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Y Gao

Publications and source records attributed to Y Gao.

At least 307 records · Page 17Linked to original sources
PubMed1997

The ordered phosphorylation of cardiac troponin I by the cAMP-dependent protein kinase--structural consequences and functional implications.

The pattern of phosphorylation of adjacent serine residues in several peptides based on the N-terminal region of human cardiac troponin I has been analysed by PAGE and 1H NMR spectroscopy to identify the products. With cAMP-dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild-type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N-terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two-dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N-terminal peptide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N-terminal to the serine pair and stabilisation of the structure in the region of residues 25-29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.

Amino Acid Sequence↗
PubMed1997

Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination.

V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities. All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR). Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function. The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme. To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression. We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels. Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70(-/-) embryonic stem cells have markedly increased sensitivity to gamma-irradiation relative to Ku70(+/-) or wild-type embryonic stem cells. Furthermore, the Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are discussed.

Animals↗
PubMed1997

Role of Gq alpha in insulin-stimulated glucose uptake by C6 glioma cells.

To investigate the possibility that insulin-stimulated glucose uptake in C6 cells is due to transactivation of a G protein-mediated pathway, the role of Gq alpha in insulin signaling was studied. Insulin stimulation of [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was time- and concentration-dependent: at a concentration of 1 microM, insulin stimulated 2DG uptake by C6 cells by about 30% (p < 0.05). Pertussis toxin treatment of C6 cells did not alter the ability of insulin (1 microM) to promote 2DG uptake, ruling out the involvement of Gion in insulin-stimulated hexose uptake. Next, C6 cells were transfected with Gq alpha cDNA for 48 h, challenged with 1 microM insulin, and 2DG uptake by the cells was determined. Insulin-stimulated 2DG uptake was 1.14 +/- 0.03 and 1.75 +/- 0.19 nmol/min/mg protein in mock- and Gq alpha-transfected cells, respectively (p < 0.05); insulin stimulated 2DG uptake in Gq alpha-transfected cells by 54%. These results suggest an involvement of Gq alpha in the transactivation of the G protein signal transduction pathway by insulin.

Deoxyglucose↗
PubMed1997

Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation.

Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

Actins↗
PubMed1997

Mutant human protein disulfide isomerase assists protein folding in a chaperone-like fashion.

Human protein disulfide isomerase with an extra 10 amino acid residues of AEITRIDPAM at the N-terminal was expressed in E. coli as a soluble protein comprising 20% of total cell proteins, and was purified to near homogeneity through one step of DEAE-Sephacel chromatography. The mutant enzyme, which had the same CD spectrum and comparable disulfide isomerase and thiol-protein oxidoreductase activities with that of the wild type human and bovine protein disulfide isomerases, also showed chaperone-like activity in stimulating the refolding of proteins containing no disulfide bond. The overall yield of the active product is about 20 mg 1-1 culture.

Amino Acid Sequence↗
PubMed1997

Nuclear factor interleukin 6 motifs mediate tissue-specific gene transcription in hypoxia.

Activation of transcription at the nuclear factor interleukin 6 (NF-IL-6) DNA binding motif modulates expression of multiple genes important in host adaptive and developmental mechanisms. Studies showing that hypoxia-induced transcription of IL-6 in cultured endothelial cells was due to transcriptional activation by the NF-IL-6 motif in the promoter (Yan, S.-F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L., and Stern, D. (1995) J. Biol. Chem. 270, 11463-11471) led us to prepare transgenic mice using 115- or 14-base pair regions of the promoter encompassing the NF-IL-6 site ligated to the lacZ reporter gene and the basal thymidine kinase promoter. On exposure to hypoxia or induction of ischemia, mice bearing either of the constructs showed prominent expression of the transgene in lung and cardiac vasculature and in the kidney but not in the liver (parenchyma or vasculature). In contrast, transgenic mice bearing a mutationally inactivated NF-IL-6 site showed no increase in transgene expression in hypoxia. Gel retardation assays revealed time-dependent, hypoxia-enhanced nuclear binding activity for the NF-IL-6 site in nuclear extracts of the heart, lung, and kidney but not in the liver; the hypoxia-enhanced band disappeared on addition of antibody to C/EBPbeta-NF-IL-6. Consistent with the specificity of hypoxia-mediated activation of C/EBPbeta-NF-IL-6, gel retardation assays showed no change in the intensity of the hypoxia-enhanced gel shift band in the presence of excess unlabeled oligonucleotide probes or antibodies related to other transcription factors, including NFkappaB, AP1, cAMP response element-binding protein, SP1, and hypoxia-inducible factor 1. These data indicate that the transcription factor NF-IL-6 is sensitive to environmental oxygen deprivation, and the tissue-specific pattern of gene expression suggests that local mechanisms have an important regulatory effect.

Animals↗
PubMed1997

Estrogen inhibits hypothalamic pro-opiomelanocortin gene expression in hypothalamic neuronal cultures.

Many in vitro studies show estrogen regulation of the hypothalamic pro-opiomelanocortin (POMC) system, including a decrease in hypothalamic POMC mRNA after estradiol treatment. Because such in vivo experiments do not allow one to determine whether peripheral, interacting systems or extra-hypothalamic brain regions are involved in this regulation, we sought to establish whether estrogen acts directly in hypothalamus to decrease POMC mRNA. Using an in vitro approach, we studied effects of estradiol (E2) on POMC/cyclophilin mRNA concentrations (RNAse protection assays) in neuronal cultures derived from day 17 fetal rat hypothalamus. Chemically defined medium was deprived of progesterone for 2 days prior to E2 treatment and for the duration of the study. E2 (10(-13)-10(-9) M) dose-dependently decreased POMC mRNA concentrations during a 2-day treatment. Whereas the lowest dose (10(-13) M) of E2 resulted in a statistically significant 44% decrease in POMC mRNA concentrations relative to control cultures, this inhibitory effect was lost because higher doses (10(-11) and 10(-9) M) did not produce statistically significant decrements (22 and 16%, respectively) in POMC mRNA concentrations. Additional time course studies revealed that this decrease in POMC mRNA can be seen as early as 4 h after E2 (10(-13) M) treatment. We conclude that E2 inhibition of POMC mRNA concentrations in hypothalamic neuronal cultures indicates that this inhibition can occur directly in hypothalamus.

Amino Acid Isomerases↗
PubMed1997

Role of nitric oxide and tumor necrosis factor on lung injury caused by ischemia/reperfusion of the lower extremities.

PURPOSE: Acute aortic occlusion with subsequent ischemia/reperfusion (I/R) of the lower extremities is known to predispose to lung injury. The pathophysiologic mechanisms of this injury are not clear. In the present study, we studied the role of tumor necrosis factor (TNF) and nitric oxide (NO) in lung injury caused by lower extremity I/R. METHODS: A rat model in which the infrarenal aorta was cross-clamped for 3 hours followed by 1 hour of reperfusion was used. The rats were randomized into five groups: group 1, aorta exposed but not clamped; group 2, aorta clamped for 3 hours, followed by 1 hour of reperfusion; group 3, 1 mg/kg dexamethasone administered before the aorta was clamped; group 4, 25 mg aminoguanidine, a specific inducible NO synthase (iNOS) inhibitor, administered before the aorta was clamped; and group 5, 2 mg/kg TNFbp, a PEG-ylated dimeric form of the high-affinity p55 TNF receptor I (RI), administered before the aorta was clamped. NO concentration in the exhaled gas (ENO) was measured, as an index of NO production by the lung, in 30 minute intervals during I/R. Serial arterial blood samples for TNF assay were obtained during the course of the experiment. At the end of the experiment, the lungs were removed and histologically examined for evidence of injury. RESULTS: ENO in group 2 increased from 0.7 +/- 0.3 ppb at baseline to 54.3 +/- 7.5 ppb at the end of ischemia and remained stable during reperfusion (54.6 +/- 8.5 ppb at the end of reperfusion). ENO production was blocked by aminoguanidine, by dexamethasone, and by TNFbp given before aortic occlusion. Serum TNF in groups 2, 3 and 4 increased rapidly during early ischemia, reaching its peak value 60 minutes after occlusion of the aorta, then gradually declined to baseline levels at the end of ischemia, and remained low during reperfusion. TNFbp decreased serum TNF concentration significantly when it was given before aortic occlusion. Histologic examination of the lungs at the end of the experiment revealed that aminoguanidine, dexamethasone, and TNFbp had a protective effect on the lungs. CONCLUSIONS: Serum TNF increases rapidly during lower extremity ischemia and causes increased production of NO from the lung by upregulating iNOS. Increased NO is associated with more severe lung injury, and iNOS blockade has beneficial effects on the lung. TNF blockade before ischemia decreases NO production by the lung and attenuates lung injury. ENO can be used as an early marker of lung injury caused by lower extremity I/R.

Animals↗
PubMed1997

The ultrastructure and computer imaging of the lymphatic stomata in the human pelvic peritoneum.

The lymphatic stomata in the pelvic peritoneum of human fetuses and mature mice were initially observed and studied quantitatively by using computer image processing (C.I.P.) attached to a scanning electron microscope (SEM). Two types of mesothelial cells were found in the pelvic peritoneum of human fetuses and mature mice, i.e. flattened and cuboidal cells. The lymphatic stomata, arranged in clusters, were only found irregularly distributed among the cuboidal cells. The divergence of stoma area in the pelvic peritoneum of human fetuses varied greatly, ranging from 0.8 micron2 to 43.4 microns2. The average area of the lymphatic stomata in human fetuses was 10.00 +/- 9.44 microns2. The variation coefficient was 94.40. The standard deviations and standard errors were 9.44 and 0.98 respectively. Most of the lymphatic stomata in human fetuses were between 1.34 microns2 and 32.11 microns2 in size (accounting for 90%), with maximum and minimum values of 43.4 microns2 and 0.8 micron2. The average distribution density of the lymphatic stomata in human fetuses was 7.2% and the maximum density was 11.6%, which means that the average and the maximum absorption rates of the human pelvic peritoneum from the peritoneal cavity were 7.2% and 11.6% respectively. Therefore, it is suggested that the lymphatic stomata in pelvic peritoneum play an important role in draining materials from the peritoneal cavity, and that the absorption effect of the pelvic peritoneum is similar to that of the diaphragmatic peritoneum.

Animals↗
PubMed1997

Molecular genetic characterization of XRCC4 function.

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.

Amino Acid Sequence↗
PubMed1997

Induction of an exceptionally high-level, nontranslated, Epstein-Barr virus-encoded polyadenylated transcript in the Burkitt's lymphoma line Daudi.

An Epstein-Barr virus transcript (designated D-HIT [Daudi high-level-inducible transcript]), constitutively expressed at low levels in the Burkitt's lymphoma (BL)-derived cell line Daudi, can be induced with tetradecanoylphorbol acetate or n-butyrate or, in combination, to about 1% of the levels of high-molecular-weight RNAs in cells. The transcript can also be induced in some other EBV-positive BL-derived cells but to a much lesser extent, particularly in lines that can give rise to productive infection. D-HIT is viral in origin and is composed largely of repetitive sequence. It is polyadenylated but mainly nuclear in location and is highly structured, sensitive only to double-strand-specific RNase. It is endogenously expressed in interferon-sensitive Daudi strains but not in an insensitive strain, Daudi 100K. D-HIT contains a part of a viral open reading frame (designated LF3, and deleted in the prototype B95-8 strain), using an internal polyadenylation (AAUAAA) sequence as a signal to specify processing of its 3' end. In Daudi cells, the promoter contains a putative hinge structure, as found in some interferon-inducible genes and c-myc. Since D-HIT lies adjacent to, probably even encompassing, one of the two viral lytic origins (D(R)) of replication, it may have a role in the regulation of DNA replication. Alternatively, or in addition via its double-stranded structure, D-HIT may play a regulatory role in interferon pathways. Its promoter could be of value for studying expression in constructions containing heterologous genes.

Base Sequence↗
PubMed1997

Antenatal betamethasone therapy augments isoproterenol and prostaglandin E2-mediated relaxation of preterm ovine pulmonary veins.

Antenatal glucocorticoid therapy improves pulmonary function in preterm newborns. We have determined the effect of antenatal glucocorticoid therapy on isoproterenol and prostaglandin (PG) E2-mediated relaxation in preterm ovine pulmonary veins after birth. Ovine fetuses (121 and 126 d of gestation; term = 150 d) received an ultrasound guided intramuscular injection of betamethasone, 0.5 mg/kg, or saline. Lambs were delivered 15 or 48 h later, ventilated for 3 h, and killed. Isolated fourth generation pulmonary veins were suspended in organ chambers filled with modified Krebs-Ringer solution (95% O2, 5% CO2) at 37 degrees C, and their isometric tension was recorded. During contractions to U46619, isoproterenol and PGE2 induced greater relaxations of pulmonary veins of betamethasone-treated lambs than those of control. Forskolin, an activator of adenylate cyclase, caused greater relaxation in veins of betamethasone-treated lambs than in those of controls. A greater relaxation of veins treated with betamethasone than that of control veins also occurred in the presence of isobutylmethylxanthine, an inhibitor of phosphodiesterases. All vessels relaxed similarly to 8-bromo-cAMP, a cell membrane-permeable analog of cAMP. When stimulated with isoproterenol, PGE2, and forskolin, adenylate cyclase activity of crude membrane preparations of pulmonary veins treated with betamethasone was greater than that of controls. These results demonstrate that antenatal betamethasone therapy potentiates isoproterenol and PGE2-mediated relaxation of pulmonary veins of preterm lambs; an enhanced adenylate cyclase activity explain in part the effect of antenatal glucocorticoid therapy on pulmonary veins of preterm lambs.

Adenylyl Cyclases↗
PubMed1997

Electrophysiologic effect of enalapril on guinea pig papillary muscles in vitro.

AIM: To study the direct effect of enalapril on cellular electrophysiology of myocardium. METHODS: Conventional microelectrodes technique was used to record the action potentials (AP) of guinea pig papillary muscles. RESULTS: Enalapril caused an increase of the AP amplitude (APA) and the resting potential (RP) in a concentration-dependent manner without any significant change of AP duration, Vmax and overshoot of AP. Superfusion of ouabain 0.5 mumol.L-1 reduced APA and RP, induced stable delayed after-depolarizations (DAD) at different basic cycle lengths (BCL) in a frequency-dependent manner. At BCL 200 ms, the amplitude of DAD was large enough to induce nonsustained triggered activity (TA). In additional presence of enalapril 10 mumol.L-1, the DAD amplitude at 500, 400, 300, and 200 ms were decreased from 5.3 +/- 2.3, 5.9 +/- 2.8, 7.4 +/- 2.1, and 8.9 +/- 1.3 to 2.6 +/- 0.7, 3.1 +/- 1.0, 3.7 +/- 1.5, and 5.3 +/- 1.1 (mV) respectively, all P < 0.01. The compensation intervals were increased in a similar frequency-dependent manner. The number of TA induced at BCL 200 ms was decreased from 3.6 +/- 0.7 to 0.8 +/- 0.2 (P < 0.05). CONCLUSION: Enalapril directly inhibits DAD and TA induced by ouabain through increasing RP and APA, which may contribute to its anti-arrhythmic effect.

Action Potentials↗
PubMed1997

[The effect of endurance training and exhaustive exercise on metallothionein in rats].

To understand physiological role of metallothionein (MT) during exercise, MT levels in liver, brain, heart, lung, blood vessel, skeletal muscle and serum were observed in rats. The results showed that the levels of MT were decreased by 13-34% in lung, liver, heart and skeletal muscle of rats trained by swimming for 10 weeks and were increased by 21%-75% in skeletal muscle, heart, brain, lung and liver of rats after exhaustive swimming respectively, compared with normal control rats. (P < 0.05 and P < 0.01). But the levels of MT in blood vessel and serum were not changed in two groups of rats by swimming for 10 weeks and acute exhaustive swimming (P > 0.05). It is suggested that the different changes of MT levels under physical training and acute exhaustive exercise may be of importance in protection against oxygen free radicals.

Animals↗
PubMed1997

[Studies of mechanism of erythrocyte deformability injury during hypobaric hypoxia in rats].

Rats were randomized into sea level group and hyoxic group. Erythrocyte filtration index (IF), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), erythrocyte [pH]i, [Mg2-]i, [Ca2+]i and [K+]i/[Na+]i were investigated in our studies. The results were as follows: 1. IF was increared in hypoxic group (0.234 +/- 0.060) 2. [Ca2+]i of hypoxic group (7.02 +/- 1.66) was evidently increased and correlated with its IF (r = 0.7386, P < 0.01), but [Mg2+]i had no obvious changes, [K+]i/ [Na+]i ratio of hypoxic group was reduced and negatively correlated with IF (r = -0.3957, P < 0.05), meanwhile, [pH] of hypoxic group increased evidently. 3. There was no statistic difference between MCHC of hypoxic and sea level group, but MCV of hypoxic group increased. These findings suggest that the changes of [Ca2+]i, [K+]i/[Na+]i ratio, [pH]i and MCV may play important role in erythrocyte deformability injury during hypoxia.

Animals↗
PubMed1997

[Aerosolized recombinant interferon-gamma prevent antigen-induced eosinophil recruitment in guinea pig trachea].

OBJECTIVE: In order to determine whether interferon-gamma (IFN-gamma) inhibits eosinphil infiltration in the trachea of asthmatic guinea pigs induced by Rhizopus nigricans. METHOD: We had administered aerosolized rIFN-gamma in the tracheas of 30 sensitized guinea pigs which had been divided into six groups, then teated animal inhaled rIFN-gamma of 5 x 10(4), 20 x 10(4), and 40 x 10(4) concentration, BDP and normal saline respectively at 24 h, 12 h, 2 h before being challenged. RESULT: (1) Provocation positive rates decreased in 40 x 10(4) rIFN-gamma and BDP group compared with that in normal saline group and before intervention (P < 0.05), airway resistence decreased (P < 0.01). (2) The administration of aerosolized rIFN-gamma (40 x 10(4)) and BDP also decreased fungus-induced eosnophils but not other cells infiltration in the trachea. (3) In BALF, Eos count and ECP level were obviously lower than those in other groups. However, eosinophil numbers did not show significant change in the peripheral blood. CONCLUSION: Local administration of rIFN-gamma (40 x 10(4)) may reduce airway inflammation and intervene asthmatic attack by inhibition of Eos, ECP infiltration in airways.

Administration, Inhalation↗
PubMed1997

[The relationship between the prognosis of retinoblastoma and proliferating cell nuclear antigen and argyrophilic protein at nucleolus organizer regions].

OBJECTIVE: To investigate the relationship between the proliferative activity of retinoblastoma (Rb) cells and the prognosis of the tumor. METHODS: Proliferating cell nuclear antigen (PCNA) and argyrophilic nucleolar organizer regions (AgNORs) in paraffin-embedded Rb specimens from 48 cases were detected by LSAB immunohistochemical method using an anti-PCNA monoclonal antibody and the technique of ploton. RESULTS: The PCNA labeling index (PCNALI) and the AgNORs counts were well correlated (r = 0.647, P < 0.001). In 29 patients of the 48 cases receiving simple eyeball extirpation and being followed up for 5 years, the mean PCNALI and the mean numbers of AgNORs per nucleus in 19 patients surviving > or = 5 years after operation were markedly lower than that in 10 patients surviving < 5 years after operation (P < 0.01, P < 0.05). CONCLUSION: It is indicated that the PCNALI or the AgNOR count is negatively related with the clinical prognosis of Rb patients, and can be useful in judging the prognosis.

Eye Enucleation↗