Search PubMed⌕ Search

Biomedical subjects

D Y Huang

Publications and source records attributed to D Y Huang.

At least 19 recordsLinked to original sources
PubMed2001

Interaction of human thymidine kinase 1 with p21(Waf1).

The overexpression of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1) can inhibit cell proliferation, which is mediated by direct binding to CDK and proliferating-cell nuclear antigen. In this study, we demonstrated that human cytosolic thymidine kinase 1 (TK1) polypeptide can form a complex with p21(Waf1). The C-terminal domain of p21(Waf1) appeared to interact with the TK1 polypeptide, but, despite the inhibitory function of p21(Waf1), their association did not alter TK1 functional activity. However, overexpression of TK1 overcame p21(Waf1)-mediated growth suppression and blocked the association of CDK2 with p21(Waf1), suggesting that TK1 interferes with the inhibitory function of p21(Waf1). Based on these results, we here propose that the molecular function of p21(Waf1) in cells can be perturbed through its interaction with another cellular protein, TK1.

Animals↗
PubMed2000

Acute renal response to the non-peptide vasopressin V2-receptor antagonist SR 121463B in anesthetized rats.

Vasopressin V2-receptor antagonists are promising agents for the use in water-retaining diseases. Potential renal mechanisms of action include effects on water permeability in the collecting duct as well as on electrolyte transport in the thick ascending limb of Henle's loop (TALH). To elucidate sites of action upstream of the distal tubule, e.g., in TALH, micropuncture experiments were performed in anesthetized rats during application of the V2-receptor antagonist SR 121463B. As compared to vehicle-treated rats, SR 121463B (0.3 mg/kg i.v.) did not affect mean arterial blood pressure (means +/- SEM, n=10 rats per group: 108+/-4 mmHg vs. 107+/-4 mmHg), whole kidney GFR (1.1+/-0.1 ml/min vs. 1.1+/-0.1 ml/min), or whole kidney fractional reabsorption (FR) of potassium (66+/-5% vs. 68+/-4%). The drug, however, reduced whole kidney FR of fluid (92+/-1% vs. 99+/-1%), increased urinary flow rate (84+/-7 microl/min vs. 8+/-1 microl/min) and electrolyte-free-water clearance (72+/-8 microl/min vs. 2+/-1 microl/min), and reduced urinary osmolality (148+/-11 mosmol/kg vs. 1,200+/-185 mosmol/kg). This pronounced diuretic response was associated with a minor reduction in whole kidney FR of sodium (99.6+/-0.1% vs. 99.9+/-0.1%) and chloride (98.3+/-0.2% vs. 98.9+/-0.1%). As compared to vehicle application, SR 121463B did not significantly alter single nephron GFR (39+/-2 nl/min vs. 39+/-1 nl/min, n=22 and 23 nephrons, respectively) or the FR up to the early distal tubule of fluid (76+/-2% vs. 76+/-1%), sodium (92+/-1% vs. 93+/-1%), potassium (91+/-1% vs. 90+/-1%) or chloride (90+/-1% vs. 91+/-1%). Together these data indicate a predominant aquaretic effect of SR 121463B which is located downstream of the early distal tubule. This response is compatible with blockade of vasopressin V2-receptors in the collecting duct and, as directly demonstrated by immunohistochemistry, subsequent retrieval of aquaporin-2 from apical plasma membrane, which inhibits water permeability and transport.

Animals↗
PubMed2000

Determinants of unexplained antepartum fetal deaths.

OBJECTIVE: To assess fetal, maternal, and pregnancy-related determinants of unexplained antepartum fetal death. METHODS: We conducted a hospital-based cohort study of 84,294 births weighing 500 g or more from 1961-1974 and 1978-1996. Unexplained fetal deaths were defined as fetal deaths occurring before labor without evidence of significant fetal, maternal, or placental pathology. RESULTS: One hundred ninety-six unexplained antepartum fetal deaths accounted for 27.2% of 721 total fetal deaths. Two thirds of the unexplained fetal deaths occurred after 35 weeks' gestation. The following factors were independently associated with unexplained fetal death: maternal prepregnancy weight greater than 68 kg (adjusted odds ratio [OR] 2.9; 95% confidence interval [CI] 1.85, 4.68), birth weight ratio (defined as ratio of birth weight to mean weight for gestational age) between 0.75 and 0.85 (OR 2.77; 95% CI 1.48, 5.18) or over 1.15 (OR 2.36; 95% CI 1.26, 4.44), fewer than four antenatal visits in women whose fetuses died at 37 weeks or later (OR 2.21; 95% CI 1.08, 4.52), primiparity (OR 1.74; 95% CI 1.26, 2.40), parity of three or more (OR 2.01; 95% CI 1.26, 3.20), low socioeconomic status (OR 1.59; 95% CI 1.14, 2.22), cord loops (OR 1.75; 95% CI 1.04, 2.97) and, for the 1978-1996 period only, maternal age 40 years or more (OR 3.69; 95% CI 1.28, 10.58). Trimester of first antenatal visit, low maternal weight, postdate pregnancy, fetal-to-placental weight ratio, fetal sex, previous fetal death, previous abortion, cigarette smoking, and alcohol use were not significantly associated with unexplained fetal death. CONCLUSION: In this study, we identified several factors associated with an increased risk of unexplained fetal death.

Adult↗
PubMed2000

Sodium reabsorption in thick ascending limb of Henle's loop: effect of potassium channel blockade in vivo.

1. Based on previous in vitro studies, inhibition of K(+) recycling in thick ascending limb (TAL) is expected to lower Na(+) reabsorption through (i) reducing the luminal availability of K(+) to reload the Na(+)-2Cl(-)-K(+) cotransporter and (ii) diminishing the lumen positive transepithelial potential difference which drives paracellular cation transport. 2. This issue was investigated in anaesthetized rats employing microperfusion of Henle's loop downstream from late proximal tubular site with K(+)-free artificial tubular fluid in nephrons with superficial glomeruli. 3. The unselective K(+) channel blocker Cs(+) (5 - 40 mM) dose-dependently increased early distal tubular delivery of fluid and Na(+) with a maximum increase of approximately 20 and 185%, respectively, indicating predominant effects on water-impermeable TAL. 4. The modest inhibition of Na(+) reabsorption in response to the 15 mM of Cs(+) but not the enhanced inhibition by 20 mM Cs(+) was prevented by luminal K(+) supplementation. Furthermore, pretreatment with 20 mM Cs(+) did not attenuate the inhibitory effect of furosemide (100 microM) on Na(+)-2Cl(-)-K(+) cotransport. 5. Neither inhibitors of large (charybdotoxin 1 microM) nor low (glibenclamide 250 microM; U37883A 100 microM) conductance K(+) channels altered loop of Henle fluid or Na(+) reabsorption. 6. The intermediate conductance K(+) channel blockers verapamil and quinine (100 microM) modestly increased early distal tubular Na(+) but not fluid delivery, indicating a role for this K(+) channel in Na(+) reabsorption in TAL. As observed for equieffective concentrations of Cs(+) (15 mM), Na(+) reabsorption was preserved by K(+) supplementation. 7. The results indicate that modest inhibition of K(+) channels lowers the luminal availability of K(+) and thus transcellular Na(+) reabsorption in TAL. More complete inhibition lowers paracellular Na(+) transport probably by reducing or even abolishing the lumen positive transepithelial potential difference. Under the latter conditions, transcellular Na(+) transport may be restored by paracellular K(+) backleak.

Adamantane↗
PubMed2000

Frequency and significance of antibodies to chromatin in autoimmune hepatitis type I.

AIMS AND METHODS: To assess the frequency and clinical significance of antibodies to chromatin (ACA) in autoimmune hepatitis (AIH), 36 Japanese patients with AIH type I were studied for serum reactivity with chromatin by using an ELISA. RESULTS: Antibodies to chromatin were detected in 19 of 36 patients with AIH type I. There was a significantly higher frequency of ACA in patients with AIH type I than in patients with primary biliary cirrhosis, chronic hepatitis C and B (52.8 vs 13.2, 5.4 and 6.7%, respectively; P<0.01). None of the 19 healthy subjects had positive reactions. Sixteen of 19 patients with seropositive sera (44.4%) had reactivities with other nuclear antigens (recombinant nucleoproteins U1RNP-A, U1RNP-70; recombinant ribonucleoprotein complexes SSA/Ro 52K, SSA/Ro 60K; recombinant centromere Cenp-B; dsDNA and histones). Adsorption with double-stranded DNA (dsDNA) and histones could not remove the majority of antichromatin reactivity as 81.9% of the antibody reactivity still remained. In five sera samples from AIH type I patients positive for anti-dsDNA and antihistones, the antibody activities for dsDNA and histones were inhibited after the absorbtion of sera with chromatin. The patterns of antinuclear antibodies (ANA) detected by using indirect immunofluorescence were similar between patients with and without ACA. Patients with ACA had significantly high serum levels of gamma-globulin and immunoglobulin G. The ACA titers dropped significantly after corticosteroid treatment (P< 0.01). CONCLUSIONS: Antibodies to chromatin are frequently present in patients with AIH type I and they are one of the dominant autoantibodies associated with ANA reactivity in AIH type I. Antibodies to chromatin cannot be used to characterize distinct clinical subgroups of AIH type I.

Adrenal Cortex Hormones↗
PubMed2000

Molecular characterization of the plasma membrane H(+)-ATPase, an antifungal target in Cryptococcus neoformans.

The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.

Amino Acid Sequence↗
PubMed1999

NF-Y-mediated trans-activation of the human thymidine kinase promoter is closely linked to activation of cyclin-dependent kinase.

Transcriptional activation is important for the elevated expression of human thymidine kinase (hTK) in tumor cells. Here, we used TK(-133/+33)-luciferase reporter gene construct and bandshift assay to study the cis-elements involved in transcriptional activation of the hTK promoter. We found that two CCAAT boxes at -71/-67 and -40/-36 and Sp1 binding site located at -118/-113 were critical for maximal expression of the hTK promoter activity. As Sp1-mediated activation of the hTK promoter was not detectable for the promoter construct with double mutations at two CCAAT boxes, we proposed that NF-Y binding to the hTK promoter sequence is a requisite step for the functional interaction with Sp1. Here, we further showed that the hTK promoter activity was reduced in HeLa cells transfected with p16 or p21, both of which are inhibitors of cyclin-dependent kinases (CDKs). Inhibition of the hTK promoter activity by p16 could be abrogated by overexpression of cyclin A, indicating that the cyclin A activating event is more directly involved in transcriptional activation of the hTK promoter. We thus proposed that NF-Y-mediated activation of the hTK promoter is closely linked to the activation of CDK2/cyclin A pathway.

Base Sequence↗
PubMed1999

Molecular cloning of retinal oxidase/aldehyde oxidase cDNAs from rabbit and mouse livers and functional expression of recombinant mouse retinal oxidase cDNA in Escherichia coli.

Retinal oxidase (EC 1.2.3.11) is a molybdenum-containing flavoenzyme with high enzymatic activity as to retinoic acid synthesis. In this study, we provide direct evidence that retinal oxidase is identical to aldehyde oxidase (EC 1.2.3.1) by cDNA cloning. Retinal oxidase and aldehyde oxidase, purified from rabbit liver cytosol using the original methods, showed completely identical HPLC patterns and amino acid sequences for three corresponding polypeptides (103 amino residues). The primary structural information obtained from the cleaved polypeptides permitted molecular cloning of the full-length cDNA of rabbit liver retinal oxidase (aldehyde oxidase). We also cloned and sequenced the full-length cDNA of mouse retinal oxidase. The cDNAs of rabbit and mouse retinal oxidase have a common sequence approximately 4.6 kb long, comprising 4-kb coding regions. The open reading frames of the cDNAs predict single polypeptides of 1334 and 1333 amino acids; the calculated minimum molecular mass of each is approximately 147,000. Northern blot analysis showed that the rabbit retinal oxidase mRNA was widely expressed in tissues. Finally, we successfully constructed a prokaryotic expression system for mouse retinal oxidase. The purified recombinant retinal oxidase from Escherichia coli showed a typical spectrum of aldehyde oxidases and a lower Km (3.8 microM) for retinal and a higher Vmax (807 nmol/min/mg protein) for retinoic acid synthesis than those of rabbit retinal oxidase (8 microM and 496 nmol/min/mg protein). This represents the first eukaryotic molybdenum-containing flavoprotein to be expressed in an active form in a prokaryotic system.

Aldehyde Oxidoreductases↗
PubMed1999

Inhibition by retinoids of benzo(A)pyrene metabolism catalyzed by 3-methylcholanthrene-induced rat cytochrome P-450 1A1.

Benzo(a)pyrene, a well-known procarcinogen, is believed to be activated by microsomal cytochrome P-450 1A1 (CYP 1A1). We recently reported that rat CYP 1A1 induced by 3-methylcholanthrene (3-MC) catalyzed the conversion of retinal to retinoic acid. In this study, we investigated retinoid inhibition of the metabolism of benzo(a)pyrene and 7-ethoxyresorufin, another specific substrate of CYP 1A1, using liver microsomes prepared from control and 3-MC-pretreated rats as the enzyme source. In 3-MC-treated rats, retinal and retinol, but not retinoic acid, inhibited benzo(a)pyrene metabolism. The 50% inhibitory concentration (IC50) of retinal was about 11.5 micromol/L and the inhibition was competitive, with a Ki value of 5.8 micromol/L. Retinol is a more potent inhibitor than retinal. The IC50 was about 5 micromol/L and the inhibition was mixed, with a Ki value of 19.2 micromol/L and a Ki' value of 4.2 micromol/L. Almost the same results were obtained for the reaction of 7-ethoxyresorufin deethylation. In contrast, the metabolic activity of both benzo(a)pyrene and 7-ethoxyresorufin was much lower in untreated versus 3-MC-treated rats, and only weak inhibition by the retinoids was observed. The results suggest that retinoids inhibit the activation of benzo(a)pyrene and that the substrate specificity of cytochrome P-450 isozymes associated with retinoid metabolism should be taken into account when studying the anticarcinogenic action of retinoids.

Animals↗
PubMed1999

Eukaliuric diuresis and natriuresis in response to the KATP channel blocker U37883A: micropuncture studies on the tubular site of action.

1. Systemic application of U37883A, a blocker of ATP sensitive potassium (KATP) channels, elicits diuresis and natriuresis without significantly altering urinary potassium excretion. 2. To elucidate tubular sites of action upstream to the distal nephron, micropuncture experiments were performed in nephrons with superficial glomeruli of anaesthetized Munich-Wistar-Frömter rats during systemic application of U37883A (1, 5 or 15 mg kg-1 i.v.). 3. The observed eukaliuric diuresis and natriuresis in response to U37883A at 15 mg kg-1 was accompanied by an increase in early distal tubular flow rate (VED) from 10 - 18 nl min(-1) reflecting a reduction in fractional reabsorption of fluid up to this site (FR-fluid) of 13%. The latter proposed an effect on water-permeable segments such as the proximal tubule which could fully account for the observed reduction in fractional reabsorption of Na+ up to the early distal tubule (FR-Na+) of 8% and the increase in early distal tubular Na+ concentration ([Na+]ED) from 35 - 51 mM whereas [K+]ED was left unaltered. 4. In comparison, furosemide (3 mg kg-1 i.v.), which acts in the water-impermeable thick ascending limb, elicited diuresis, natriuresis and kaliuresis which were associated with a fall in FR-Na+ of 10% with no change in FR-fluid, and a rise in [Na+]ED from 42 - 117 mM and [K+]ED from 1.2 - 5.7 mM with no change in VED. 5. Direct late proximal tubular fluid collections confirmed a significant inhibition of fluid reabsorption in proximal convoluted tubule in response to systemic application of U37883A. 6. These findings suggest that the diuretic and natriuretic effect upstream to the distal tubule in response to systemic application of U37883A involves actions on water-permeable segments such as the proximal convoluted tubule.

Adamantane↗
PubMed1998

Serine 13 is the site of mitotic phosphorylation of human thymidine kinase.

It has been reported that the polypeptide of thymidine kinase type 1 (TK1) from human and mouse cells can be modified by phosphorylation. Our laboratory has further shown that the level of human TK phosphorylation increases during mitotic arrest in different cell types (Chang, Z.-F., Huang, D.-Y., and Hsue, N.-C. (1994) J. Biol. Chem. 269:21249-21254). In the present study, we demonstrated that a mutation converting Ser13 to Ala abolished the mitotic phosphorylation of native TK1 expressed in Ltk- cells. Furthermore, we expressed recombinant proteins of wild-type and mutated human TK1 with fused FLAG epitope in HeLa cells, and confirmed the occurrence of mitotic phosphorylation on Ser13 of hTK1. By using an in vitro phosphorylation assay, it was shown that wild-type hTK1, but not mutant TK1(Ala13), could serve as a good substrate for Cdc2 or Cdk2 kinase. Coexpression of p21(waf1/cip1), which is a universal inhibitor of Cdk kinases, in Ltk- fibroblasts also suppressed mitotic phosphorylation of hTK1 expressed in this cell line. Thus, Cdc2 or related kinase(s) is probably involved in mitotic phosphorylation on Ser13 of the hTK1 polypeptide. We also found that mutation on Ser13 did not affect the functional activity of hTK1. As the sequences around Ser13 are highly conserved in vertebrate TK1s, we speculate that phosphorylation of Ser13 may play a role in the regulation of TK1 expression in the cell cycle.

Animals↗
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↗