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Biomedical subjects

J Yu

Publications and source records attributed to J Yu.

At least 451 records · Page 25Linked to original sources
PubMed1998

Studies of the cytochrome subunits of menaquinone:cytochrome c reductase (bc complex) of Bacillus subtilis. Evidence for the covalent attachment of heme to the cytochrome b subunit.

The menaquinone:cytochrome c reductase, or bc complex, of Bacillus subtilis belongs to a third class of bc-type complex, distinct from the bc1 and b6f classes. Using a mutagenesis approach, we demonstrate that the cytochrome b (QcrB) and c (QcrC) subunits of the complex give rise to bands at 22 and 29 kDa, respectively, after denaturing electrophoresis; that both subunits are required for proper complex assembly and/or stability; and that both subunits retain one heme molecule under denaturing conditions. This unusual property of a b-type cytochrome was investigated further. We present evidence for the existence of a covalent linkage between the polypeptide and heme bH and of an important role for Cys43 in binding of heme bH. It is proposed that heme is also covalently attached to the cytochrome b subunit of b6f complexes of chloroplasts and cyanobacteria.

Amino Acid Sequence↗
PubMed1998

Influence of copper depletion on iron uptake mediated by SFT, a stimulator of Fe transport.

We recently identified a novel factor involved in cellular iron assimilation called SFT or Stimulator of Fe Transport (Gutierrez, J. A., Yu, J., Rivera, S., and Wessling-Resnick, M. (1997) J. Cell Biol. 149, 895-905). When stably expressed in HeLa cells, SFT was found to stimulate the uptake of both transferrin- and nontransferrin-bound Fe (iron). Assimilation of nontransferrin-bound Fe by HeLa cells stably expressing SFT was time- and temperature-dependent; both the rate and extent of uptake was enhanced relative to the activity of control nontransfected cells. Although the apparent Km for Fe uptake was unaffected by expression of SFT (5.6 versus 5.1 microM measured for control), the Vmax of transport was increased from 7.0 to 14.7 pmol/min/mg protein. Transport mediated by SFT was inhibitable by diethylenetriaminepentaacetic acid and ferrozine, Fe3+- and Fe2+-specific chelators. Because cellular copper status is known to influence Fe assimilation, we investigated the effects of Cu (copper) depletion on SFT function. After 4 days of culture in Cu-deficient media, HeLa cell Cu,Zn superoxide dismutase activity was reduced by more than 60%. Both control cells and cells stably expressing SFT displayed reduced Fe uptake as well; levels of transferrin-mediated import fell by approximately 80%, whereas levels of nontransferrin-bound Fe uptake were approximately 50% that of Cu-replete cells. The failure of SFT expression to stimulate Fe uptake above basal levels in Cu-depleted cells suggests a critical role for Cu in SFT function. A current model for both transferrin- and nontransferrin-bound Fe uptake involves the function of a ferrireductase that acts to reduce Fe3+ to Fe2+, with subsequent transport of the divalent cation across the membrane bilayer. SFT expression did not enhance levels of HeLa cell surface reductase activity; however, Cu depletion was found to reduce endogenous activity by 60%, suggesting impaired ferrireductase function may account for the influence of Cu depletion on SFT-mediated Fe uptake. To test this hypothesis, the ability of SFT to directly mediate Fe2+ import was examined. Although expression of SFT enhanced Fe2+ uptake by HeLa cells, Cu depletion did not significantly reduce this activity. Thus, we conclude that a ferrireductase activity is required for SFT function in Fe3+ transport and that Cu depletion reduces cellular iron assimilation by affecting this activity.

Biological Transport↗
PubMed1998

Mutations of mitotic checkpoint genes in human cancers.

Genetic instability was one of the first characteristics to be postulated to underlie neoplasia. Such genetic instability occurs in two different forms. In a small fraction of colorectal and some other cancers, defective repair of mismatched bases results in an increased mutation rate at the nucleotide level and consequent widespread microsatellite instability. In most colorectal cancers, and probably in many other cancer types, a chromosomal instability (CIN) leading to an abnormal chromosome number (aneuploidy) is observed. The physiological and molecular bases of this pervasive abnormality are unknown. Here we show that CIN is consistently associated with the loss of function of a mitotic checkpoint. Moreover, in some cancers displaying CIN the loss of this checkpoint was associated with the mutational inactivation of a human homologue of the yeast BUB1 gene; BUB1 controls mitotic checkpoints and chromosome segregation in yeast. The normal mitotic checkpoints of cells displaying microsatellite instability become defective upon transfer of mutant hBUB1 alleles from either of two CIN cancers.

Amino Acid Sequence↗
PubMed1998

A glycosidase antibody elicited against a chair-like transition state analog by in vitro immunization.

Antibodies were generated against the positively charged chair-like glycosidase inhibitor nojirimycin by in vitro immunization. A number of catalytic antibodies were isolated, one of which catalyzes the hydrolysis of p-nitrophenyl beta-D-glucopyranoside 3 with a rate enhancement (kcat/kuncat) of 10(5) M over the HOAC-catalyzed reaction. The antibody discriminates modifications in the pyranoside ring of substrate 3 at the C2, C4, and the anomeric positions. The pH dependence of the reaction and chemical modification studies suggest the presence of an active-site Asp or Glu residue that may function as a general acid. This study further defines those requirements necessary to generate antibodies that efficiently cleave glycosidic bonds.

1-Deoxynojirimycin↗
PubMed1998

A simplified system for generating recombinant adenoviruses.

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.

Adenoviridae↗
PubMed1998

Metabolic depletion inhibits the uptake of nontransferrin-bound iron by K562 cells.

The effect of metabolic inhibitors on nontransferrin bound iron transport by K562 cells was investigated. Incubation with 1 microM rotenone, 10 microM antimycin, or 0.5 mM 2,4-dinitrophenol effectively reduced ATP levels by approximately 50%. Both the rate and extent of Fe+3 uptake were impaired in ATP-depleted cells, which display a reduced Vmax for uptake. K562 cell ferrireductase activity was also lowered by metabolic inhibitors, suggesting that the apparent energy requirements for transport reside in the reduction of Fe+3 to Fe+2. However, ATP depletion was found to inhibit the rate and extent of Fe+2 uptake as well. Thus, the transbilayer passage of Fe+2 and/or Fe+3 appears to be an energy-requiring process. These features possibly reflect properties of the transport mechanism associated with a recently identified K562 cell transport protein, called SFT for "Stimulator of Fe Transport," since exogenous expression of its activity is also affected by ATP depletion.

2,4-Dinitrophenol↗
PubMed1998

C112R, W323S, N317K mutations in the vasopressin V2 receptor gene in patients with nephrogenic diabetes insipidus. Mutations in brief no. 165. Online.

Nephrogenic diabetes insipidus (NDI) is a rare, mostly X-linked recessive disorder characterized by renal tubular resistance to the antidiuretic effect of arginine vasopressin. The gene responsible for the X-linked NDI, the G-protein-coupled vasopressin V2 receptor, has been localized on the Xq28 region. In this study we present three NDI families from Hungary with three different missense mutations in the vasopressin V2 receptor gene. After the mutations in the affected probands in each family had been characterized, other family members were screened by restriction enzyme analysis. The N317K and W323S mutations have not been detected previously. The C112R is an already known mutation. The N317K was a de novo mutation in the patient. The C112R and the W323S were found in the mothers of the patients as carriers and in all other patients, but not in the unaffected members of the families. Segregation of the mutations was consistent with the clinically observed symptoms as well as their severity. As conclusion, these findings further evidence that X-linked NDI results from defects in the V2 receptor gene.

Diabetes Insipidus, Nephrogenic↗
PubMed1998

Differential modulation of dopamine D1-receptor binding and mRNA expression in the basal ganglia by the D1-receptor antagonist, SCH-23390.

Dopamine D1-receptor binding in the basal ganglia is differentially regulated by subtype nonspecific dopamine antagonists such as the antipsychotic, Fluphenazine. The purpose of the present study was to determine the relative contributions of D1 and D2 receptor systems in the regulation of basal ganglia D1-receptor binding. Rats were injected twice daily for 21 days with saline, the D1-receptor antagonist, SCH-23390, the D2-receptor antagonist, Raclopride, or both SCH-23390 and Raclopride. Dopamine D1-receptor levels (as indicated by [125I]SCH-23982 binding) and mRNA expression were measured using receptor autoradiographic and in situ hybridization histochemical techniques. [125I]NCQ-298 binding to D2-receptors was also measured as a positive control for the effects of Raclopride. SCH-23390 administration independently increased [125I]SCH-23982 binding in a region-dependent manner with the greatest increases occurring in the entopeduncular nucleus. SCH-23390 also increased D1-receptor mRNA expression in specific striatal subregions suggesting that increases in binding were related to changes in receptor synthesis. In addition, Raclopride independently enhanced D2 binding with comparable increases observed in extrastriatal regions and increases of a lesser magnitude in the striatum. These data show that the modulation of basal ganglia D1-receptor binding observed in animals treated with nonselective antagonists is due primarily to the blockade of D1-receptors. The differential enhancement in basal ganglia D1 binding observed after D1-receptor blockade may be due to anatomical or phenotypic heterogeneity within the population of striatal D1-receptor synthesizing neurons. Similarly, the differential enhancement in striatal and extrastriatal D2-receptor binding may be due to differences in the regulation of striatal and extrastriatal D2-receptor synthesizing neurons.

Animals↗
PubMed1998

Contrasting effects of inflammatory cytokines and glucocorticoids on the production of activin A in human marrow stromal cells and their implications.

Human marrow stromal cells were analysed with immunocytochemical staining, Northern blot, and functional bioassay for production of activin A. Although Northern blot and immunocytochemical staining did not detect the alpha subunit of inhibin in human marrow stromal cells, RT-PCR analyses confirmed its presence, along with the expected activin beta A PCR products. Present studies showed that human marrow fibroblastoid cells were reactive with anti-activin A antibodies and that the production of beta A RNA was upregulated by pro-inflammatory cytokines/regulators like interleukin 1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) or 12-O-tetradecanoylphorbol 13-acetate (TPA). IL-1 alpha or TNF-alpha stimulated-marrow stromal cells accumulated beta A RNA after 2 h of incubation, reaching a peak stimulation at approximately 8 h. Biologically active activin A molecules were detected in the conditioned media by a bioassay, and their activity was specifically inhibited by a blocking antibody or an activin-binding protein, follistatin. Accumulation of bioactive activin A in conditioned medium of human marrow stromal cells increased after incubation with IL-1 alpha or TNF-alpha. Nuclear run-off assays with TNF-alpha stimulated marrow stromal cells showed that the enhanced expression of activin A was related to an increase in its rate of transcription. In contrast to the stimulatory effect of pro-inflammatory cytokines, hydrocortisone and dexamethasone at 1 x 10(-7) to 1 x 10(-6) M inhibited both the constitutive and the cytokine-stimulated expression of activin beta A RNA, and also the production of bioactive activin A protein. The upregulation of activin A production by cytokines and its suppression by glucocorticoids imply that activin A may also act as a moderator in diverse functions including host defences.

Activins↗
PubMed1998

Complete coding sequence of inducible nitric oxide synthase from human heart and skeletal muscle of patients with chronic heart failure.

The presence of the inducible nitric oxide synthase (iNOS) has previously been reported in heart and skeletal muscle of patients with chronic heart failure, where it is thought to be involved in the pathogenesis of dilated cardiomyopathy or of exercise intolerance observed in patients with chronic heart failure. To identify the iNOS of these human tissues the cDNA sequence was determined using reverse transcription polymerase chain reaction (RT-PCR). The deduced amino acid sequence of human heart and skeletal muscle shows a 99% identity between each other. The amino acid sequence was distinct from the constitutively expressed nitric oxide synthases. A 98% identity at the protein level was detected to rat vascular smooth muscle iNOS, whereas the homology to human hepatocyte iNOS was only 79%. The cofactor binding sites in the reported iNOS from human heart and skeletal muscle are highly conserved. The iNOS sequence described in this report is the first one isolated directly from human tissue expressing iNOS in the circumstances of chronic heart failure.

Amino Acid Sequence↗
PubMed1998

Experimental study on the correlation of nitric oxide with portal hypertensive enteropathy.

To explore the role of nitric oxide (NO) in the pathogenesis of portal hypertensive enteropathy (PHE), cirrhotic portal hypertension was induced in Wistar rats by subcutaneous administration of carbon tetrachloride. At the end of 12th week, NADPH-diaphorase staining was performed on ice frozen sections with the sample of fresh colonic tissues. NO synthase (NOS) activities and NOS mRNA expression of colonic tissues were investigated with chemoluminescence method and reverse transcription-polymerase chain reaction (RT-PCR), respectively. After NADPH-diaphorase histochemical staining, the computer image analysis and paired t test showed that NOS staining intensities of submucosal vascular endothelial cells and nerve fibers were all significantly higher in PHE group than those in normal group (P < 0.01 and P < 0.05, respectively), but the intensities of superficial epithelial cells were significantly lower than those of controls (P < 0.01). Chemoluminescence method demonstrated that general NOS activity of colonic mucosa was significantly higher in PHE group than that in control group (P < 0.01). Moreover, the degree of iNOS activity increase was greater than that of cNOS (104% vs 35%). RT-PCR revealed that NOS mRNA expressions were dramatically higher in PHE group than those in control group (P < 0.01). The results suggested that NO, with its property of vasodilatation, may be involved in the pathogenesis of vascular lesions of PHE in cirrhotic rats, and may also has something to do with mucosal lesions of colon in PHE.

Animals↗
PubMed1998

Phosphorylated insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) inhibits while non-phosphorylated IGFBP-1 stimulates IGF-I-induced amino acid uptake by cultured trophoblast cells.

The effects of phosphorylated insulin-like growth factor-binding protein (pIGFBP-1) and non-phosphorylated (npIGFBP-1) IGFBP-1 on amino acid uptake induced by IGF-I were studied using cultured trophoblast cells. Trophoblast cells obtained from term pregnancy were incubated with indicated concentrations of pIGFBP-1 or npIGFBP-1 for 24 h and further incubated with 10 nM IGF-I for 3 h. Cells were then incubated with 3H-alpha-amino isobutyric acid (3H-AIB) for 30 min. Both pIGFBP-1 and npIGFBP-1 alone had no effect on 3H-AIB uptake; however, pIGFBP-1 inhibited IGF-I-stimulated 3H-AIB uptake with an ED50 of 0.26 nM while npIGFBP-1 potentiated 3H-AIB uptake with an ED50 of 0.27 nM. Maternal IGF-I promotes fetal growth by stimulating nutrient transport in the placenta. As shown in this study, pIGFBP-1 inhibits while npIGFBP-1 stimulates this IGF-I action in the placenta. Thus, it is suggested that IGFBP-1 phosphoisoforms are also involved in fetal growth by modulating IGF-I action in the placenta.

Amino Acids↗