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Biomedical subjects

M Xu

Publications and source records attributed to M Xu.

At least 271 records · Page 15Linked to original sources
PubMed1996

Modulatory effects of peroxovanadates on insulin receptor binding.

The insulin-mimetic effects exhibited by vanadate, hydrogen peroxide, and some peroxovanadates have recently been shown to occur, at least in part, through an activation of the insulin receptor tyrosine kinase activity. In this study, we examine the effects of these compounds on insulin receptor binding using receptor preparations from human placental membranes. Among the 16 vanadium(V)-peroxo complexes studied, the [VO(O2)2(bipy)]- ion, where bipy = 2,2'-bipyridine, was found to increase insulin receptor binding by 24%, whereas the [VO(O2)2(en)]- ion, where en = ethylenediamine, was found to reduce insulin receptor binding by about the same amount under steady-state conditions. Scatchard analysis of the binding data indicates that the observed effect of the [VO(O2)2(bipy)]- ion on insulin receptor binding is exerted mainly at the high-capacity low-affinity sites. Furthermore, this modulatory effect is reversible and requires a continuous presence of the compound. By perturbing the membrane environment of the insulin receptor, we have shown that an intact membrane structure is essential for an observable effect. The observed modulation of insulin receptor binding by peroxovanadates is interpreted in terms of a ternary complex model in which the peroxovanadate acts as an allosteric effector modulating the binding equilibrium between insulin and its receptor.

Humans↗
PubMed1996

Direct study of DNA-protein interactions in repressed and active chromatin in living cells.

Current methods for analysis of chromatin architecture are invasive, utilizing chemicals or nucleases that damage DNA, making detection of labile constituents and conclusions about true in vivo structure problematic. We describe a sensitive assay of chromatin structure which is performed in intact, living yeast. The approach utilizes expression of SssI DNA methyltransferase (MTase) in Saccharomyces cerevisiae to provide an order-of-magnitude increase in resolution over previously introduced MTases. Combining this resolution increase with the novel application of a PCR-based, positive chemical display of modified cytosines provides a significant advance in the direct study of DNA-protein interactions in growing cells that enables quantitative footprinting. The validity and efficacy of the strategy are demonstrated in mini-chromosomes, where positioned nucleosomes and a labile, operator-bound repressor are detected. Also, using a heterologous system to study gene activation, we show that in vivo hormone occupancy of the estrogen receptor is required for maximal site-specific DNA binding, whereas, at very high receptor-expression levels, hormone-independent partial occupancy of an estrogen-responsive element was observed. Receptor binding to a palindromic estrogen-responsive element leads to a footprint with strand-specific asymmetry, which is explicable by known structural information.

Chromatin↗
PubMed1996

A genetic approach to study mechanisms of cocaine action.

The brain dopamine system is thought to be the major target for the neuropharmacological actions of psychomotor stimulants such as cocaine. To investigate the mechanisms of cocaine action, we used a genetic approach, the gene-targeting technique, and generated D1 dopamine receptor mutant mice. Locomotor activity analysis in response to cocaine indicates that, in contrast to control mice which showed a dose-dependent increase in locomotion, D1 receptor mutant mice exhibited a dose-dependent decrease, suggesting that D1 receptors play an essential role in mediating such effects. Extracellular single unit recording of dopamine sensitive nucleus accumbens neurons in the D1 receptor mutant mice and control mice revealed a marked reduction in the inhibitory effects of cocaine and dopamine on the generation of action potentials, suggesting that D1 receptors play a fundamental role in cocaine- and dopamine-mediated neurophysiological effects within the nucleus accumbens. From these analyses, we conclude that the D1 dopamine receptor plays essential roles in mediating these effects of cocaine. In the future, the use of this powerful genetic approach will be essential for elucidating the molecular components of the signal transduction pathway leading to anatomical, cellular and behavioral changes upon cocaine administration and dopamine neurotransmission.

Animals↗
PubMed1996

Identification of a new member of the MNP transcription factor family in differentiated HL60 cells.

MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells, MIP-1 alpha is expressed in transformed B cells and can also be induced during cellular activation of CD4+ T-cells and monocytes. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. In this communication we show evidence identifying a new member of the MNP transcription family, MNP-3, in PMA differentiated HL60 cells.

Base Sequence↗
PubMed1996

Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains.

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins.

Amino Acid Sequence↗
PubMed1996

HIV-1 tat induces the expression of a new hematopoietic cell-specific transcription factor and downregulates MIP-1 alpha gene expression in activated T-cells.

MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells. The expression of MIP-1 alpha is induced during cellular activation of CD4+ T-cells and monocytes. It is also expressed in transformed B-cells. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation and in transformed B cells. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. Recently, we reported that HIV-1 tat downregulates MIP-1 alpha expression in Jurkat T-cells. In this report we show induction of MNP-1 in Jurkat T-cells expressing HIV-1 tat. Expression of neither HTLV-1 tax in Jurkat T-cells nor EBV in B-cells had any effect on MNP-1 or MNP-2 expression, showing that the effect is specific for HIV-1 tat. We propose that HIV-1 tat may inhibit MIP-1 alpha expression by inducing MNP-1 expression in T-cells, probably by either competing with MNP-2 for binding to the MIP-1 alpha promoter or by sequestering it into inactive forms.

B-Lymphocytes↗
PubMed1996

Importance of the form of topical vitamin E for prevention of photocarcinogenesis.

With increasing solar ultraviolet (UV)-B radiation reaching the Earth's surface and the incidence of skin cancer rising steadily, there is an ever-increasing need to determine agents that modulate photocarcinogenesis and to understand the mechanisms underlying this modulation. Our laboratory has demonstrated that topical application of the dl-alpha-tocopherol form of vitamin E to mice prevents skin cancer and the immunosuppression induced by UVB irradiation. However, dl-alpha-tocopherol has limited stability at room temperature. The current study was designed to ask whether the thermostable esters of vitamin E, alpha-tocopheryl acetate, or alpha-tocopheryl succinate prevent skin cancer and immunosuppression induced in mice by UV radiation. In the alpha-tocopheryl acetate study, skin cancers developed in 70% of UVB-irradiated control mice and in 90%, 73%, and 90% of mice receiving topical applications of 12.5, 25, and 50 mg of dl-alpha-tocopheryl acetate, respectively. In the alpha-tocopheryl succinate study, skin cancer developed in 59.3% of control UVB-irradiated mice and in 82%, 100%, and 81.5% of mice treated with 2.5, 12.5, and 25 mg d-alpha-tocopheryl succinate, respectively. Thus neither alpha-tocopheryl acetate nor alpha-tocopheryl succinate prevented photocarcinogenesis. At 12.5 and 25 mg/treatment, alpha-tocopheryl acetate and alpha-tocopheryl succinate, respectively, enhanced photocarcinogenesis (p = 0.0114 and 0.0262, respectively, log rank test). On the basis of high-performance liquid chromatography analysis at 16-17 weeks after the first vitamin E treatment, the esterified forms of vitamin E applied epicutaneously accumulated in the skin, but the levels of free alpha-tocopherol remained low. Neither alpha-tocopheryl acetate nor alpha-tocopheryl succinate prevented the induction by UV radiation of immunosusceptibility to implanted syngeneic antigenic UV-induced tumor cells. Thus alpha-tocopheryl acetate or alpha-tocopheryl succinate not only failed to prevent photocarcinogenesis, but may have enhanced to process. Considering that alpha-tocopherol esters are included in many skin lotions, cosmetics, and sunscreens, further studies are needed to determine the conditions under which topical alpha-tocopheryl acetate and alpha-tocopheryl succinate enhance photocarcinogenesis.

Administration, Topical↗
PubMed1996

Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit.

The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of gamma-aminobutyric acid (GABA) to the GABA(A) receptors induces the opening of an anion-selective channel that remains open for tens of milliseconds before it closes. To understand how the structure of the GABA(A) receptor determines the functional properties such as ion conduction, ion selectivity and gating we sought to identify the amino acid residues that line the ion conducting channel. To accomplish this we mutated 26 consecutive residues (250-275), one at a time, in and flanking the M2 membrane-spanning segment of the rat alpha1 subunit to cysteine. We expressed the mutant alpha1 subunit with wild-type beta1 and gamma2 subunits in Xenopus oocytes. We probed the accessibility of the engineered cysteine to covalent modification by charged, sulfhydryl-specific reagents added extracellularly. We assume that among residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction through the channel. We infer that nine of the residues, alpha1 Val257, alpha1 Thr26l, alpha1 Thr262, alpha1 Leu264, alpha1 Thr265, alpha1 Thr268, alpha1 Ile27l, alpha1 Ser272 and alpha1 Asn275 are exposed in the channel. On a helical wheel plot, the exposed residues, except alpha1 Thr262, lie on one side of the helix in an arc of 120 degrees. We infer that the M2 segment forms an alpha helix that is interrupted in the region of alpha1 Thr262. The modification of residues as cytoplasmic as alpha1 Val257 in the closed state of the channel suggests that the gate is at least as cytoplasmic as alpha1 Val257. The ability of the positively charged reagent methanethiosulfonate ethylammonium to reach the level of alpha1 Thr261 suggests that the charge-selectivity filter is at least as cytoplasmic as this residue.

4-Chloromercuribenzenesulfonate↗
PubMed1996

Strategies to improve 3D whole-body PET image reconstruction.

An algorithm is described for three-dimensional whole-body (3DWB) image reconstruction in positron emission tomography. For whole-body applications, improvements to the popular fixed-axial-acceptance-angle technique are achieved by combining axially adjacent projection data available with a long-axis data set. Time-consuming reprojection of unmeasured oblique lines of response is reduced or eliminated by axial overlap of bed positions, while pixel variance and reconstructed axial resolution are made more uniform by the overlap. Improvements in noise and resolution uniformity are accompanied by gains in reconstruction efficiency, and may be optimized against increased acquisition-time due to overlapping acquisition segments and reduced axial coverage. An 11-detector-ring overlap improves axial uniformity in coronal images of a long, uniform cylinder from 23% to 8% with uniform axial resolution. Associated with a 37% improvement in reconstruction time is a 34% reduction in axial coverage for four bed positions. A smaller degree of overlap is found to provide the best trade-off between image uniformity, total scan duration, and reconstruction time because of a proportionally greater reduction in reprojected lines of response. Using a sample optimization scheme, we find a three-ring overlap is best for a 60 cm axial field of view and a five-ring overlap for an 80 cm axial field of view.

Algorithms↗
PubMed1996

Protection by green tea, black tea, and indole-3-carbinol against 2-amino-3-methylimidazo[4,5-f]quinoline-induced DNA adducts and colonic aberrant crypts in the F344 rat.

Male F344 rats were exposed for 8 weeks to extracts of green tea (2% w/v) or black tea (1% w/v), or to 0.1% dietary indole-3-carbinol (I3C). In weeks 3 and 4 of the study, rats were given 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) every other day by oral gavage (50 mg/kg body wt) in order to induce aberrant crypt foci (ACF) in the colon. Compared with controls given IQ alone, all three inhibitors reduced the number of total aberrant crypts per colon, and green tea and I3C inhibited significantly the mean number of ACF (P < 0.05). Rats pre-treated with green tea, black tea, or I3C and given a single p.o. injection of 50 mg IQ/kg body wt 24-48 h before sacrifice had reduced levels of IQ-DNA adducts in the liver, and excreted lower amounts of IQ and other promutagens in the urine and feces. Inhibitors also reduced the excretion of IQ-sulfamate in the urine, but increased the relative amounts of IQ-5-O-sulfate and IQ-5-O-glucuronide. Western blotting together with assays for 7-ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase established that I3C preferentially induced cytochrome P4501A1 over 1A2, consistent with the altered profile of urinary metabolites. However, both teas caused slight induction of cytochrome P4501A2 versus 1A1, which would be predicted to enhance the activation of IQ. Thus, green tea and black tea are likely to protect against IQ-DNA adducts and ACF by mechanisms other than induction of cytochromes P450, such as inhibition of enzymes which activate IQ or the scavenging of reactive intermediates.

Animals↗
PubMed1996

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Amino Acid Sequence↗
PubMed1996

Sodium-glucose cotransporters display sodium- and phlorizin-dependent water permeability.

Expression of Na(+)-glucose cotransporters of the SGLT-1 type by Xenopus laevis oocytes increased the osmotic water permeability (Pf) of oocytes by a factor of 1.9-2.8, in the presence and in the absence of 5 mM extracellular glucose. The Pf increase was correlated with the amount of SGLT-1 cRNA injected. In oocytes expressing SGLT-1, either addition of phlorizin to the medium or the replacement of Na+ by choline inhibited the uptake of methyl-alpha-D-glucopyranoside, a specific substrate for SGLT-1, and returned oocyte Pf to its level in uninjected oocytes. Phlorizin inhibited the SGLT-1-attributable increase in Pf with an inhibition constant (Ki) of 6.1 microM, a value analogous to the Ki for phlorizin inhibition of sugar uptake. However, neither the presence of phlorizin nor the absence of extracellular Na+ significantly affected the increase in Pf elicited in oocytes expressing GLUT-1, a facilitative glucose transporter. These findings suggest that SGLT-1 forms a pore that allows the transmembrane passage of water and that water and glucose traverse the protein through this pore. The finding that removal of extracellular Na+ abolishes the increase in Pf attributable to SGLT-1 suggests that extracellular Na+ is required to maintain patency of this transporter's water-permeable transmembrane pore.

Animals↗
PubMed1996

Vasoactive intestinal peptide stimulates prolactin mRNA expression in turkey pituitary cells: effects of dopaminergic drugs.

It is well documented that vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor and that dopamine (DA) is an inhibitory neurotransmitter in avian species. However, the roles of VIP and DA in the regulation of PRL gene expression are unclear. In this study, primary anterior pituitary cells cultured from laying turkeys were utilized to investigate the influence of VIP and dopaminergic D1 and D2 receptors on PRL secretion, PRL mRNA, and PRL synthesis. Incubation of pituitary cells with VIP increased PRL secretion up to 3.5-fold within 3 hr. Prolactin mRNA was undetectable during the first 2 hr of pituitary cell treatment; thereafter, the PRL mRNA content response to VIP increased with 24-48 h (P < 0.05). Total PRL content (media + cellular) increased over time in the presence of VIP. The response of cells incubated in the presence of a dopaminergic D1 receptor agonist (SKF38393) was variable and inconclusive. However, cells incubated with a dopaminergic D2 receptor agonist (quinpirole) inhibited VIP-induced PRL secretion (P < 0.05) and PRL mRNA levels (P < 0.05) in a dose-related fashion without effect on the basal levels of PRL release and PRL mRNA. These observations suggest that VIP, in addition to acting as a PRL-releasing peptide, also plays a role in the regulation of PRL gene expression. Moreover, the results of this study also indicate that a drug that can selectively stimulate dopamine D2 receptors can also regulate PRL secretion and PRL mRNA in turkey pituitary cells in culture.

Animals↗