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Biomedical subjects

T Wei

Publications and source records attributed to T Wei.

At least 73 records · Page 4Linked to original sources
PubMed1997

Identification of the major casein kinase I phosphorylation sites on erythrocyte band 3.

Human erythrocyte band 3 is a major substrate of two red blood cell protein kinases, casein kinase I and p72syk protein tyrosine kinase. Although the phosphorylation sites and physiologic consequences of p72syk phosphorylation have been characterized, little is known regarding casein kinase I phosphorylation. In this report, we identify the major phosphorylation site of casein kinase I. Using isolated components, casein kinase I was found to phosphorylate the cytoplasmic domain of band 3 (CDB3), primarily on Thr residues. Classical peptide mapping narrowed the major phosphorylation site to a peptide encompassing residues 24-91. Computer-assisted evaluation of this sequence not only showed two consensus casein kinase I phosphorylation sites, but also provided information on how to proteolytically separate and isolate the candidate sites. Following the suggested protocols, a heptapeptide containing the major phosphorylation site was isolated, subjected to amino acid sequencing, and found to be phosphorylated on Thr 42. A minor phosphorylation site was similarly identified as Ser 303. Because Thr 42 is situated near the binding sites on CDB3 of ankyrin, protein 4.1, protein 4.2, and the glycolytic enzymes, phosphorylation of CDB3 by casein kinase I could conceivably impact erythrocyte structure and/or function.

Amino Acid Sequence↗
PubMed1997

Resistance of actin to cleavage during apoptosis.

A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1beta-converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

Actins↗
PubMed1997

Probing the stability of class I major histocompatibility complex (MHC) molecules on the surface of human cells.

We used binding of a fluorescent adduct of beta2-microglobulin, fluorescein beta2m, to probe the stability of class I HLA molecules on the surface of human cells. The weight of the literature suggests that this ligand binds to heavy chains that have lost beta2m and possibly peptide as well. Hence Fl-beta2m reports on the stability of the class I HLA trimer. A small fraction of HLA molecules, approximately 5%, binds Fl-beta2m on both resting and activated T cells. A larger fraction of all HLA molecules binds Fl-beta2m in FO-1 cells, beta2m-deficient cells, transfected with various B2m genes. HLA molecules of FO-1 cells are more stable when expressed with human beta2m, than when expressed with mouse beta2m. The non-covalent association of HLA heavy chains, beta2m and peptide implies that eventually every molecule of HLA trimer ought to dissociate and bind Fl-beta2m. In fact, the extent of exchange is limited by the lifetime of a given molecule at the cell surface. beta2m exchange decreases as cell concentration increases, suggesting that some density-dependent process acts to enhance degradation or denaturation of beta2m-free HLA heavy chains.

Animals↗
PubMed1997

Stimulation of liver regeneration and function after partial hepatectomy in cirrhotic rats by continuous infusion of recombinant human hepatocyte growth factor.

BACKGROUND/AIMS: Radical resection is accepted as one of the most curative treatments for hepatocellular carcinoma. However, most patients have coexisting cirrhosis and their liver function is usually impaired. It is therefore important to stimulate the regeneration and function of the remnant cirrhotic liver after hepatectomy. Hepatocyte growth factor is a potent mitogen that has been suggested to play a crucial role in liver regeneration. METHODS: In this study, we performed 45% hepatectomy in rats with cirrhosis induced by thioacetamide, and administered recombinant human hepatocyte growth factor (rhHGF) with dextran sulfate by continuous infusion into the jugular vein with an infusion pump. RESULTS: rhHGF stimulated an increase in the wet weight of the remnant liver compared with untreated control rats. The proliferating cell nuclear antigen labeling index showed that this increase resulted from the stimulation of DNA synthesis. Serum levels of liver enzymes increased after hepatectomy, but returned to the prehepatectomy level more rapidly in rhHGF-treated rats than in controls. rhHGF increased hepatic protein synthesis above prehepatectomy levels and also markedly increased the serum levels of hepatic lipid metabolites. CONCLUSIONS: These results demonstrate that continuous intravenous infusion of rhHGF enhances the growth and function of the remnant liver in rats with cirrhosis after partial hepatectomy. Therefore, rhHGF may be useful after hepatic resection in patients with cirrhosis.

Animals↗
PubMed1997

Activation of cyclin A-dependent kinases associated with WAF1 degradation during radiation-induced apoptosis.

We describe here the further characterisation of the radiation response of a pair of isogenic Burkitt's lymphoma cell lines which differ significantly in their susceptibility to radiation-induced apoptosis. In both cases a marked inhibition of cyclin A-dependent kinase activity was observed at 4 h post-irradiation which recovered to normal levels in the susceptible line by 12 h but remained inhibited in the resistant cell line. Under these conditions the cellular abundance of p58cyclinA and p33cdk2 did not significantly change in the two cell types and there was no evidence for phosphorylation changes in p33cdk2 which might account for the activity differences. In parallel with the changes in activity, p21WAF1 increased initially in both cell lines, declined in the sensitive cell line as the activity recovered but remained high in the resistant cell line. This appears to be explained by a more rapid turn-over of p21WAF1 in the sensitive cell line and an increased association of p21WAF1 with cyclin kinase as determined by immunoprecipitation. These results implicate p21WAF1 in the regulation of cyclin-dependent kinases during radiation-induced apoptosis, with persistence of induced p21WAF1 being associated with a more resistant phenotype.

Journal Article↗
PubMed1997

The neuroendocrine axis in patients with multiple sclerosis.

We investigated the basal and dynamic regulation of the hypothalamo-pituitary-adrenal (HPA), hypothalamo-pituitary-thyroid (HPT) and hypothalamo-pituitary-gonadal axes and prolactin secretion in 52 patients with clinically definite multiple sclerosis. These patients also had gadolinium enhanced brain MRI scans and were divided into relapsing-remitting, secondary progressive and primary progressive subgroups. These subgroups were compared with healthy controls and a group of patients with other neurological diseases. The cortisol diurnal rhythm was preserved in all groups of patients. The time-integrated cortisol response to human corticotropin-releasing hormone (CRH) stimulation was lower in the patients with secondary progressive multiple sclerosis, compared with patients with primary progressive multiple sclerosis and healthy subjects. The time-integrated beta-endorphin response to CRH was greater in the patients with relapsing-remitting multiple sclerosis compared with the others. Feedback regulation assessed by dexamethasone suppression was normal. Serum testosterone was low in 24% of male multiple sclerosis patients and oestradiol was low in 25% of pre-menopausal female multiple sclerosis patients, whereas prolactin and the HPT function were normal. Correlations with C-reactive protein (CRP) and MRI suggest that activation of the HPA axis in multiple sclerosis patients is secondary to an active inflammatory stimulus.

Adult↗
PubMed1997

Mitogenic response and steroid sensitivity in MS lymphocytes.

OBJECTIVES: We investigated mitogenic response and steroid sensitivity in multiple sclerosis (MS) lymphocytes to establish if MS lymphocytes were less steroid responsive. MATERIAL AND METHODS: We compared mitogenic response to phytohaemagglutinin (PHA) and inhibition by dexamethasone (DEX) in circulating lymphocytes from both MS patients and healthy subjects. RESULTS: We found a range of responses in each group but no significant differences between the two groups, nor in patients with and without enhancement on magnetic resonance imaging. The mid-inhibitory concentration of DEX in response to 1 microg/ml PHA was significantly lower than that for 2.5 microg/ml PHA in the patients. The mid-inhibitory concentrations of DEX in response to 2.5 microg/ml PHA negatively correlated with endogenous serum cortisol concentrations. CONCLUSION: These data imply a spectrum of glucocorticoid response that is similar in normal and MS lymphocytes and can partly explain why response to steroid therapy is variable.

Adult↗
PubMed1996

Interaction of the IciA protein with AT-rich regions in plasmid replication origins.

A set of AT-rich repeats is a common motif in prokaryotic replication origins. We have screened for proteins binding to the AT-rich repeat region in plasmids F, R1 and pSC101 using an electrophoretic mobility shift assay with PCR-amplified DNA fragments from the origins. The IciA protein, which is known to bind to the AT-rich repeat region in the Escherichia coli origin of chromosome replication, oriC, was found to bind to the corresponding region from plasmids F (oriS) and R1, but not to pSC101. DNase I footprint analysis showed that IciA interacted with the AT-rich region in both F and R1. When the IciA gene was deleted, the copy number of plasmid F increased somewhat, whereas there was no major effect on the replication of pSC101 and R1, or on the E. coli chromosome.

Bacterial Proteins↗
PubMed1996

Structure and characterization of a putative drought-inducible H1 histone gene.

A drought- and abscisic acid (ABA)-inducible gene, His1, was isolated from Lycopersicon pennellii, a drought-resistant relative of cultivated tomato, and the gene structure was defined experimentally. The nucleotide sequence of His1 predicts a protein of 202 amino acid residues, with a significant sequence homology to plant H1 histones. Consensus sequences for both H1 histone-specific promoter elements as well as an ABA-responsive element were identified in the 5'-flanking region of His1. Transcripts of this gene accumulate in leaf tissue in response to drought in three tomato species including cultivated tomato (L. esculentum), L. pennellii, and L. chilense, as well as in tobacco. Transcripts for His1 are constitutively expressed in roots; transcript abundances in tomato root tips were equivalent to transcript abundances in more mature regions of the seedling root. The accumulation in leaves of transcripts for His1 preceded visible symptoms of drought stress in the plants. Transcript accumulation was detected in both drought-sensitive and drought-resistant species at similar leaf water potentials, psi W -1.3 to -1.4 MPa.

Abscisic Acid↗
PubMed1996

Reduced IL-10 secretion by CD4+ T lymphocytes expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR).

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non-transformed, antigen-responsive CD4+ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the delta F508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca(2+)-mediated Cl- current; however, TCC from patients with CF but not controls displayed defective cAMP-mediated Cl-current. Although CF-derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted approximately 45% less IL-10 compared with control TCC after activation with concanavalin A (Con A) (624 +/- 101 versus 1564 +/- 401 pg/ml per 10(6) cells, respectively; P = 0.04) or anti-CD3/phorbol ester (5148 +/- 1634 versus 11788 +/- 2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon-gamma, IL-2, and IL-4 was comparable in CF and control TCC after both forms of activation, while IL-5 was reduced in CF TCC following anti-CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL-10 secretion after polyclonal activation. It is possible that disruption of IL-10-mediated anti-inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways.

Animals↗
PubMed1995

Luminescence quenching by long range electron transfer: a probe of protein clustering and conformation at the cell surface.

Quenching of luminescence from fluorescent and phosphorescent probes by nitroxide spin labels with a long range electron transfer (LRET) mechanism (44,45) has been tested as a tool to monitor association/clustering and conformational changes of cell surface proteins. The membrane proteins were labeled with monoclonal antibodies or Fab fragments conjugated with luminescent probes or water-soluble nitroxide spin labels. The method was tested as a probe of 3 different aspects of protein-protein association involving class I MHC molecules: (1) interaction between the heavy and light chains of the MHC molecules, (2) clustering, self-association of MHC molecules, (3) proximity of MHC molecules to transferrin receptors of fibroblasts or surface immunoglobulin molecules of B lymphoblasts. The extent of quenching upon increasing the fractional density of the quencher was sensitive for protein association in accordance with earlier immunoprecipitation and flow cytometric Förster-type energy transfer (FCET) data obtained on the same cells. These data suggest that the LRET quenching can be used as intra- or intermolecular ruler in a 0.5-2.5 nm distance range. This approach is simpler (measurements only on donor side) and faster than many other experimental techniques in screening physical association or conformational changes of membrane proteins by means of spectrofluorimetry, flow cytometry, or microscope based imaging.

Antibodies, Monoclonal↗
PubMed1995

[Clinical and experimental study on composite wuzi dihuang liquor in treating male infertility].

Composite Wuzi Dihuang Liquor (CWDL) in treating male infertility was used. The results showed that the effective rate was 84%. CWDL is indicated in mild and medium idiopathic oligozoospermia. There was some effect in the male infertility which was complicated by prostatitis, varicocele, antisperm antibody positive patients, but was ineffective in severe oligozoospermia, azoospermia, tesicular volume 15ml, endocrinologic and chromatic abnormality. Experimental study revealed that the drug could significantly increase the percentage of reproductivity in mice and could directly safeguard the sperm of male infertility patients.

Adult↗
PubMed1994

Analysis of the structure of empty and peptide-loaded major histocompatibility complex molecules at the cell surface.

We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.

Animals↗
PubMed1994

Clustering of class I HLA molecules on the surfaces of activated and transformed human cells.

We surveyed cells for the clusters of class I HLA molecules, HLA-I, which we have previously found on JY lymphoblasts. Two fluorescence techniques, fluorescence resonance energy transfer and electron exchange quenching, detected clustered HLA-I molecules on activated normal B and T cells, on cells of B and T lymphoblast lines, and on transformed fibroblasts. No HLA-I clusters were detectable in the surfaces of resting B or T cells or normal fibroblasts. HLA clustering correlates perfectly with the presence of the HC-10 epitope of beta 2-microglobulin (beta 2m)-free heavy chains at the cell surface although not with the amount of this epitope expressed. Clustering was reversed by exogenous beta 2m, but this did not change the amount of HC-10 bound. This suggests that a form of beta 2m-free heavy chain in equilibrium with both native HLA molecules and fully denatured HC-10-positive heavy chains is involved in HLA-I cluster formation.

B-Lymphocytes↗
PubMed1994

Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*0201. Effect on maturation and peptide presentation.

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.

Amino Acid Sequence↗
PubMed1993

Human erythrocyte casein kinase II: characterization and phosphorylation of membrane cytoskeletal proteins.

Casein kinase II activities were purified from human erythrocyte membrane and cytosolic fractions to apparent homogeneity. The kinases isolated from the membrane and cytosolic fractions exhibited the same subunit composition and the ability to utilize ATP and GTP as phosphoryl donors. Antibodies against the alpha and alpha' subunits of human casein kinase II cross reacted with the corresponding subunits of both erythrocyte casein kinases. Spermine, spermidine, putrescine, and polylysine stimulated to varying degrees the activities of erythrocyte casein kinase II, whereas heparin inhibited the kinase activities. Both kinases were found to catalyze the phosphorylation of several erythrocyte membrane cytoskeletal proteins, including spectrin, ankyrin, adducin, protein 4.1, and protein 4.9. Unlike casein kinase I, casein kinase II did not phosphorylate band 3 appreciably. A preliminary estimate indicates that both human erythrocyte membrane and cytosolic casein kinase II catalyze the incorporation of approximately 1.2 and 3.5 moles of phosphate into each mole of spectrin and ankyrin, respectively. An analysis of the phosphopeptide maps of ankyrin indicates that both membrane and cytosolic kinases phosphorylate the same domains within ankyrin. These data, taken together, suggest that the type II casein kinases isolated from human erythrocyte membrane and cytosol are either identical or closely related and may play a role in the regulation of cytoskeletal protein interactions.

Adenosine Triphosphate↗