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Biomedical subjects

Y Fu

Publications and source records attributed to Y Fu.

At least 55 records · Page 3Linked to original sources
PubMed1997

Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.

The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2(CHK) to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2(CHK)-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2(CHK). These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2(CHK)-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2(CHK) to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2(CHK) binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2(CHK). This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.

Animals↗
PubMed1997

CDC2-related kinase PITALRE phosphorylates pRb exclusively on serine and is widely expressed in human tissues.

Mammalian cell cycle progression is regulated by sequential activation and inactivation of cyclin-dependent kinases (cdks). Recently, several new members of the cdk family were cloned, and some of these were shown to complex with different cyclins and to be active at discrete stages of the cell cycle. PITALRE, a new member of this family, was cloned by our laboratory and was shown to be able to phosphorylate pRb protein in vitro. In the current work, we found that PITALRE kinase activity phosphorylated pRb at sites similar to those phosphorylated by the CDC2 kinase, which itself is known to mimic, in vitro, the in vivo phosphorylation of pRb. Phosphorylation of pRb by the PITALRE-associated kinase activity was on Ser residues exclusively. Moreover, we investigated the expression pattern of PITALRE in normal human tissues, using immunohistochemical techniques so as to gain additional data on the characteristics of this new cdk family member. The protein was widely expressed, although a different tissue distribution and/or level of expression was found in various organs. Some specialized tissues such as blood, lymphoid tissue, ovarian cells, and the endocrine portion of the pancreas showed a high expression level of PITALRE. The specific expression pattern found suggests that PITALRE may be involved in specialized functions in certain cell types.

Antibodies↗
PubMed1997

Angiotensinogen gene variation in a population case-control study of preeclampsia/eclampsia in Australians and Chinese.

Preeclampsia/eclampsia (PE/E) is a common disease of human pregnancy with a strong genetic component. The etiology of PE/E is unknown. Two recent reports indicated that the angiotensinogen gene (AGT) could be involved in susceptibility to PE/E. We performed a population-based case-control study in Australian and Chinese populations to investigate whether AGT is a good candidate gene for PE/E. A microsatellite polymorphism within AGT was typed as well as a molecular variant T235 (Met-->Thr) of AGT using allele-specific PCR and allele-induced restriction site PCR. The allele distributions of the microsatellite and the variant T235 of AGT were significantly different between the two ethnic groups. However, no significant allele associations were found with disease when comparing PE/E patients and controls in Australian or Chinese populations, which is in contrast to the two earlier reports. The results suggest that the contribution of AGT to the occurrence of PE/E is small, if anything, and is not constant across populations.

Alleles↗
PubMed1997

Does a hypertonic saline load predict fluid retention in pacing induced heart failure?

OBJECTIVE: We examined the response to hypertonic saline challenge (SC) as a potential predictor of fluid retention during heart failure induced by rapid ventricular pacing. METHODS: Twelve dogs (22 +/- 4 kg) were given an intra-arterial bolus of 30 ml of 20% saline after establishing baseline fluid intake and urine output (24 h). Dogs were classified according to whether they drank more (Group A) or less (Group B) than the amount required to dilute the s.c. to isotonicity. Fluid retention was then assessed during heart failure after rapid ventricular pacing according to a graded ordinal scale and correlated with the responses to s.c.. RESULTS: No difference was noted in baseline fluid intake (1112 +/- 236 ml in Group A vs. 809 +/- 129 ml in Group B). Five hours after s.c. cumulative water intake was significantly greater in Group A than in Group B (1018 +/- 136 vs. 591 +/- 17 ml) (P < 0.01). Urine sodium concentration was 113 +/- 11 and 124 +/- 28 mmol/l at baseline in Group A and B, respectively; increased to 190 +/- 21 and 295 +/- 59 mmol/l at 5 h and remained elevated 24 h after s.c., 177 +/- 60 and 274 +/- 55 mmol/l (both P < 0.01 for within-group comparisons vs. baseline). Urine sodium concentration was less in Group A than in Group B at 5 and 24 h (P < 0.05). The fluid retention score was greater in Group A (3.6 +/- 0.5) than in Group B (0.8 +/- 0.4) (P < 0.01). Fluid retention in heart failure correlated with water intake after the pre-pacing s.c. (r = 0.68, P < 0.025) and inversely with urine concentrating ability (r = -0.58, P < 0.05). Furthermore, water intake and urine concentrating ability following the s.c. were inversely related (r = -0.67, P < 0.02). CONCLUSIONS: We conclude that normal dogs may be classified according to their fluid intake after s.c.. Those dogs that drank excessively and produced a dilute urine were more likely to retain fluid during pacing-induced heart failure. Hence, fluid intake and the ability to excrete a concentrated urine after a saline challenge may be useful variables to predict fluid retention in pacing-induced heart failure.

Animals↗
PubMed1997

Rabies virus infection of IMR-32 human neuroblastoma cells and effect of neurochemical and other agents.

IMR-32 human neuroblastoma cells are a continuous nerve cell line expressing neuronal nicotinic acetylcholine receptors. These cells were found to be susceptible to infection by rabies virus (CVS strain). After infection, viral antigen accumulated in the cell body in puncta and larger masses and spread out into the processes until at 3-4 days the entire cell was filled with antigen and lysed. A variety of chemical agents including cholinergic agonists and antagonists were tested for ability to inhibit infection of IMR-32 cells in a fluorescent focus assay. Agents found to inhibit infection were antibodies against the viral glycoprotein, gangliosides, a synthetic peptide of the neurotoxin-binding site of Torpedo acetylcholine receptor alpha1 subunit, alpha-bungarotoxin, and lysosomotropic agents. All other agents tested including other cholinergic ligands and synthetic peptides were not effective. Except for lysosomotropic agents, the agents which inhibited infection also inhibited attachment of virus to the cell surface. These results indicate that IMR-32 cells are a useful model in studying the interaction of a neurotropic virus with human neurons. The ability of alpha-bungarotoxin to inhibit infection suggests that neuronal alpha-bungarotoxin-binding receptors might serve as central nervous system receptors for rabies virus.

Antigens, Viral↗
PubMed1997

Transforming region of 243R E1A contains two overlapping but distinct transactivation domains.

Conserved regions 1 and 2 as well as the amino terminus of E1A are required for the transforming activity of the E1A oncoprotein. We show here that the amino terminus of 243R E1A has transactivation activity when brought to a promoter in yeast. Recruitment to a specific promoter is essential. Mutagenesis studies correlated the transactivation function with the extreme amino terminus and the conserved region 1 of E1A. Cotransfection assays in rodent cells confirmed that two overlapping but distinguishable domains, amino acids 1-65 and 37-80, can transactivate independently when targeted to a promoter. We also observed that when recruited to the proliferating cell nuclear antigen (PCNA) promoter, the amino-terminal region was sufficient to transactivate the PCNA promoter. On the other hand, deletion of the amino terminus of E1A resulted in failure to induce PCNA expression. Fusion of VP16 with the amino-terminal-deleted E1A mutant was able to restore the ability to induce the PCNA promoter. We further show that the amino-terminal region also is required for 243R E1A to repress the transactivation mediated by a universal transactivator DBD.VP16 and DBD.E1A. This repression could be specifically relieved by overexpression of TBP but not TFIIB. In addition, we show that the amino terminus of E1A is involved in in vitro interaction with the TATA binding protein (TBP). Thus the amino-terminal transforming region of E1A may regulate cellular gene expression in species that are distant in evolution via a common mechanism, functionally targeting TBP.

3T3 Cells↗
PubMed1997

Spontaneous spinal subarachnoid hematoma of unknown pathogenesis: case reports.

OBJECTIVE AND IMPORTANCE: The occurrence of spontaneous spinal subarachnoid hematoma of unknown pathogenesis is extremely rare. In the cases reported to date, the hematoma, located dorsal to the spinal cord (dorsal type), has caused paraplegia and has required emergency surgical intervention. CLINICAL PRESENTATION: We examined two patients who sustained spontaneous spinal subarachnoid hematoma. Both experienced sudden back pain, but there were no symptoms of spinal cord compression. Magnetic resonance imaging revealed spinal subarachnoid hematoma located ventral to the spinal cord (ventral type). INTERVENTION: Both patients were treated conservatively, and follow-up examinations have revealed that they have remained neurologically normal for 7 years and 6 months, respectively. CONCLUSION: We postulate that there are two types of spontaneous spinal subarachnoid hematoma of unknown pathogenesis (ventral and dorsal types), each of which presents a distinct clinical picture and prognosis. Ventral type hematoma may be one of the causes of acute back pain, and because of its benign prognosis, surgical treatment may not be necessary.

Adult↗
PubMed1997

Human TAP1 polymorphisms detected by denaturing gradient gel electrophoresis.

Presentation of endogenous peptides by major histocompatibility complex class I (MHC) molecules is controlled, in part, by the Tap1 and Tap2 genes in the MHC class II region that encode a heterodimeric peptide transporter. Polymorphisms of human Tap1 in normal individuals have now been investigated systematically by denaturing gradient gel electrophoresis (DGGE) analysis of fragments of genomic DNA generated by the polymerase chain reaction. Polymorphisms identified by distinctive DGGE band patterns were confirmed by DNA sequencing. In addition to four previously described polymorphisms in the open reading frame, DGGE detected three new polymorphisms: a G-->T substitution in the promoter region, a 10-bp insert in intron 9, and a G-->T substitution 80-bp downstream of the translation termination codon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Screening of the TAP1 gene by denaturing gradient gel electrophoresis in insulin-dependent diabetes mellitus: detection and comparison of new polymorphisms between patients and controls.

New protective or disease-associated polymorphisms in the TAP1 gene were sought in insulin-dependent diabetes mellitus (IDDM) patients with the use of denaturing gradient gel electrophoresis (DGGE) screening of genomic DNA. The TAP1 gene is located in the human leukocyte antigen (HLA) class II region of the genome and encodes components of a peptide transporter essential for antigen presentation by HLA class I molecules. Fragments of TAP1 corresponding to the 5' promoter, each of the 11 exons (with portions of adjacent intronic regions) and the 3' flanking region were amplified by the polymerase chain reaction and then subjected to DGGE. DNA fragments of TAP1 yielded DGGE bands with patterns whose frequencies differed between IDDM patients and controls. Specific DGGE band patterns with fragments corresponding to the promoter, exons or introns 3, 6, 7, 8, 9 or 10 of TAP1 were detected exclusively in either patients or controls. Sequencing of TAP1 fragments encompassing exon 7 gave rise to a DGGE band pattern exclusively observed in an IDDM patient and sequencing revealed a previously unidentified polymorphisms at codon 518 (GTC-->ATC, Val-->Ile). Another unique polymorphism uncovered by DGGE revealed by sequencing a polymorphism in intron 2 in a diabetic patient. The genotypes of additional HLA class II matched patients and controls were determined with regard to five exonic and one intronic TAP1 polymorphism. A 10 base pair intronic insertion in intron 9 was exclusively identified in controls and missing from patients (P = 0.017). Further large population-based studies may reveal whether these newly identified at risk or protective TAP1 variants confer markers of statistical risk in diverse population groups.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Singlet oxygen-mediated inactivation of acetylcholinesterase: a comparison of purified enzyme in solution and enzyme bound to K562 leukemia cells.

We have compared the singlet oxygen-mediated inactivation of acetylcholinesterase (ACE) in solution with the inactivation of ACE on the surface of K562 leukemia cells. In solution, the actions of the singlet-oxygen quenchers, methionine, azide, disodium [N,N'-ethylenebis (5-sulfosalicylideneimminato)]nickelate(II) (Ni-chelate 1) and disodium [(N,N'-2,3-propionic acid)bis(5-sulfosal-icylideneimminato)] nickelate(II) (Ni-chelate 2) could be explained quantitatively by assuming their only mechanism of action was to quench singlet oxygen. The singlet oxygen quenchers, azide, Ni-chelate 1 and Ni-chelate 2, caused smaller inhibitions in the rate of singlet oxygen-mediated inactivation of ACE on K562 cells than ACE in solution. The effects of these quenchers and of deuterium oxide were interpreted using a mathematical model of singlet-oxygen quenching and diffusion to estimate the lifetime of singlet oxygen near the cell surface. The azide quenching data and the deuterium-oxide data gave lifetimes of 0.9 +/- 0.2 microsecond and 0.45 +/- 0.15 microsecond, respectively. The increases in ACE inactivation lifetime caused by the nickel chelates were anomalously large. The unexpectedly large quenching due to the nickel chelates may have been due to a nonuniform distribution of the chelates in the cytoplasm with a large concentration of the chelate near the cell membrane.

Acetylcholinesterase↗
PubMed1997

Adrenocorticotropin response and nicotine-induced norepinephrine secretion in the rat paraventricular nucleus are mediated through brainstem receptors.

Nicotine is a potent stimulus for the secretion of ACTH, and norepinephrinergic neurons originating in the brainstem are involved. Prior reports using in vivo microdialysis in alert rats have shown that nicotine, administered i.p. or into the fourth ventricle, stimulated the release of norepinephrine (NE) into the hypothalamic paraventricular nucleus (PVN), the site of neurons containing CRH. In the present studies, rats received an i.v. infusion of nicotine into the jugular vein on alternate days during their active (dark) phase; therefore, direct correlations between the levels of NE microdialyzed from the PVN and plasma ACTH could be made in each animal. Nicotine administered i.v. (0.045-0.135 mg/kg) elicited dose-dependent increases in both NE and ACTH (P < 0.01). A significant correlation was found between nicotine-stimulated NE release in the PVN and ACTH secretion (r = 0.91, P < 0.01). To address whether the site(s) of action of nicotine was on presynaptic receptors on NE terminals in the PVN or on receptors on neurons in brainstem regions accessible from the fourth ventricle, the nicotinic cholinergic antagonist, mecamylamine (0.1-4.8 microg), was microinjected directly into the PVN or into the fourth ventricle before nicotine infusion. Fourth-ventricular administration of mecamylamine (1.6 microg) or higher, before i.v. nicotine (0.09 mg/kg), completely blocked both NE release in the PVN (IC50 = 0.64 microg) and ACTH secretion (IC50 = 0.40 microg) (P < 0.01, compared with vehicle before nicotine), whereas it was ineffective when injected directly into the PVN. The results demonstrate that the nicotinic cholinergic receptors in the brainstem, rather than presynaptic receptors within the PVN itself, mediate nicotine-stimulated PVN NE release and ACTH secretion.

Adrenocorticotropic Hormone↗
PubMed1997

New monoclonal antibody diagnostic reagents for type I diabetes: differential lymphocyte surface antigen expression related to disease.

New cellular-based reagents are needed to diagnose type I diabetes as well as to monitor the outcomes of clinical trials at early time points. Four new monoclonal antibodies (mAbs) have been shown to demonstrate reduced binding to lymphocytes from identical twins with long-term type I diabetes relative to that observed with lymphocytes from their twin partners without diabetes or from control subjects. Biochemical analysis revealed mAb 3G12EG recognized an unidentified 45-kDa protein, whereas mAb 2E8F1 and 5B6E11 did not appear to precipitate specific proteins as detected by SDS-PAGE. Electrophoresis under reducing and nonreducing conditions and peptide mapping revealed that mAb 8F410 recognizes a novel dimeric form of HLA class I molecule. Predictions from crystallography studies suggested previously this class I dimer as the optimal activation of a single CD8 T-cell. In B-cells from both normal and diabetic individuals, the class I dimer was minimally associated with beta2-microglobulin rapidly formed in the endoplasmic reticulum. These new reagents appear to be able to identify new lymphocyte surface phenotypes associated with diabetes expression in both fresh blood samples and Epstein-Barr virus-established cell lines.

Adult↗
PubMed1997

Novel methodology for echocardiographic quantification of cardiac shape.

BACKGROUND: Left ventricular (LV) shape is a major determinant of cardiac physiology with important prognostic implications. Current techniques are limited in their ability to characterize shape. Regional curvature analysis is a recently developed method that is free of idealized shape assumptions and is applicable globally or regionally. OBJECTIVE: To measure the change in regional curvature from diastole to systole to determine whether modifications of this methodology can be applied to study LV shape noninvasively, and to compare this method with other shape analysis methods. ANIMALS: Seventeen normal dogs underwent transthoracic echocardiography. LV dimensions were measured at end-diastole and end-systole. LV endocardial outlines were traced from apical four-chamber views. LV short to long axis (D/L) ratios and eccentricity index (E) were calculated. Regional curvatures were determined using customized software. Reproducibility of regional curvature was also determined. MAIN RESULTS: LV regional curvature values were consistent among animals at diastole and systole in all areas. Regional curvature showed divergence in different areas. D/L ratios showed a 7% decrease from diastole to systole, whereas E demonstrated a 10% increase. However, regional curvature exhibited a 39% change, suggesting that this methodology is more sensitive. Duplicate curvature values from same or different cardiac cycles were highly reproducible. CONCLUSIONS: Quantitative regional curvature analysis can be applied with echocardiography to study LV shape. This methodology is more sensitive than current methods, is reproducible and may be applicable to the noninvasive study of shape changes in cardiac disease.

Analysis of Variance↗
PubMed1997

[The relationship between insulin resistance and abnormalities of cellular calcium metabolism and cell membrane abnormalities in patients with essential hypertension].

OBJECTIVE: To investigate the relationship between the insulin resistance (IR) and the abnormalities of cellular calcium metabolism and cell membrane in patients with essential hypertension. METHODS: Plasma insulin and glucose before and during OGTT, membrane ATPase activity, cholesterol (C) and phospholipid (P), lipid peroxides (MDA), intracellular total calcium concentration [(Ca2+)i] and calcium binding capacity of inner surface of membranes were determined in 26 normotensive (NT) subjects, 17 hypertensives (HT), and 16 hypertensives associated with IR (HT + IR). RESULTS: The HT + IR group had higher levels of plasma glucose and insulin than that in the NT group and HT group with significant differences. The HT + IR group consistently demonstrated depressed activity of each Na(+)-K(+)-ATPase and Ca(2+)-ATPase studied with significant differences when compared with the NT group, but no difference was found if compared with the HT group. There was a higher [(Ca2+)i] in the HT + IR group than those in the NT group and even in the HT group with significant differences, respectively. Insulin sensitivity in the HT + IR group was significantly lower than that in both groups of HT and NT, respectively. The membrane C/P molar ratio and MDA content in the HT + IR group and HT group were significantly higher than those in the NT group, respectively. CONCLUSIONS: It is likely that there is a causal relation between IR and abnormalities of cellular calcium metabolism and membrane lipids in patients with essential hypertension.

Aged↗
PubMed1997

Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer.

Two proteins, p107 and pRb2/p130, which are structurally and functionally similar to the product of the retinoblastoma gene (pRb), were cloned by taking advantage of their ability to bind transforming proteins of DNA tumor viruses through a particular region called the "pocket domain." Like pRb, both proteins play a fundamental role in growth control. Using immunocytochemical techniques, we examined a variety of normal human tissues for the expression of pRb2/p130 and p107. Both proteins were expressed ubiquitously, although a different tissue distribution and/or level of expression were found in various organs. Terminally differentiated cells, such as neurons and skeletal muscle, showed high expression levels for Rb2/p130, whereas p107 was expressed at higher levels in other cell types such as epithelia of the breast and prostate. We then examined the expression pattern of Rb2/p130 in 158 specimens of human lung cancer and found an inverse correlation between the histological grading of the tumors, the development of metastasis, and the level of expression of Rb2/p130.

Blood Cells↗
PubMed1997

[Artificial biological skin for preventing adhesion of repaired tendon in white rats].

To study the effect of tendon healing and adhesion after tendon repaired using the artificial biological skin (Biofill) produced in Brazil, we divided randomly 46 rats [correction of mice] into groups, compared the pelvic limbs with themselves, observed by eyes and light microscopy, and made biomechanical and histological detection in 1st, 2nd, 3rd and 4th weeks postsurgically. The results showed that the speed and strength of tendon healing had light affect in three weeks after operation, and had no difference between experimental groups and control groups after 4 weeks. It is indicated that artificial skin "Biofill" can effectively prevent tendon adhesion after tendon repaired and have not obviously influenced in the intrinsic self-healing process of tendon.

Achilles Tendon↗
PubMed1996

Dynamics and equilibrium behavior of cytonuclear disequilibria under genetic drift, mutation, and migration.

A simple two-locus drift model for cytonuclear systems is developed, in which the stochastic dynamics of cytonuclear genotypic frequencies are specified. Random union of zygotes is assumed. Trajectories for the first two moments of both genotypic and allelic disequilibria are given under three scenarios: (i) random drift alone; (ii) random drift with mutation; and (iii) random drift with migration. Steady state solutions for the cytonuclear disequilibria are reported. The utility of this simple two-locus drift model in testing the neutrality of mitochondrial DNA markers in artificial hybrid zones is briefly illustrated

DNA, Mitochondrial↗