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Biomedical subjects

X Yang

Publications and source records attributed to X Yang.

At least 1,081 records · Page 60Linked to original sources
PubMed1993

Molecular cloning of the murine JAK1 protein tyrosine kinase and its expression in the mouse central nervous system.

Degenerate oligonucleotide primers were employed in PCRs to clone protein tyrosine kinases that may play potential roles in the development of the mammalian CNS. Using one PCR clone to screen a mouse eye cDNA library, a full-length cDNA of a cytoplasmic tyrosine kinase, the homolog of human JAK1, was obtained. The murine JAK1 kinase belongs to a new family of cytoplasmic kinases that contain two tandem catalytic domains. Northern analyses indicated that murine JAK1 mRNA is expressed in a variety of tissues and cell lines. In the adult mouse eye, in situ hybridization and immunohistochemistry showed that JAK1 mRNA and protein were expressed in the retinal ganglion cell layer and the inner part of the inner nuclear layer, presumably in amacrine cells. JAK1 protein was also detected in horizontal cells and in the two synaptic layers of the adult retina. During retinal development, JAK1 protein was first detected in retinal ganglion cells and in their axons as early as embryonic day 14. Expression of JAK1 protein in amacrine cells and horizontal cells occurred only postnatally. This pattern of expression was also observed in the chick retina, suggesting an evolutionarily conserved function of JAK1 kinase in vertebrate retinal development and/or function. Immunohistochemical staining against JAK1 was detected in two areas of the adult mouse brain, the olfactory bulb and a group of cells in the hypothalamus. Together, these expression studies suggest a role for JAK1 kinase in the differentiation or function of a subset of CNS neurons.

Aging↗
PubMed1993

[Study on determination of gentamycin by enhanced chemiluminescent immunoassay].

Gentamycin was determined by solid antigen competition inhibition method of chemiluminescet immunoassay, in which luminol-(p-hydroxydiphenyl)-H2O2 was used a the chemiluminescence system and p-hydroxydiphenyl as the chemiluminescent enhancer. Solid antigen was synthesized by a method in which gentamycin was immobilized covalently on microcrystalline cellulose previously activated by 1,1'-carbonyldiimidazole. The rabbit anti-gentamycin IgG and the HRP-donkey anti-rabbit IgG were used as the first antibody and labelled second antibody, respectively. The linear range of the gentamycin (with blank serum) was 0.03-1 micrograms/tube and the correlation coefficient of the log-log regression equation [r[ was 0.981 +/- 0.016 with a limit of detection 3.3-11.4 ng/tube and the recovery of gentamycin 88.2 +/- 4.5%.

Biphenyl Compounds↗
PubMed1993

[A comparative study of "angioblastic meningioma", hemangiopericytoma and cerebellar hemangioblastoma].

Forty-nine cases of the so-called "angioblastic meningioma", six cases of cerebellar hemangioblastoma, and four cases of soft tissue hemangiopericytoma were studied with HE, reticulin stainings, and immunohistochemical reaction with Vimentin, EMA, GFAP, S-100, and UEA-1 antibodies. The results showed that among the three subtypes of "angioblastic meningioma", namely, the hemangiomatous (H), the hemangioblastic (AB), and the hemangiopericytic (P) types, (1) type H belongs to true meningioma; (2) most of type P are almost identical with soft tissue hemangiopericytoma and different from true meningiomas, therefore belong to hemangiopericytoma arising from pericytes; however (3) some true meningiomas may appear as hemangiopericytoma histologically; (4) a few of AB are very similar to cerebellar hemangioblastoma and thus should be considered as supratentorial hemangioblastoma, while most of AB are still true meningiomas.

Adolescent↗
PubMed1993

Warm versus cold blood cardioplegia--is there a difference?

This experimental study sought to compare the effectiveness of warm blood cardioplegia versus cold blood cardioplegia in protecting areas of ischemic myocardium during urgent coronary revascularization. In 40 adult pigs, the second and third diagonal vessels were occluded with snares for 90 minutes. All animals were then placed on cardiopulmonary bypass and underwent 45 minutes of cardioplegic arrest followed by 3 hours of reperfusion during which time the coronary snares were released. During the period of cardioplegic arrest, 10 pigs received antegrade continuous warm blood cardioplegic solution (37 degrees C) at 100 ml/min; 10 animals received retrograde warm blood cardioplegic solution at 100 ml/min; 10 received intermittent, antegrade cold blood cardioplegic solution (4 degrees C), and 10 animals received intermittent, antegrade/retrograde cold blood cardioplegic solution. Hearts protected with antegrade warm blood cardioplegic solution had the lowest pH values in the area at risk (6.59 +/- 0.10 antegrade warm blood cardioplegia versus 6.80 +/- 0.10 retrograde warm blood cardioplegia versus 6.72 +/- 0.18 antegrade cold blood cardioplegia versus 6.85 +/- 0.15 antegrade/retrograde cold blood cardioplegia and the highest area of necrosis (42% +/- 3% antegrade warm blood cardioplegia versus 26% +/- 2% [p < 0.05 from antegrade warm blood cardioplegia] retrograde warm blood cardioplegia versus 31% +/- 2% [p < 0.05 from antegrade warm blood cardioplegia] antegrade cold blood cardioplegia versus 21% +/- 2% [p < 0.05 from antegrade warm blood cardioplegia] antegrade/retrograde cold blood cardioplegia). We conclude that in the presence of an acute coronary occlusion with ischemic myocardium, warm blood cardioplegic solution should be given in a continuous retrograde fashion and does not result in myocardial protection superior to the protection that can be achieved with antegrade/retrograde cold blood cardioplegic solution.

Animals↗
PubMed1993

Concomitant defect in internal release and influx of calcium in patients with congenital platelet dysfunction and impaired agonist-induced calcium mobilization. Thromboxane production is not required for internal release of calcium.

The rise in cytoplasmic ionized calcium concentration ([Ca2+]i) on platelet activation is a combination of Ca2+ release from internal stores and influx of extracellular Ca2+. To understand the underlying mechanisms, we studied internal release and influx of Ca2+ in platelets from four patients with impaired agonist-induced Ca2+ mobilization and abnormal platelet aggregation and secretion responses. In normal platelets, thrombin caused a dose-dependent increase in internal release and influx; aspirin inhibited the total rise in [Ca2+]i and influx but not internal release, indicating that internal release occurs independent of cyclooxygenase products. In the four patients, both internal release and influx of Ca2+ induced by thrombin and adenosine diphosphate were diminished; the defect was more striking at lower agonist concentrations. To determine whether the Ca2+ storage organelles of these platelets had a diminished responsiveness to inositol 1,4,5-triphosphate (IP3) we studied IP3 (0.05 to 5 mumol/L) induced Ca2+ release and found it to be normal in all patients. We conclude that the impaired Ca2+ mobilization in our patients is due to abnormalities in both internal release and influx and that it is unlikely to be due to impaired platelet responsiveness to IP3 or defective thromboxane production. The impaired Ca2+ mobilization may be due to defects in phospholipase C activation and IP3 production. These patients provide direct evidence that internal release and influx of Ca2+ on platelet activation are closely interrelated.

Blood Platelet Disorders↗
PubMed1992

Molecular cloning of rat Wilms' tumor complementary DNA and a study of messenger RNA expression in the urogenital system and the brain.

In an effort to study the molecular basis of kidney development and carcinogenesis, we isolated complementary DNA clones of the rat homologue of the human Wilms' tumor gene, WT-1. When compared to the predicted sequence of the human WT-1 polypeptide, the rat WT-1 amino acid sequence is highly conserved (> 97%), except for the loss of one amino acid. In situ mRNA hybridization experiments localized WT-1 expression to the glomerular cells in the kidney during embryogenesis and the Sertoli cells of the testis. Similar to expression in humans and mice, WT-1 mRNA expression in the rat kidney and testis is developmentally regulated. In addition, two novel sites of specific high level WT-1 mRNA expression were seen. One is an area in the spinal cord where high level expression occurs throughout embryonic development. The second is the area postrema in the brain where localized expression continues through adulthood, suggesting a functional role for WT-1 in rat brain.

Amino Acid Sequence↗
PubMed1992

A protein kinase substrate identified by the two-hybrid system.

A genetic method, the two-hybrid system, was used to identify four genes encoding proteins that interact with the SNF1 protein kinase from yeast. One of the genes, SIP1, was independently isolated as a multicopy suppressor of defects caused by reduced SNF1 kinase activity, and genetic evidence supports its function in the SNF1 pathway. The SIP1 protein co-immunoprecipitated with SNF1 and was phosphorylated in vitro. Thus, the two-hybrid system, which is applicable to any cloned gene, can be used to detect physical interactions between protein kinases and functionally related substrate proteins.

AMP-Activated Protein Kinases↗
PubMed1992

Inhibition of the amplification reactions of blood coagulation by site-specific inhibitors of alpha-thrombin.

Hirudin and hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(54-65)] abrogate the enzyme activities of alpha-thrombin by binding the enzyme simultaneously at its catalytic centre and fibrin(ogen)-recognition exosite. In contrast, hirugen [hirudin-(54-65)] binds alpha-thrombin solely at the fibrin(ogen)-recognition exosite, and competitively inhibits fibrinopeptide A release. To investigate the extent to which the fibrin(ogen)-recognition exosite is involved when alpha-thrombin catalyses the amplification reactions of coagulation, we compared the abilities of hirudin, hirulog-1 and hirugen to inhibit simultaneously Factor X, Factor V and prothrombin activation. Whereas 0.1 microM-hirudin and 0.1 microM-hirulog-1 (i.e. less than 10% of the concentration of prothrombin in plasma) inhibited Factor X, Factor V and prothrombin activation, 10 microM was the minimum concentration of hirugen to achieve a similar anticoagulant action. Concentrations of hirudin and hirulog-1 equimolar to and 5 times greater than those of alpha-thrombin respectively abrogated Factor V activation by exogenous alpha-thrombin. In contrast, a 500-fold molar excess of hirugen could not. The inability of hirugen to inhibit the activation of the three clotting factors effectively suggests that the fibrin(ogen)-recognition exosite does not play a mandatory role when thrombin activates Factor V.

Amino Acid Sequence↗
PubMed1992

Guanosine 5'-0-(3-thiotriphosphate) induced calcium release in human platelets is mediated by inositol 1,4,5-triphosphate.

Guanosine 5'-triphosphate (GTP) and its nonhydrolyzable analogs, such as guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S), induce several responses in platelets including secretion, production of inositol 1,4,5-triphosphate (IP3) and mobilization of Ca2+ from intracellular sites. Because IP3 is well established as a second messenger in mobilizing Ca2+ from intracellular stores it has been generally assumed that Ca2+ release by GTP/GTP gamma S in platelets is mediated by IP3. However, studies in neuronal, hepatic and smooth muscle cells have suggested that IP3 and GTP/GTP gamma S activate Ca2+ release by distinct mechanisms and that IP3-independent mechanisms mediate GTP/GTP gamma S-induced Ca2+ release. In several tissues heparin inhibits binding of IP3 and blocks IP3-stimulated Ca2+ release in a competitive and specific manner. In the present studies, IP3 and GTP gamma S induced Ca2+ release and their relationship was examined in human platelets using heparin as a probe. In saponin permeabilized platelets, IP3 (0.05-5 microM) induced a prompt, dose-dependent release of Ca2+ (EC50 0.5 microM). GTP gamma S (1-50 microM) released Ca2+ in a dose-dependent manner with EC50 of 2 microM but with a time lag of 30-90 seconds. Exposure of platelets to 1 microM IP3 following a submaximal response with GTP gamma S (1 microM) resulted in a further increase in Ca2+ release but no further increase was noted on adding 1 microM IP3 following a maximal response with GTP gamma S (10 microM); similar findings were noted on reversing the order of addition of GTP gamma S and IP3 suggesting that these effectors release Ca2+ from the same source. IP3 (0.5 microM) induced Ca2+ release was blocked by low molecular weight (4000-6000) heparin (IC50 30 micrograms/ml). More importantly, heparin abolished GTP gamma S (2.5 microM) induced Ca2+ release (IC50 10 micrograms/ml). These results indicate that, in contrast to the findings in some other cells, in human platelets GTP gamma S-induced Ca2+ release is mediated largely by a mechanism involving IP3.

Blood Platelets↗
PubMed1992

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects.

The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as "tail stump defect." No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasm. SPZ were treated with 0.1 microM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at -25 degrees C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39 degrees C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively.

Animals↗
PubMed1992

Fertilization of bovine oocytes after microsurgical injection of spermatozoa.

The objective of this study was to compare the fertilization rate of bovine oocytes matured in vitro (22, 25 or 28 hours) and in vivo (30 to 35 hours after standing estrus) following the microinjection of a single spermatozoon. A single motile spermatozoon was injected into the perivitelline space (Experiments 1 to 9), and a single immotile spermatozoon was injected into the ooplasm (Experiments 10 to 15). A single ejaculate of frozen-thawed semen was used throughout. The spermatozoa were injected either without treatment or after treatment with heparin (100 microg/ml), or Ca ionophore A23187 (0.1 microM), or co-cultured for 5 hours with bovine oviduct epithelial cells (BOEC), or they were co-cultured for 5 hours with BOEC and immobilized by freezing and thawing twice without cryoprotectant, or they remained untreated. Oocytes were placed in a droplet of hyperosmotic solution of 0.1 M sucrose in PBS to enlarge the perivitelline space (Experiments 1 to 9) or in PBS (Experiments 10 to 15). Small amounts of polyvinyl pyrrolidone (PVP) without spermatozoa were injected as a control for parthenogenetic activation. After injection, oocytes were incubated in Medium 199 for 22 hours at 39 degrees C, and they were stained with 1% aceto-orcein and examined for evidence of fertilization or parthenogenetic activation. Low rates (9 to 11%) of fertilization resulted from injection into the perivitelline space of oocytes matured for 22 hours in vitro irrespective of spermatozoa treatment. Fertilization rates were higher in oocytes matured in vivo after injection into either perivitelline space (66%) or ooplasm (74%) than in oocytes matured in vitro (9 to 44% fertilization). Surprisingly, in oocytes matured in vivo, there was no difference in the proportions fertilized by spermatozoa injection into ooplasm and parthenogenetically activated by injection of medium alone (74 and 66%, respectively).

Journal Article↗
PubMed1992

Micromanipulation of mammalian embryos: Principles, progress and future possibilities.

Numerous advances in development of techniques for manipulating mammalian embryos outside the maternal environment have been made over the past decade. Some techniques were developed primarily for use in research; others were developed in response to problems of practical livestock production but have proven useful in research as well. Embryo micromanipulation procedures are used often in conjunction with embryo transfer, and interest in these procedures was stimulated by growth of the embryo transfer industry. Included in this review are discussions of procedures for manipulation of gametes and embryos, including sperm injection into oocytes, pronuclear and nuclear transfer, embryo biopsy and splitting, experimental chimera production and isolation of embryonic stem cells.

Journal Article↗
PubMed1992

Organization and expression of the gene encoding chick kainate binding protein, a member of the glutamate receptor family.

The gene encoding chick cerebellar Bergmann glia-specific kainate binding protein (chKBP), has been isolated, characterized and expressed in heterologous systems. The structural gene spans 11.2 kb and contains 11 exons and 10 introns. Several of the exons encode specific receptor domains, including each of the predicted transmembrane regions. Exon/intron boundaries flanking the second, putative channel-forming transmembrane domain are conserved between chKBP and other glutamate/kainate receptor subunits. The putative promoter region 5' to the first exon displays high GC content and TATA, CAAT and AP1 consensus sequences. Transcription of the chKBP gene is evident prior to full cerebellar cortical maturation. Transcripts are abundant in cells consistent with Bergmann glia, as revealed by in situ hybridization. Transfection of 293 kidney cell cultures with chKBP cDNA or chKBP gene expression constructs confers CNQX-sensitive kainate binding with the pharmacological specificity displayed by both chKBP and kainate receptors. However, expression of the same constructs in Xenopus oocytes fails to yield detectable agonist-activated currents.

Amino Acid Sequence↗
PubMed1992

Heterogeneity of radiosensitivity in a human glioma cell line.

Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.

Aged↗
PubMed1992

N-terminal mutations modulate yeast SNF1 protein kinase function.

The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.

AMP-Activated Protein Kinases↗
PubMed1992

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.

Animals↗