Biomedical subjects
Publications and source records attributed to J Wu.
12 patients with gastric eosinophilic granuloma (GEG) were diagnosed mistakenly to be stomach cancer before operations, including 3 patients with acute massive hemorrhage from upper alimentary tract and 2 patients with pyloric obstruction. Operations were given to all. No relapses were noted after operation.
The large ribosomal subunit of the thermophilic fungus Thermomyces lanuginosus was treated with 2.96 M NH4Cl to remove specific complements of ribosomal proteins, and the core particles thereby derived were imaged by bright field transmission electron microscopy, and recurring views computed by single particle electron image analysis. A new characteristic projection was elucidated which showed a large depression or channel passing through the subunit. Such a channel has been perceived in the prokaryotic large ribosomal subunit under certain conditions and has been postulated to be the exit pathway for the nascent polypeptide chain, but its existence has not hitherto been demonstrated in eukaryotes.
Shc is a ubiquitously expressed Src homology 2 (SH2) domain protein that can transform fibroblasts and differentiate PC12 cells in a Ras-dependent fashion. Shc binds a variety of tyrosine-phosphorylated growth factor receptors presumably via its carboxyl-terminal SH2 domain. We cloned a fragment of Shc when screening a bacterial expression library with tyrosine-phosphorylated epidermal growth factor (EGF) receptor. Surprisingly, this fragment encodes the amino terminus of Shc, a region that has no significant similarity to an SH2 domain. When expressed as a glutathione S-transferase fusion protein, this amino-terminal domain binds to autophosphorylated EGF receptor, as well as HER2/neu and TrkA receptors. This fragment acts like an SH2 domain in that it does not bind non-phosphorylated EGF receptor or EGF receptor with all tyrosine phosphorylation sites mutated or deleted. Our data define a novel domain in Shc that has the potential to interact with growth factor receptors and other tyrosine-phosphorylated proteins.
Protein phosphorylation is an important posttranslational modification process that plays a crucial role in signal transduction. There are many protein kinases involved in cell signaling. However, substrate motifs of many protein kinases in signal transduction are not well-known. Traditional methodologies for identifying these motifs are often difficult and inefficient. In the present study, we developed a novel approach for discovering linear substrate motifs of protein kinases. This method is based on the screening of random synthetic combinatorial peptide libraries on beads where each bead expresses only one peptide entity [Lam et al. (1991) Nature 354, 82-84]. We chose cyclic AMP-dependent protein kinase (cAPK) as a model system in the present study since it is a well-studied enzyme. Random pentapeptide and heptapeptide libraries were screened with the addition of [gamma-32P]ATP and cAPK. 32P-Labeled peptide-beads were then isolated for microsequencing. The identified peptide motif for cAPK was RRXS and is identical to that reported in the literature. Kinetic studies of the best three peptides indicate that they are efficient substrates for cAPK discovered from random synthetic combinatorial peptide libraries. Our results also suggest that this method is potentially useful for identifying substrate motifs of various protein kinases with high sensitivity and specificity. In addition, this method can also be used as a general method for identifying linear substrate motifs for various posttranslational modifications.
By using site-directed fluorescence spectroscopy, we have carried out structure/function studies on lactose permease purified from Escherichia coli in dodecyl beta, D-maltoside. Initially, permease containing a single native Cys at position 148 (helix V) was studied, since this residue is protected against alkylation by substrates of the permease. In the absence of ligand, Cys 148 permease reacts rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), a fluorophore whose quantum yield increases dramatically upon reaction with a thiol, indicating that this residue is readily accessible to the probe. Various ligands of the permease block the reaction, and the concentration dependence is commensurate with the affinity of each ligand for the permease (i.e., beta, D-galactopyranosyl 1-thio-beta, D-galactopyranoside << lactose < galactose), but neither sucrose nor glucose has any effect whatsoever. Thus, the permease retains the ability to bind ligand specifically when the molecule is in dodecyl beta, D-maltoside. Permease containing single Cys substitutions in the vicinity of Cys 148 was also studied. Interestingly, labeling of Cys 145 which is presumed to be one helical turn removed from Cys 148 exhibits properties similar to those observed with Cys 148 permease, but the effects of ligand are far less dramatic. On the other hand, permease with a single Cys residue at position 146 or 147 behaves in a completely different manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Growth factors or serum can induce transcription and translation of a dual specificity MAP (mitogen-activated protein) kinase phosphatase, MKP-1 (MAP kinase phosphatase-1). The role of induction of MKP-1 (formerly 3CH134) in the rapid phase of MAP kinase deactivation was studied in rat pheochromocytoma (PC12) cells. MAP kinase was nearly completely deactivated in PC12 cells by 10 min after stimulation with epidermal growth factor (EGF) whereas MAP kinase activity remained elevated at 30% of the maximal response after stimulation with nerve growth factor. Protocols for treating cells with actinomycin D and cycloheximide were established that eliminate detection of MKP-1 mRNA and protein in PC 12 cells. Treatment of PC12 cells with actinomycin D and cycloheximide did not affect the rapid deactivation of MAP kinase. Thus, the rapid phase of MAP kinase deactivation in PC12 cells is not dependent on the induction of the MAP kinase phosphatase MKP-1.
Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.
This study was undertaken to establish a correlation between prothrombin activation fragment F 1 + 2 and one-stage prothrombin time (PT) ratios in patients receiving oral anticoagulant therapy. One hundred consecutive patients on warfarin treatment were utilized for this study. The patients had received warfarin for not less than four days prior to entry into the study. F 1 + 2 levels and PT ratios were found to be 0.28 +/- 0.24 nM/L (mean +/- SD) ranging from 0.01 to 1.5 nM/L and 1.62 +/- 0.46 (mean +/- SD) ranging from 0.97 to 3.11, respectively. Most patients on oral anticoagulants with PT ratios between 1.2 - 1.7 exhibited decreased concentrations of F 1 + 2. Normal control values of F 1 + 2 were established for this study in 40 healthy individuals; they were 0.40 +/- 0.23 nM/L (median +/- SD) ranging from 0.11 to 1.19 nM/L. Mean plasma levels of F 1 + 2 were significantly lower in the anticoagulated patients as compared to the healthy controls (t = 2.377, p < 0.05). The relationship between F 1 + 2 levels and PT ratios in the 100 anticoagulated patients was analyzed by linear regression. No significant correlation (r = -0.208) was found between F 1 + 2 levels and PT ratios. It is concluded that the degree of reduction in F 1 + 2 levels is not proportional to the intensity of therapy reflected by the PT ratios in anticoagulated patients.
The hepatitis B virus X gene encodes a transcriptional trans-activator that appears essential for viral replication in the infected liver. It has been shown by others that at least two X protein products, initiating at different in-frame ATG codons within the X open reading frame, are necessary for full trans-activating function. Yet, previous studies have demonstrated only a full-length X gene transcript that extends 5' of the first ATG codon in the X open reading frame. In this communication, we present evidence for the existence of shorter X gene transcripts, whose heterogeneous 5' ends straddle the second ATG codon of the X open reading frame. These transcripts are not degradation products of the full-length X gene transcripts but rather arise from an independent promoter. Furthermore, immunofluorescence and functional analyses reveal that active X proteins are translated from these transcripts. Therefore, there appears to be a previously undescribed promoter embedded within the X open reading frame that can give rise to functionally important transcripts.
The transcription of the ars operon is negatively controlled by the ArsR repressor and induced by arsenite and antimonite. Using hydroxylamine mutagenesis, four arsR mutants were isolated; three were selected for inability to respond to inducers: C32Y, C32F, and C34Y. Each of the three altered proteins still bound specifically to the ars operator, but inducers were less effective in effecting the release of the altered proteins from the DNA. Each of the mutant arsR genes in trans with a reporter gene controlled by the ars promoter repressed expression of the reporter gene, and addition of inducer did not relieve repression. These results suggest that the altered ArsR proteins are defective in the inducer binding site. The fourth arsR mutation, resulting in a H50Y alteration, produced constitutive expression. His-50 is located within a putative helix-turn-helix region of the ArsR protein. We propose that cysteines Cys-32 and Cys-34 in the ArsR protein comprise part of a metal binding motif found in members of the ArsR family of metalloregulatory proteins.
Recently, there has been renewed interest in propyl gallate, a preservative in foods and fuels. This compound, which exhibits antimicrobial activity, has been found to be toxicologically safe after almost 30 years of evaluation. In the present study, we examined whether propyl gallate is a hepatoprotective antioxidant, and investigated some of its bases of action vis-à-vis Trolox, a vitamin E analogue. In isolated rat hepatocytes, propyl gallate prolonged substantially cell survival against oxyradicals generated with xanthine oxidase-hypoxanthine. The protection was dose dependent and excelled that of Trolox, mannitol, or ascorbate, each at or near its optimum level in the same system. In rats undergoing an 80-min partial hepatic ischemia, infusion of propyl gallate at 20 mumol/kg body weight just before a 24-hr reperfusion salvaged the organ by 80.0 +/- 11.5%, an extent comparable to that with Trolox. Mechanistically, we found that propyl gallate (a) protected hepatocytes against the cascade of oxyradicals produced by xanthine oxidase-hypoxanthine; (b) protected hepatocytes against superoxide radicals generated specifically by menadione; (c) protected the functionally important hepatic vascular endothelial cells more effectively than Trolox against xanthine oxidase-hypoxanthine, and (d) approximately halved the amount of lipid conjugated dienes (a more specific marker of oxyradical damage than malondialdehyde) formed in tissues after oxidant damage. Therefore, there are fundamental reasons why propyl gallate is an effective antioxidant-based hepatoprotector, both in vitro and in vivo.
A capillary isoelectric focusing instrument with an on-line optical absorption imaging detector was used to analyse protein samples. The separation column was a 4 cm x 100 microns I.D. capillary. The light source of the imaging detector was a argon ion laser. The light beam from the laser was focused into the capillary by a cylindrical lens. An 1024-pixel charge-coupled device (CCD) measured intensity of light beam passing through the capillary. The optical alignment of the detector was optimized to eliminate interference produced by the refractive index gradient created by sample zones inside the capillary. The signal-to-noise ratio of the detector was enhanced by averaging 30 scans of the CCD every 3 s. The on-line imaging detector allows simultaneous separation and detection so that the analysis time for a sample is only 2-4 min. Several protein samples were analyzed by the instrument, including human hemoglobin variants, cytochrome c, myoglobin and transferrin.
Capillary isoelectric focusing with universal concentration gradient imaging detection was used to separate and detect tryptic peptides from bovine and chicken cytochrome c. For a desalted sample of peptide angiotensin 2, the isoelectric point (pI) measured by the instrument agreed well with the pI calculated from amino acid pK values. For the cytochrome digests, correlations between measured and calculated pI values were imprecise because peak positions shifted slightly from test to test. This problem is thought to be caused by the inefficient desalting process used on the samples, leaving salt residues which caused distortion in the pH gradient during the focusing process. However, this system differentiated between the two cytochrome c's. The concentration gradient imaging detected peptides which contain no tyrosine and no tryptophan amino acids, which a UV absorption detector operating at 280 nm could not. The separation and detection steps took only 5-7 min because no mobilization was necessary after the focusing process.
CD36 has been implicated in several intracellular signalling events, including platelet and monocyte activation, and receptor-mediated internalization of bound ligands such as oxidized low-density lipoprotein and apoptotic neutrophils. These processes are presumably mediated by the intracytoplasmic domain(s) of the molecule. By analysis of hydrophobicity plots and by analogy to rat LIMPII, which has a 60% homology to CD36, a two-transmembrane domain model has been proposed. To characterize the structure-function relationships of CD36 involved in transducing the signal, we have defined the number of transmembrane and intracellular domains experimentally using a mutagenesis approach. A truncated CD36 cDNA was constructed that encodes a protein that terminates just proximal to the putative C-terminal transmembrane domain. This mutant was cloned into eukaryotic expression plasmid vectors to generate short-term and stable transfected cells. Our results indicate that the truncated mutant is secreted by the transfectants into the postculture medium, indicating that there is only one transmembrane domain in CD36, which is present at the C-terminal end. The soluble secreted protein from all of these cells is functional as indicated by its binding to thrombospondin.
The electrophysiological properties of sevoflurane (Sev)-induced current (ISev) were investigated in CA1 pyramidal neurons freshly dissociated from the rat hippocampus by using the nystatin perforated patch recording configuration under voltage-clamp condition. Within the range of Sev concentrations from 3 x 10(-4) to 2 x 10(-3) M, ISev was an inward current which consisted of an initial transient peak component and a successive steady-state plateau component. The peak current component increased in a concentration-dependent manner with a conductance increase. The application of Sev over 2 x 10(-3) M, however, suppressed the peak and steady-state current components with a concomitant decrease in conductance and elicited a transient inward current ('hump' current) immediately after wash out. The current-voltage relationship for ISev showed some outward rectification suggesting a slight voltage-dependency of the ISev. The reversal potential of ISev (ESev) was close to the ECl and shifted by 52 mV for a 10-fold change in extracellular Cl- concentrations, indicating that ISev is passing through Cl- channels. The single channel conductance obtained from the analysis of the variance of ISev fluctuations was 15.3 +/- 1.3 pS.
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Cadmium metallothionein (CdMT) was injected subcutaneously into obese hyperglycaemic Umeå ob/ob mice or their lean litter mates (normal mice) at doses of 0, 0.1 and 0.4 mg Cd/kg. Proteinuria and calciuria were induced in both types of mice, but in the ob/ob mice this condition developed at a lower dose of CdMT (0.1 mg Cd/kg) than in the normal mice (0.4 mg Cd/kg). These results show, therefore, that Umeå ob/ob mice are particularly susceptible to CdMT-induced nephrotoxicity. The mechanism underlying this phenomenon needs to be further investigated. After the administration of CdMT, a dose-related increase in glycosuria was observed in both types of mice, in spite of decreased levels of serum insulin and glucose. It is suggested that such glycosuria induced by CdMT could be one of the signs of cadmium nephrotoxicity. The results of the present study thus indicate that metabolic changes like those in diabetes may increase susceptibility to cadmium-induced renal tubular damage.
SH2 domain proteins are important components of the signal transduction pathways activated by growth factor receptor tyrosine kinases. We have been cloning SH2 domain proteins by bacterial expression cloning using the tyrosine phosphorylated C-terminus of the epidermal growth factor receptor as a probe. One of these newly cloned SH2 domain proteins, GRB-7, was mapped on mouse chromosome 11 to a region which also contains the tyrosine kinase receptor, HER2/erbB-2. The analogous chromosomal locus in man is often amplified in human breast cancer leading to overexpression of HER2. We find that GRB-7 is amplified in concert with HER2 in several breast cancer cell lines and that GRB-7 is overexpressed in both cell lines and breast tumors. GRB-7, through its SH2 domain, binds tightly to HER2 such that a large fraction of the tyrosine phosphorylated HER2 in SKBR-3 cells is bound to GRB-7. GRB-7 can also bind tyrosine phosphorylated SHC, albeit at a lower affinity than GRB2 binds SHC. We also find that GRB-7 has a strong similarity over > 300 amino acids to a newly identified gene in Caenorhabditis elegans. This region of similarity, which lies outside the SH2 domain, also contains a pleckstrin homology domain. The presence of evolutionarily conserved domains indicates that GRB-7 is likely to perform a basic signaling function. The fact that GRB-7 and HER2 are both overexpressed and bound tightly together suggests that this basic signaling pathway is greatly amplified in certain breast cancers.