
PubMed1992

Crystal structure of four-stranded Oxytricha telomeric DNA.

The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

Animals↗
PubMed1992

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

The role of histidine in catalysis by prostaglandin H synthase has been investigated using chemical modification with diethyl pyrocarbonate (DEPC), an agent that has been found to rather selectively derivatize histidine residues in proteins under mild conditions. Incubation of the synthase apoprotein with DEPC at pH 7.2 resulted in a progressive loss of the capacity for both cyclooxygenase and peroxidase catalytic activities. The kinetics of inactivation of the cyclooxygenase activity were dependent on the concentration of DEPC; a second-order rate constant of 680 M-1 min-1 was estimated for reaction of the apoenzyme at pH 7.2 and 0 degrees C. The kinetics of inactivation of the cyclooxygenase by DEPC exhibited a sigmoidal dependence on the pH, indicating that deprotonation of a group with a pKa of 6.3 was required for inactivation. The presence of the heme prosthetic group slowed, but did not prevent, inactivation by DEPC. The stoichiometry of histidine modification of apoenzyme during inactivation determined from absorbance increases at 242 nm agreed well with the overall stoichiometry of derivatized residues determined with [14C]DEPC, indicating that modification by DEPC was quite selective for histidine residues on the synthase. Although modification of several histidine residues by DEPC was observed, only one of the histidine residues was essential for cyclooxygenase activity. Modification of the holoenzyme with DEPC altered the EPR signal of the hydroperoxide-induced tyrosyl free radical from the wide doublet (35 G, peak-to-trough) found with the native synthase to a narrower singlet (28 G, peak-to-trough) quite like that found in the indomethacin-synthase complex. Reaction of the indomethacin-synthase complex with DEPC was found to increase the cyclooxygenase velocity by 9 times its initial value, to about one-third of the uninhibited value, without displacement of the indomethacin; the peroxidase was significantly inactivated under the same conditions. Histidyl residues in the synthase are thus likely to have important roles not only in cyclooxygenase and peroxidase catalysis but also in the interaction of the synthase with indomethacin.

Acetylation↗
PubMed1992

Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in transgenic mice.

Myelin basic protein (MBP) is the second most abundant protein in CNS myelin. We have used transgenic mice to investigate the ability of the 5' flanking sequence of the mouse MBP gene to regulate the cell-type-specific- and temporal expression of a heterologous gene under its control. Transgenic mice were produced with a construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene down-stream of the MBP 5' flanking sequence and CAT expression was monitored both enzymatically and histochemically. The results indicate that 1323 bp of 5' flanking sequence is sufficient to direct CAT expression specifically to the tissue and cell-type, in which MBP is normally synthesized. Additionally, this length of sequence also retains the ability to temporally regulate CAT levels in a manner analogous to endogenous MBP levels.

Animals↗
PubMed1992

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.

Adenosine Triphosphate↗
PubMed1992

Aberrant expression of the c-erbB-2/neu protooncogene in ovarian cancer.

Overexpression of the c-erbB-2/neu protooncogene has recently been shown in ovarian tumors collected from the United States. It is known that environmental and cultural factors may contribute to certain types of cancer, therefore, we examined expression of c-erbB-2/neu in ovarian tumors collected from China by immunohistochemical staining. Out of 81 tumor specimens, 57 (70.4%) were found to be immunopositive, whereas only one out of 17 (5.9%) normal ovarian tissue samples was slightly positive. Our results indicate that overexpression of c-erbB-2/neu is a general phenomenon for ovarian cancer regardless of different population. To search for a c-erbB/neu overexpressing cell line for future study on molecular mechanism, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-2/neu. The c-erbB-2/neu RNA was found to be overexpressed at least 100-fold in one of the four ovarian cancer cell lines examined. An aberrant c-erbB-2/neu RNA was also found to be overexpressed in this cell line. Southern blot analysis indicated that the c-erbB-2/neu was amplified 2-4-fold in this line, and some of these alleles have structural alteration which may account for expression of the aberrant c-erbB-2/neu RNA. Since the 2-4-fold gene amplification is not proportional to the greater than 100-fold overexpression in RNA, other mechanisms such as transcriptional or posttranscriptional control must be involved in overexpression of this gene in ovarian cancer.

Blotting, Northern↗
PubMed1992

T cells from atopic individuals produce IgE-inducing activity incompletely blocked by anti-interleukin-4 antibody.

We investigated peripheral blood B and T lymphocyte functions in atopic individuals. B cells were co-cultured with mutant EL4 thymoma cells in the presence of a standard T cell supernatant (T-SN) with or without exogenous interleukin (IL)-4. IgE secretion in this assay was found to be IL-4 dependent, but not significantly different for atopic patients (n = 25) vs. normal controls (n = 25). Phytohemagglutinin plus phorbol 12-myristate 13-acetate (PHA+PMA)- induced T-SN from patients or controls was tested on normal B cells in the same assay system (in the absence of exogenous IL-4). Compared to the controls, the IgE-inducing activity was significantly increased for patients with asthma or allergic rhinitis (n = 12; p less than 0.005) but not for patients with atopic dermatitis (n = 13). The difference between the asthma or allergic rhinitis vs. the atopic dermatitis groups was significant (p greater than 0.05). Since the assay was not inhibited by interferon (IFN)-gamma, this difference can not be attributed to IFN-gamma concentrations. Other T cell activities may be different between the patient groups or atopic T cells from the respiratory mucosa may recirculate more than those from the skin. In any case, the T cells rather than the B cells were found to be abnormal in atopic individuals. If atopic T cells were stimulated with PHA+PMA not as immediately but after a resting period of 48 h in culture medium alone, the IgE-inducing activity, but not the total Ig-inducing activity or the IL-2 secretion, disappeared. In addition, a mean of 37% of the IgE-inducing activity (range of 13% to 79% for five very active T-SN) was not inhibited by an anti-IL-4 antibody which neutralized exogenous IL-4, indicating a participation of factors capable of bypassing the requirement for IL-4 for the IgE response.

Antibodies, Monoclonal↗
PubMed1992

Evaluation of a new generation synthetic peptide combination assay for detection of antibodies to HIV-1, HIV-2, HTLV-I, and HTLV-II simultaneously.

A new generation combination test (Detect-Plus, IAF BioChem, Montreal, Canada) based on synthetic peptides for HIV-1, HIV-2, HTLV-I, and HTLV-II was compared with three routine commercial screening assays and confirmatory assays to determine its sensitivity and specificity and to evaluate it as a substitute screening method. Samples from 356 sexually transmitted disease (STD) patients were tested by the four screening tests. All initially reactive samples were retested in duplicate by the corresponding EIA and repeatedly reactive samples were confirmed by Western blots for HIV-1, HIV-2, and HTLV-I/II. The confirmed positives detected by each screening assay were HIV-1 (23/356, 6.46%), HIV-2 (11/356, 3.09%), and HTLV-I/II (5/356, 1.4%). The new generation Detect-Plus test produced only two results (2/356, 0.56%) that were presumed to be false-positives in comparison to the screening tests, but the OD/CO values were just slightly high (1.5 and 1.9). There were no false-negative results, indicating that the sensitivity of the new combination test was excellent (100%). Compared with routine retroviral EIA assays, the test is easy to perform--the total time requirement is only 2 hr and there is no need for incubation equipment. The OD/CO values were very high when samples were positive, making even visual interpretation possible. We conclude that this new combination assay is an excellent screening method for detection of antibodies to the human retroviruses, and may be particularly useful for screening blood for transfusion and in epidemiological investigations.

Deltaretrovirus↗
PubMed1992

Release of tissue-type plasminogen activator by activated rat eggs and its possible role in the zona reaction.

The resumption of meiosis results in synthesis of tissue-type plasminogen activator (tPA) in the rat and mouse oocytes (Haurte et al., Cell 43:551-558, 1985). The present study demonstrates that freshly ovulated rat oocytes released their tPA into the surrounding medium upon in vitro activation by sperm penetration or treatment with a calcium ionophore. The presence of a neutralizing monoclonal anti-tPA antibody during in vitro activation by the calcium ionophore inhibited the activation-induced zona hardening and also preserved the ability of the oocyte to be penetrated by sperm subsequent to activation. Rat oocytes undergo zona hardening during in vitro maturation in the absence of serum, presumably as a result of spontaneous cortical granule release, based on findings in mice and hamsters. In the present study, the anti-tPA antibody prevented the zona hardening and enhanced partition by spermatozoa of rat oocytes that were matured in vitro without serum. Collectively, the observations reported have suggest a possible role of tPA released during the cortical granule reaction in the zona reaction, which contributes to the block to polyspermy.

Animals↗