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D Y Huang

Publications and source records attributed to D Y Huang.

At least 37 records · Page 2Linked to original sources
PubMed1998

Intratubular application of sodium azide inhibits loop of Henle reabsorption and tubuloglomerular feedback response in anesthetized rats.

Sodium azide (NaN3, AZ) is a potent inhibitor and uncoupler of oxidative phosphorylation as well as a nitrovasodilator after being converted to nitric oxide (NO). We studied the effect of intratubular application of AZ on loop of Henle reabsorption and tubuloglomerular feedback (TGF) employing renal micropuncture experiments in nephrons with superficial glomeruli of anesthetized Munich-Wistar-Fromter rats. During perfusion of Henle's loop downstream from an obstructing wax block, AZ (3x10(-5) mol/l and 3x10(-4) mol/l) concentration-dependently increased early distal tubular flow rate and sodium and potassium ion concentration (V(ED), [Na+]ED, [K+]ED). In comparison, application of furosemide (10(-4) mol/l), the action of which is restricted to the water-impermeable thick ascending limb of Henle's loop (TALH) and the macula densa, similarly increased [Na+]ED and [K+]ED, but did not affect V(ED). The effect of AZ on loop of Henle reabsorption appeared to be predominantly localized upstream to the TALH since (1) AZ significantly inhibited net fluid reabsorption (the latter being completely abolished at 3x10(-4) mol/l), (2) the effect of AZ on [Na+]ED and [K+]ED could be mimicked by perfusing the Henle's loop at a flow rate that caused a comparable increase in V(ED) (reflecting a comparable load to TALH), and (3) the effects of AZ and furosemide were additive. In spite of the increase in [Na+]ED and [K+]ED, intratubular application of AZ caused a concentration-dependent inhibition of TGF response, the latter being assessed as the fall in early proximal tubular stop flow pressure during perfusion of Henle's loop at increasing flow rate. Like AZ and furosemide, the NO donor sodium nitroprusside (10(-4) mol/l) blunted the TGF response, but in contrast to furosemide or AZ, it caused a minor decrease in V(ED), without changing [Na+]ED or [K+]ED. The inhibitory effect of AZ on TGF was abolished by the NO scavenger carboxy PTIO. In summary, AZ inhibits both reabsorption in the water-permeable segment of Henle's loop and the TGF response. The effect on reabsorption may be linked to metabolic inhibition rather than NO release, whereas the blunted TGF response appears to involve conversion to NO.

Absorption↗
PubMed1998

Purification and characterization of sucrose synthase isozymes from etiolated rice seedlings.

Presence of homo- and hetero-tetrameric rice sucrose synthase (RSS) isoforms in etiolated rice seedlings was demonstrated by immunoprecipitation using monospecific antibodies. Three RSS isozymes with various pI values were purified by ammonium sulfate fractionation, Sepharose CL-6B gel filtration, DEAE-Sephacel and Mono-Q ion exchange chromatographies. They were characterized as heterotetramers composed of RSS1 and RSS2 subunits. All of them used UDP as the best substrate. The presence of divalent metal ions increased the activities of synthesis but inhibited the cleavage of sucrose.

Blotting, Western↗
PubMed1998

The murine Hoxb1 3' RAIDR5 enhancer contains multiple regulatory elements.

Homeobox genes play key roles in specifying body part identity during vertebrate embryonic development. Retinoids are signaling molecules involved in the regulation of expression of homeobox genes. We have previously identified an retinoic acid (RA)-inducible enhancer (RAIDR5) located approximately 6.5 kb 3' of the coding region of the murine Hoxb1 gene. This 3' enhancer contains three sequences that are highly conserved in similar RA-inducible enhancers identified in the murine and human Hoxa1 genes and in the chicken Hoxb1 gene. One element, a DR5 RA response element, contributes to the RA inducibility of a Hoxb1 reporter gene construct in F9 cells. In this report, further analysis of the other two elements of the Hoxb1 3' enhancer is reported. The two other sequences, conserved element (CE) 1 and CE2, act as negative elements in cultured F9 cells; when either is mutated, an increase in the beta-galactosidase activity of a Hoxb1 reporter gene construct results. A single Hoxb1 CE2 DNA element:protein binding complex was detected in F9 stem cells, and experiments suggest that this is the same binding protein that recognizes the CE2 element of Hoxa1. In a variant F9 cell line in which both allelic copies of the RA receptor gamma (RARgamma) gene are disrupted, the CE2 binding complex is absent, and this absence correlates with the inability of the CE2 element to function as a repressor of Hoxb1 reporter gene expression in these cells. A single Hoxb1 CE1 binding complex is also detected by gel shift assays in nuclear extracts prepared from both stem and RA-treated F9 cells. This complex contains an Mr approximately 200,000 protein as shown by UV cross-linking. Although the sequences of the CE1 elements of Hoxb1 and Hoxa1 are highly conserved, they differ by two nucleotides. Gel shift analysis shows that either of these nucleotide changes prevents binding of F9 cell protein extracts. When gel shift assays were performed using nuclear extracts prepared from mouse embryos at a time when Hoxb1 mRNA is expressed, i.e., day 9.0, CE1 and CE2 binding complexes identical in mobility to those detected in F9 cells were observed. This suggests roles for both the CE1 and CE2 elements in regulating Hoxb1 gene expression during development.

Animals↗
PubMed1997

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower Km of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.

Alcohol Oxidoreductases↗
PubMed1996

Identification of an essential cis-element near the transcription start site for transcriptional activation of the proliferating cell nuclear antigen gene.

Interleukin 2 (IL-2) stimulates T lymphocyte proliferation and induces the expression of proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerase delta. Previously, deletion analysis suggested cis-element(s) in the proximal region of the PCNA promoter (-40 to +143) are required for IL-2 induction in cloned T lymphocytes. The sequence 5'-TTGCGGGC-3' located at +10 to +17 is similar to the E2F consensus binding site and is required for optimal PCNA promoter activity. In IL-2-stimulated T cells, nuclear proteins are induced to bind to this sequence as demonstrated using electrophoretic mobility shift assay (EMSA), competition EMSA, and methylation interference analysis. A 180-kDa polypeptide was detected by UV cross-linking to bind specifically to the PCNA E2F-like sequence. Our data indicate that the protein bound to the PCNA E2F-like site is not one of the transcription factor E2F proteins. Our results demonstrate that the E2F-like sequence and the protein(s) binding to it are required for optimal PCNA promoter activity and IL-2 induction of PCNA expression.

Animals↗
PubMed1996

Crosslinking of apolipoprotein E by products of lipid peroxidation.

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.

Adult↗
PubMed1996

Orthogonal analysis of aggravating effects of alpha-, beta-agonists and leukocytes on reperfusion-induced arrhythmias and ventricular fibrillation in Langendorff's rat heart.

AIM: To study the effect of leukocyte (Leu), alpha-agonist (alpha-Ago), and beta-agonist (beta-Ago) on the arrhythmias induced by ischemia and reperfusion to determine which of the 3 factors was the most important one in exacerbating arrhythmias. METHODS: Arrhythmias were induced by the reduction and subsequent resumption of perfused flow in Langendorff's perfused rat hearts. Ventricular tachycardia (VT) and ventricular fibrillation (VF) were recorded on ECG, and the results were orthogonally analyzed. RESULTS: When Leu was present, the incidence of VF induced by ischemia-reperfusion was 80%. The incidence in Leu-depleted hearts was 20%, alpha-Ago and beta-Ago elevated it to 60% and 100%, respectively. The results by orthogonal analysis demonstrated Leu or alpha-Ago+ beta-Ago increased VF incidence. With regard to arrhythmias, arrhythmia score was remarkedly increased by all of 3 factors and various combinations except beta-Ago + Leu. CONCLUSION: Among these 3 factors, Leu was the most important one in facilitating reperfusion-induced arrhythmias.

Adrenergic alpha-Agonists↗
PubMed1995

Different regulation of the human thymidine kinase promoter in normal human diploid IMR-90 fibroblasts and HeLa cells.

Transcriptional activation of the human thymidine kinase (hTK) promoter plays an important role in the cell cycle control of thymidine kinase expression. Using the luciferase reporter cotransfection assay, we found that the activity of the hTK promoter in IMR-90 normal human diploid fibroblasts was increased by the constitutively over-expressed cyclin A or cyclin E but not by cyclin D, suggesting that the former two cyclins may act as positive regulators for the hTK promoter. The sequence responsible for the transcriptional activation by cyclin E was identified to be located between -133 and -92 of the hTK promoter. Regulation of the hTK promoter in HeLa cells appeared to be different from that in IMR-90 fibroblasts. Firstly, the hTK promoter in HeLa was already highly activated and could not be further activated by ectopically expressed cyclin A or E. Secondly, the -133 to -92 region of the hTK promoter was important for the promoter strength in HeLa cells but not in IMR-90 cells. The steady-state levels of cyclins A and E were readily detected in HeLa cells but not in normal IMR-90 fibroblasts. Based on these results, we propose that the cellular environment of the HeLa cell allows the hTK promoter to stay fully activated for transcription regardless of ectopically expressed cyclin A or E and that transcriptional activation of thymidine kinase gene is deregulated in these tumor cells.

Cell Line↗
PubMed1995

ApoE3 binding to tau tandem repeat I is abolished by tau serine262 phosphorylation.

The risk of Alzheimer's disease is determined, in part, by inheritance of specific alleles of ApoE. Isoform specific interactions of ApoE have been shown with the microtubule-associated protein tau, which forms the neurofibrillary tangle in this disease. Synthetic peptides representing each of the four microtubule-binding domains of tau more avidly bind ApoE3 than ApoE4. Phosphorylation of serine262 in domain I of tau decreases tau binding to microtubules and also abolishes binding by ApoE3. Understanding the molecular mechanisms of the high avidity, isoform-specific interactions of ApoE with tau may help in developing approaches for disease intervention.

Alzheimer Disease↗
PubMed1995

Identification of essential lysyl and cysteinyl residues, and the amino acid sequence at the substrate-binding site of retinal oxidase.

Retinal oxidase, which synthesizes all-trans and 9-cis retinoic acid from all-trans and 9-cis-retinal, has been purified from rabbit liver cytosol. The substrate-binding site of the retinal oxidase was investigated with some chemical modification reagents. Lysyl-specific pyridoxal 5'-phosphate (PLP) and cysteinyl-specific p-chloromercuribenzoate (PCMB) competitively inhibited the activity of the retinal oxidase, and the inhibition could be prevented by the presence of all-trans-retinal or its derivatives. Treatment with sodium borohydride (NaBH4) resulted in covalent attachment of PLP to the lysyl residue of the retinal oxidase and the PLP modified-retinal oxidase was cut with cyanogen bromide, and the polypeptides modified with PLP were further digested with trypsin. Two of the peptides modified with PLP were purified from the digested polypeptide mixture and their amino acid sequences were determined.

Aldehyde Oxidoreductases↗
PubMed1995

CCAAT-box contributions to human thymidine kinase mRNA expression.

In order to examine the role of two inverted CCAAT boxes near the start of transcription of the human thymidine kinase (TK) gene, a series of constructs were prepared in which one or both CCAAT boxes were deleted or mutated. These altered promoters (1.2 kb of 5'-flanking sequence) were used to express a TK minigene containing the first two exons and introns followed by the remainder of the cDNA. RNA blots were prepared from stable cell lines of ts13 cells containing these constructs under three conditions: 1) serum deprived cells, 2) serum stimulated cells, and 3) cells that had been stimulated with serum, but were arrested in the G1 phase of the cell cycle by the temperature sensitive mutation carried by these cells. TK mRNA expression from each construct was suppressed by the temperature sensitive block to cell cycle progression. Measurement of protein expression from the various altered TK promoters indicated that both CCAAT boxes contribute to promoter strength. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true.

Animals↗
PubMed1995

Interaction of apolipoprotein E with laminin increases neuronal adhesion and alters neurite morphology.

The extracellular matrix protein laminin profoundly affects neuronal adhesion, spreading, differentiation, and growth by binding integrin-type cell surface receptors. Laminin binds other basement membrane components, including heparan sulfate proteoglycans. Apolipoprotein E (apoE) also binds basement membrane and heparan sulfate proteoglycans and colocalizes with s-laminin in the neuromuscular junction. We sought to determine whether apoE interacts with laminin and whether, as a result, apoE alters the regulation of neuronal adhesion and differentiation by laminin. We demonstrate high-avidity interaction between apoE and laminin in vitro. Compared to laminin alone, a laminin-apoE substrate produces increased numbers of live, attached hippocampal neurons in culture. In addition, neurons grown on laminin-apoE substrates have larger growth cones, increased neuritic branching, and flattened cell bodies compared to neurons grown on laminin alone. ApoE may be important in the development and maintenance of neurons in the central nervous system by regulating interactions between the neuron and the extracellular matrix.

Apolipoproteins E↗
PubMed1994

Two different enzymes are primarily responsible for retinoic acid synthesis in rabbit liver cytosol.

Retinoic acid biosynthesis in rabbit liver was catalyzed by cytosolic NAD(+)-dependent dehydrogenase and oxygen-dependent oxidase, with an activity ratio of 59% and 41% in the presence of 2 mM dithiothreitol under aerobic conditions. The two enzymes could be well separated by fractionation involving ammonium sulfate precipitation. Purification of the enzymes indicated that the oxygen-dependent enzyme was a flavoenzyme, retinal oxidase (EC 1.2.3.11), composed of two 135 kDa subunits; and the NAD(+)-dependent enzyme was a basic pI retinal dehydrogenase composed of four 55-kDa subunits. A high concentration (1-2 mM) of DTT was required to stabilize the activity of retinal dehydrogenase during the purification procedures and storage, but inhibited the activity of retinal oxidase by 13-38%. The physiological roles of the two retinoic acid synthases in liver cytosol were discussed.

Aerobiosis↗
PubMed1994

Isoform-specific interactions of apolipoprotein E with the microtubule-associated protein MAP2c: implications for Alzheimer's disease.

The apolipoprotein E type 4 allele is a susceptibility gene for late-onset Alzheimer's disease. Apolipoprotein E is found in neurons, some of which contain paired helical filaments made of the microtubule-associated protein tau. Previous studies have demonstrated that the apoE3 isoform, but not the apoE4 isoform, binds tau with high avidity. Because the microtubule-associated protein MAP2c also effects microtubule assembly and stability, we examined interactions between apoE isoforms and MAP2c. Similar to the tau-binding results, apoE3, but not apoE4, bound MAP2c. Binding was detectable down to 10(-9) M MAP2c and 10(-8) M apoE3. Isoform-specific interactions of apoE with the microtubule-associated proteins MAP2c and tau might affect intracellular maintenance of microtubules and could contribute to a time-dependent pathogenesis of Alzheimer's disease.

Alzheimer Disease↗
PubMed1994

Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: implications for Alzheimer disease.

The apolipoprotein E (apoE) type 4 allele (APOE4) is a susceptibility gene for late-onset familial and sporadic Alzheimer disease. ApoE is found in some neurofibrillary tangle-bearing neurons, one of the major pathologic hallmarks of the disease. Neurofibrillary tangles contain paired helical filaments formed from hyperphosphorylated microtubule-associated protein tau. In vitro, tau binds avidly to apoE3, but not to apoE4, forming a bimolecular complex. Tau phosphorylated with a brain extract does not bind either isoform. ApoE3 binds to the microtubule-binding repeat region of tau, which is also the region that is thought to cause self-assembly into the paired helical filament. Binding studies with fragments of ApoE demonstrate that the tau-binding region of apoE3 corresponds to its receptor-binding domain and is distinct from the region that binds lipoprotein particles or beta/A4 peptide. Isoform-specific interactions of apoE with tau may regulate intraneuronal tau metabolism in Alzheimer disease and alter the rate of formation of paired helical filaments and neurofibrillary tangles.

Alzheimer Disease↗
PubMed1994

Differential phosphorylation of human thymidine kinase in proliferating and M phase-arrested human cells.

The expression of cytosolic human thymidine kinase (TK) occurs in a cell cycle-dependent manner. Here, we show that TK is hyperphosphorylated during the M phase in several human cell lines. Our data from characterizing TK phosphorylation in proliferating and M phase-arrested HeLa cells suggest that the polypeptide of TK is differentially phosphorylated during the progression of the cell cycle. TK in the M phase-arrested HeLa cells was found to have a 10-fold lower affinity for its substrate, thymidine, than in the proliferating cells. We propose that phosphorylation of TK by the mitotic kinase(s) may provide an attenuating mechanism to prevent unnecessary synthesis of dTTP at the time of mitosis.

Alkaloids↗