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Biomedical subjects

M Hu

Publications and source records attributed to M Hu.

At least 91 records · Page 5Linked to original sources
PubMed1999

Cell type-specific activation of neuronal nicotinic acetylcholine receptor subunit genes by Sox10.

The regulatory factor Sox10 is expressed in neural crest derivatives during development as well as in the adult CNS and peripheral nervous system. Mutations of the human Sox10 gene have been identified in patients with Waardenburg-Hirschsprung syndrome that is characterized by defects in neural crest development. Previous studies suggested that Sox10 might function as an important transcriptional regulator of neural crest development. No natural target genes of Sox10 have yet been identified. Although human Sox10 activates a synthetic promoter consisting of a TATA box and multiple Sox consensus sequences, no transcriptional activity of the rat Sox10 homolog has been detected. Here we report that the neuronal nicotinic acetylcholine receptor beta4 and alpha3 subunit gene promoters are transactivated by rat Sox10 in a cell type-specific manner. The alpha3 and beta4 subunits, in combination with the alpha5 subunit, make up the predominant nicotinic receptor subtype expressed in the peripheral nervous system. Transfections using Sox10 mutants indicate that the C-terminal region is dispensable for its ability to activate the beta4 and alpha3 promoters. Rat Sox10 was originally identified as an accessory protein of the POU domain protein Tst-1/Oct6/SCIP in glial cells. Tst-1/Oct6/SCIP was shown previously to activate the alpha3 promoter. We now demonstrate that it can transactivate the beta4 promoter as well. However, we were unable to detect any synergistic effects of Sox10 and Tst-1/Oct6/SCIP on beta4 or alpha3 promoter activity. Finally, we present data suggesting that recombinant Sox10 protein can directly interact with a previously characterized regulatory region of the beta4 gene.

3T3 Cells↗
PubMed1999

Discordant effects of glucosamine on insulin-stimulated glucose metabolism and phosphatidylinositol 3-kinase activity.

The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling.

Animals↗
PubMed1999

Rat pregnane X receptor: molecular cloning, tissue distribution, and xenobiotic regulation.

An orphan nuclear receptor, termed the pregnane X receptor (PXR), has recently been cloned from mouse and human and defines a novel steroid signaling pathway (Cell 92, 73-82, 1998; Proc. Natl. Acad. Sci. USA 95, 12208-122313, 1998). Transient cotransfection experiments demonstrate that the PXR responds to structurally dissimilar compounds and confers the induction of cytochrome P4503A (CYP3A), a subfamily of enzymes that involve the metabolism of two-thirds of drugs and other xenobiotics. In this report, we describe the molecular cloning, tissue distribution, and xenobiotic regulation of a rat PXR designated rPXR-1. rPXR-1 exhibits a 95% sequence identity with the mouse PXR, but only 79% identity with the human PXR, providing the molecular basis that rats and mice have a similar CYP3A induction profile but differ from humans. rPXR-1 gene was expressed abundantly in liver, intestine, and, to a lesser extent, kidney, lung, and stomach. The tissue distribution and the relative abundance of rPXR-1 mRNA among these tissues resemble those of CYP3A, suggesting that PXR is important not only for induction but also for constitutive expression of these enzymes. Xenobiotics known to induce liver microsomal enzymes showed differential effects on the rPXR-1 expression as determined by Northern blot analysis. Dexamethasone, for example, increased the accumulation of rPXR-1 mRNA, whereas troleandomycin slightly suppressed it. Compounds that increase PXR expression (inducers) and compounds that interact with PXR (ligands) likely have synergistic effects on CYP3A induction, which provides a novel molecular explanation for drug-drug interactions.

Amino Acid Sequence↗
PubMed1999

Reconstruction of the dihydropyridine site in a non-L-type calcium channel: the role of the IS6 segment.

Mutations of eight to nine amino acids of IIIS5, IIIS6 and IVS6 segments were shown to reconstruct the dihydropyridine (DHP) interaction site in the non-L-type alpha1E or alpha1A calcium channels. The reconstructed site enabled enantiomer-selective inhibition and activation of the expressed chimeras by DHPs but failed to transfer voltage dependence of the current inhibition. Here we show that transfer of four non-conserved amino acids from the IS6 segment to the DHP-sensitive alpha1E chimera increased the inhibition by (+)isradipine at the hyperpolarized membrane potential of -100 mV and enhanced the voltage-dependent block.

Animals↗
PubMed1999

Retinal neurogenesis: the formation of the initial central patch of postmitotic cells.

We have investigated the relationship between the birthdate and the onset of differentiation of neurons in the embryonic zebrafish neural retina. Birthdates were established by a single injection of bromodeoxyuridine into embryos of closely spaced ages. Differentiation was revealed in the same embryos with a neuron-specific antibody, zn12. The first bromodeoxyuridine-negative (postmitotic) cells occupied the ganglion cell layer of ventronasal retina, where they formed a small cluster of 10 cells or less that included the first zn12-positive cells (neurons). New cells were recruited to both populations (bromodeoxyuridine-negative and zn12-positive) along the same front, similar to the unfolding of a fan, to produce a circular central patch of hundreds of cells in the ganglion cell layer about 9 h later. Thus the formation of this central patch, previously considered as the start of retinal neurogenesis, was actually a secondary event, with a developmental history of its own. The first neurons outside the ganglion cell layer also appeared in ventronasal retina, indicating that the ventronasal region was the site of initiation of all retinal neurogenesis. Within a column (a small cluster of neuroepithelial cells), postmitotic cells appeared first in the ganglion cell layer, then the inner nuclear layer, and then the outer nuclear layer, so cell birthday and cell fate were correlated within a column. The terminal mitoses occurred in three bursts separated by two 10-h intervals during which proliferation continued without terminal mitoses.

Animals↗
PubMed1999

Polypeptide resurfacing method improves fibroblast's adhesion to hyaluronan strands.

Hyaluronic acid (hyaluronan, HyA) is a matrix component that takes part in cell adhesion and growth in normal and repaired tissues. Since it is soluble in water, HyA has been of limited use in tissue engineering of artificial matrices. Recent studies demonstrate that polypeptides have the twin advantages of reducing solubility of HyA and improving cellular attachment via cell surface adhesion molecule receptors. This paper describes a new approach of using a polypeptide resurfacing method to enhance the attachment of cells to HyA strands. HyA strands were crosslinked by glutaraldehyde and then resurfaced with poly-D-lysine, poly-L-lysine, glycine, or glutamine. After inoculation with fibroblasts in vitro, modified HyA was evaluated with histological and immunohistochemical staining methods for cell adhesion and proliferation. Modified HyA with fibroblast cells also were implanted in vivo. The results show that (1) both polylysines enhanced fibroblast adhesion to crosslinked HyA strands; (2) HyA strands were able to be crosslinked well by 3 days of treatment in glutaraldehyde, and as a biomaterial they could resist biodegradation; (3) modified HyA has good biocompatibility, both in vitro and in vivo. The results demonstrate that HyA material resurfaced by polypeptides has positive advantages for cellular adhesion. Resurfaced HyA has much potential as an improved biomaterial for clinical usage.

Animals↗
PubMed1999

The fit-1 common integration locus in human and mouse is closely linked to MYB.

The fit-1 locus was originally identified as a common insertion site for feline leukemia virus (FeLV) in thymic lymphosarcomas induced by FeLV-myc recombinant viruses, suggesting that it harbors a gene that cooperates with Myc in T-cell leukemogenesis. We have previously mapped the fit-1 locus to feline Chromosome (Chr) B2. We have now identified conserved sequences that allow the mapping of the murine homolog using the European Interspecific Backcross (EUCIB). This shows that fit-1 is located on mouse Chr 10, 1cM proximal to Ahi-1, a murine retroviral integration locus that is closely linked to Myb. Moreover, the physical linkage to MYB is maintained in the human genome, as shown by cloning of the human homolog of fit-1 from a Chr 6 cosmid library and a series of overlapping PAC clones. Generation of a contig map around the human homolog of fit-1 reveals that it is approximately 100-kb upstream of MYB. In addition to fit-1 and Ahi-1, two other common insertion sites, Mis-2 and Mml-1, have also been mapped adjacent to Myb on mouse Chr 10. Previous analysis of tumors carrying insertions at fit-1, Mml-1, Mis-2 and Ahi-1 showed no obvious abnormalities in Myb expression. However, the cluster of viral insertion loci in this region suggests either the presence of a closely linked activation target or that subtle effects on Myb have been overlooked.

Animals↗
PubMed1999

Influence of streptozotocin-induced diabetes in rats on the lithium content of tissue and the effect of dietary lithium supplements on this diabetic condition.

To study the effects of lithium supplementation on the diabetic condition, we measured the lithium concentration in the liver, kidney, and muscle from streptozotocin (STZ)-induced diabetic male Sprague-Dawley (SD) rats that were either treated or untreated with peroral lithium carbonate (0.3 mg/mL). The data showed that the lithium content of the liver and muscle was significantly lower in STZ rats than in normal control rats (0.22 +/- 0.05 v 1.30 +/- 0.15, P < .01, and 0.79 +/- 0.30 v 2.48 +/- 2.00 microg/g, respectively). After 4 weeks of lithium carbonate supplementation, we found that (1) the lithium content of the liver and muscle returned to the normal range, (2) the extent of STZ-mediated destruction of beta cells in the pancreas decreased, (3) fasting blood glucose (FBG) and 2-hour postprandial blood glucose (PBG) decreased (P < .05), (4) among the indicators of oxidative stress and antioxidant defenses, blood lipid peroxidate (LPO) decreased and erythrocyte superoxide dismutase (RBC-SOD) and glutathione (GSH) returned to normal, and (5) hepatic LPO decreased and glutathione peroxidase (GSH-Px) increased. These results suggest that the restoration of lithium to control levels in the liver and muscle of diabetic animals is associated not only with decreased blood glucose but also with reduced oxidative stress, and consequently with the protection of insulin-secreting pancreatic islet cells.

Animals↗
PubMed1999

NMR study of pradimicin derivative BMY-28864 and its interaction with calcium ions in D2O.

The dynamic structure of the antifungal antibiotic pradimicin BMY-28864 in D2O and its interaction with calcium ions were analyzed using one- and two-dimensional 1H nuclear magnetic resonance (NMR). Spectra indicate extensive self-association of molecules in the solution. Two-component spectra were observed simultaneously in a very dilute solution, suggesting equilibrium of two aggregative states. The addition of CaCl2 caused a number of changes in NMR spectra. Therefore we concluded that pradimicin BMY-28864 could form a complex with the Ca2+ ion, causing a movement of the equilibrium. The position of the bound calcium ion is determined indirectly by observing how the NMR shift affects protons that are close to the binding site. The stoichiometry of Ca2+ ion to the Pradimicin molecule for the Ca(2+)-saturated complex is verified to be 1:2. Signal broadening and changes in chemical shift in the 1H NMR spectroscopy of BMY-28864 are assumed to be related to changes in the molecular aggregate conformation.

Anthracyclines↗
PubMed1999

Transport and metabolic characterization of Caco-2 cells expressing CYP3A4 and CYP3A4 plus oxidoreductase.

PURPOSE: To further characterize CYP3A4-transfected Caco-2 cells with regard to morphological, transport, and metabolic properties, and to evaluate a different Caco-2 cell strain transfected with both CYP3A4 and oxidoreductase (OR). METHODS: Transfected Caco-2 cells, Caco-2 TC7 cells, and wild-type Caco-2 cells grown onto Millicell were used. We determined the morphological characteristics of transfected cell monolayers using light and transmission electron microscope. We determined the transport and metabolic capabilities of the transfected cells, TC7 cells, and wild-type cells with a variety of drugs, nutrients, and marker compounds. RESULTS: The transfected Caco-2 cells formed a tight monolayer with TEER values and mannitol transport similar to the untransfected parent cell strain (wild type). However, the transfected cells (grown onto Millicell) reached maturity approximately 33% faster than the wild-type cells. Permeabilities of propranolol, nifedipine, testosterone, linopirdine, mannitol, and cephalexin were similar in transfected and wild-type Caco-2 cells. On the other hand, the transfected cells of early passages were much more metabolically active, and metabolized standard CYP3A4 substrates (e.g., testosterone and nifedipine) as much as 100 times faster than untransfected cells. In addition, metabolism of standard substrates was inhibitable by ketoconazole and TAO. Using comparable data, the transfected cells metabolized testosterone the fastest, followed by linopirdine and nifedipine (approximate ratio: 10:6:2). The metabolites of standard substrates were generally preferably excreted to the apical membrane. CONCLUSION: The monolayers of newly transfected cells (CYP3A4 + OR) have a significantly increased level of CYP3A4 activities compared to untransfected cells. These cell monolayers also have desirable morphological and transport characteristics that are similar to untransfected cells.

Anticoagulants↗
PubMed1999

Expression of GABA transporter subtypes (GAT1, GAT3) in the adult rabbit retina.

PURPOSE: GABA transporters (GATs) are of importance for GABA signal systems. They have previously not been examined in rabbit retina, nor has their correlation with neurotransmitter GABA and GABA receptors been examined in the retina of any species. METHODS: The distribution of GATs, GABA and GABA receptors was examined with immunohistochemical methods. RESULTS: Both GAT1 and GAT3 immunoreactivities were found in the inner plexiform layer and in amacrine cells. GAT3 was also present in Müller cells. GAT1 appeared in amacrine cells that also had a high GABA concentration, but not in cells with moderate to low GABA concentration. GAT1 was also present in amacrine cells that did not show GABA immunoreactivity, possibly indicating a postsynaptic GABA uptake system. CONCLUSION: GAT3 is probably involved in both neuronal and glial GABA uptake whereas GAT1 is involved in predominantly neuronal uptake, and possibly also into non-GABA-ergic amacrine cells. Further, there may be at least two populations of GABA containing neurons.

Animals↗
PubMed1999

Expression of GABA transporter subtypes (GAT1, GAT3) in the developing rabbit retina.

PURPOSE: Little is known about the expression of GABA transporters (GATs) in the developing retina. We have therefore examined the expression of GABA transporters (GAT1 and GAT3) in the developing rabbit retina. METHODS: The distribution of GATs was examined with immunohistochemical methods. RESULTS: GAT3 immunoreactivity appeared at PN0, whereas GAT1 immunoreactivity appeared first at PN3, both in the inner plexiform layer. From PN5 and onwards, both GAT1 and GAT3 immunoreactivity gradually appeared in numerous amacrine cell somas. At PN10, the immunostaining patterns and the distribution of both GAT1 and GAT3 were similar to that found in the adult rabbit retina. Full staining intensity was reached at PN20. CONCLUSION: GABA neurotransmission starts to develop already the first one to three days after birth, reaches the mature neuron pattern at about PN9 to PN10 but is not fully developed until about PN20. Neither GAT1 nor GAT3 appears to be involved in trophic actions by GABA in the prenatal retina.

Aging↗
PubMed1999

Cytokine mRNA in BALB/c mouse corneas infected with herpes simplex virus.

PURPOSE: To investigate cytokine mRNA expression and the influence of acyclovir and tetrandrine on that expression in the corneas of mice infected with herpes simplex virus type 1 (HSV-1). METHODS: Male BALB/c mice were infected in the right cornea with HSV-1. The corneas were harvested from control normal mice and from untreated, acyclovir-treated and tetrandrine-treated mice 14 days after infection. The infected corneas of each group were divided into inflamed and uninflamed depending on clinical observation. After total mRNA extraction from the corneas, gene expression of interleukin 1 beta (IL-1 beta), IL-6, tumour necrosis factor alpha (TNF-alpha) and transforming growth factor (beta (TGF-beta) was analysed by reverse transcription polymerase chain reaction. RESULTS: No mRNA expression of the cytokines was found in normal corneas. IL-1 beta and TNF-alpha mRNA was seen in inflamed corneas, while mRNA expression of IL-6 and relatively weaker TGF-beta mRNA expression were found both in inflamed corneas and in infected but uninflamed corneas treated with acyclovir. TNF-alpha mRNA was present in the uninflamed corneas of tetrandrine-treated mice. No influence of either agent was found on TGF-beta gene expression. CONCLUSIONS: The results suggest that local IL-1 beta and TNF-alpha gene expression is required for corneal inflammation, whereas IL-6 and TGF-beta may exert antiviral and inflammation regulatory activities in HSV corneal infection.

Acyclovir↗
PubMed1999

Use of human intravenous immunoglobulin in lower motor neuron syndromes.

OBJECTIVE: To determine whether patients with the clinical phenotype of multifocal motor neuropathy but without the electrophysiological criteria for conduction block would respond to intravenous immunoglobulin (IVIg). METHODS: Ten patients were selected with a slowly progressive, asymmetric, lower motor neuron disorder, and were treated prospectively with IVIg at a dose of 2g/kg over 5 days. All subjects had neurophysiological testing to look for evidence of conduction block before treatment. Muscle strength was assessed by MRC grades and hand held myometry, measuring pinch and grip strength. A 20% increase in both pinch and grip myometry was considered a positive response. RESULTS: In no patient was conduction block detected. Four of the 10 patients showed a positive response to IVIg, with the best response occurring in two patients who presented with weakness but without severe muscle wasting. Three of the four responders have continued to receive IVIg for a mean period of 17 months (range 15-24 months), with continued effect. The response to IVIg was not related to the presence of anti-GM1 antiganglioside antibodies, but responders had a selective pattern of muscle weakness and normal (>90% predicted) vital capacity. CONCLUSION: The findings suggest that a course of IVIg should be considered in patients with the clinical phenotype of multifocal motor neuropathy but without neurophysiological evidence of conduction block.

Adult↗
PubMed1999

Decreased visceral adiposity accounts for leptin effect on hepatic but not peripheral insulin action.

Leptin decreases visceral fat (VF) and increases peripheral and hepatic insulin action. Here, we generated similar decreases in VF using leptin (Lep), beta(3)-adrenoreceptor agonism (beta3), or food restriction (FR) and asked whether insulin action would be equally improved. For 8 days before the in vivo study, Sprague-Dawley rats (n = 24) were either fed ad libitum [control (Con)], treated with Lep or beta3 (CL-316,243) by implanted osmotic mini-pumps, or treated with FR. Total VF was similarly decreased in the latter three groups (Lep, 3.11 +/- 0.96 g; beta3, 2.87 +/- 0.48 g; and FR, 3.54 +/- 0.77 g compared with 6.91 +/- 1.41 g in Con; P < 0.001) independent of total fat mass (by (3)H(2)O) and food intake. Insulin (3 mU. kg(-1). min(-1)) clamp studies were performed to assess hepatic and peripheral insulin sensitivity. Decreased VF resulted in similar and marked improvements in insulin action on glucose production (GP) (Lep, 1.19 +/- 0.51; beta3, 1.46 +/- 0.68; FR, 2.27 +/-0.71 compared with 6.06 +/- 0.70 mg. kg(-1). min(-1) in Con; P < 0.001). By contrast, reduction in VF by beta3 and FR failed to reproduce the stimulation of insulin-mediated glucose uptake ( approximately 60%), glycogen synthesis ( approximately 80%), and glycolysis ( approximately 25%) observed with Lep. We conclude that 1) a moderate decrease in VF uniformly leads to a marked increase in hepatic insulin action, but 2) the effects of leptin on peripheral insulin action are not due to the associated changes in VF or beta3 activation.

Adipose Tissue↗