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J C Hsieh

Publications and source records attributed to J C Hsieh.

At least 73 records · Page 4Linked to original sources
PubMed1991

Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function.

The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC)--namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-beta, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.

Amino Acid Sequence↗
PubMed1991

Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3.

Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.

Amino Acid Sequence↗
PubMed1991

Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.

DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.

Amino Acid Sequence↗
PubMed1991

The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies.

To characterize the 40-kilodalton (kD) major allergen of Candida albicans (C. albicans), six monoclonal antibodies (MoAbs) against this allergen were generated. In SDS-polyacrylamide gel electrophoresis and immunoblot analysis, these MoAbs showed four different reaction patterns to antigens of six different Candida species. With the exception of one MoAb, other MoAbs were resistant to periodate treatment indicating non-carbohydrate epitopes were probably being recognized by these MoAbs. These MoAbs were used in the molecular cloning and immunological analysis of the gene coding for the 40-kD allergen. Nucleotide sequence determination of the two lambda gt11 cDNA clones obtained showed that the 40-kD allergen is an alcohol dehydrogenase (ADH) which shares a 70% amino acid sequence homology with the ADH isozyme I of Saccharomyces cerevisiae. This finding was confirmed by positive immunological response of the lysates of the clones obtained and a preparation of ADH of Saccharomyces cerevisiae to various MoAbs and to IgE antibodies in sera of allergic patients.

Alcohol Dehydrogenase↗
PubMed1991

Intraoperative localization of sensorimotor cortex by cortical somatosensory evoked potentials.

Intraoperative cortical somatosensory potentials were evoked to localize the sensorimotor cortex under general anesthesia by contralateral median nerve stimulation in 12 patients. Eight patients underwent an excision of the epileptogenic focus for intractable complex partial seizures and the other 4 patients underwent tumor surgery following successful localization of the sensorimotor cortex. The relationships between the somatosensory evoked potentials and the anatomy of the sensorimotor cortex allowed rapid and accurate localization of the sensorimotor hand area: (1) the P2 recorded from the postcentral gyrus had a positivity of 49.0 microV on the average with a mean latency of 26.9 msec; (2) the N1 and P2 consistently showed phase-reversal across the Rolandic fissure; and (3) maximum SEP amplitudes were recorded from the hand representation area of the sensorimotor cortex. The neurophysiological and neurosurgical results demonstrate the feasibility of recording cortical SEP's under general anesthesia and the improvement of safety in surgical excisions near the sensorimotor cortex.

Adolescent↗
PubMed1990

Characterization of the basement membrane glycoprotein entactin synthesized in a baculovirus expression system.

A cDNA clone encoding the entire open reading frame of mouse entactin was constructed. This cDNA was inserted into Autographa californica multiple nuclear polyhedrosis virus and expressed in Spodoptera frugiperda, Sf9, insect cells. The recombinant entactin was produced in large quantities under the control of the polyhedrin promoter. Amino terminus sequence analysis indicated that the signal peptide in the preprotein was correctly cleaved. Polyclonal antibodies prepared against either the naturally occurring or recombinant entactin cross-reacted with both molecules in the native and denatured states. The recombinant molecule promoted cell attachment and exhibited a calcium- and temperature-dependent self-aggregation. These properties reflect those of the naturally occurring mouse molecule. Although entactin is an extracellular matrix component that is normally exocytosed, the vast majority of the molecules were localized inside the cell by immunoelectron microscopy where the molecules formed insoluble aggregates.

Animals↗
PubMed1990

Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel.

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.

Amino Acid Sequence↗
PubMed1990

An essential arginine residue for initiation of protein-primed DNA replication.

A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins). We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources. The location of this sequence motif is also similar among the different terminal-proteins. To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system. The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants. Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein. Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein. Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP. On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP.

Amino Acid Sequence↗
PubMed1990

Clinical application of infrared thermography in diagnosis and therapeutic assessment of vascular ischemic pain.

Temperature is a very important and useful manifestation of various disease entities. The importance of body temperature as an indicator of disease has been known for centuries but in recent years attention has also been paid to how to conveniently and effectively make use of skin temperature as a diagnostic tool. Skin temperature can be measured with thermocouples, electronic thermistor-thermometers, electronic integrators, liquid crystal thermography, and infrared thermography. The temperature of extremities is largely dependent on the blood flow through peripheral vessels, and in the study of vascular diseases thermography has been, therefore, found to be useful. Blood flow can be assessed by many methods including washout techniques or laser Doppler flowmetry. Of these, infrared thermography has the advantages of being noninvasive, remote from the patient when in use, and capable of producing multiple recordings at short time intervals. Here we present a case of vascular ischemic pain which was diagnosed and therapeutically assessed by thermography.

Aged↗
PubMed1990

Activation of epileptogenic activities by etomidate in electrocorticoencephalography (ECoG) during operation for epilepsy.

Epileptic surgery is a radical and ablative treatment for medically refractory epilepsy. Electrocorticoencephalography (ECoG) obtained by subdural strip electrodes should always be used during operation for precise localization of epileptic focus and mapping the extent of its involvement. But difficulties and ambiguities exist when the patient's ECoG is suppressed, either owing to the anticonvulsant used, being at the stage of posterictal period, or the effects of anesthetics during operation. Then, intentional activation of epileptogenic activity should be performed to locate the pathological focus in order to accomplish a successful surgical treatment. Etomidate has been considered as an abnormal electroencephalogram (EEG) activator and its use is not recommended in patients with epilepsy. But lesser dose of etomidate as an activator for ECoG has not been investigated. The study reported here established that etomidate as an hypnotic has not only anesthetic properties but, paradoxically, also activates epileptogenic activity. With the latter pharmacologic characteristic, etomidate can be used to deliberately activate the spikes of the potentially epileptogenic tissue, the delineation and localization of which may help the surgeon determine to what extent the pathological cortex be resected in surgical treatment of a refractory epilepsy. The current recommended dosage of etomidate at veterans General Hospital-Taipei for ECoG in epileptic surgery is 0.1-0.15 mg/kg/iv.

Adolescent↗
PubMed1990

Early detection of cerebral dysfunction by quantitative EEG during cardiopulmonary-bypass period for open-heart surgery.

Neuropsychiatric complications are well known risks of open heart surgery. In this regard cerebral damage is caused either by microembolism or insult from low perfusion pressure as a consequence of extracorporeal circulation. It seems probable that monitoring of the quantitative electroencephalography (QEEG) during the surgical procedure can help minimize the incidence of neurological sequelae through early detection. Moreover, it seems important to establish whether the pre-operative EEG has a predictive value for the possible neurological outcome and whether the post-operative EEG has conclusive value for the neurological outcome and, above all to ascertain whether the peri-operative QEEG contains information valuable on the diagnosis of final neurological outcome. To elucidate the feasibility of QEEG as a means to monitor the cerebral function a study was thus undertaken. Twenty-five ASA II-IV patients with age ranging from 25-79 y/o undergoing cardiac surgery under extracorporeal circulation were enrolled for study. The types of surgery varied from coronary artery by-pass graft (CABG), valvular replacement, and excision and grafting for aortic dissecting aneurysm. 16-channel EEGs were recorded with a Cadwell Spectrum 32 in accordance with the international 10-20 system. 48 epochs (1 epoch = 2.5 seconds) of EEG recordings of pre-bypass and post-bypass periods were measured and quantitatively analyzed. Pre- & post-operatively, psychoneurological assessments were performed and correlated with the QEEG. The parameters employed in the study were absolute power, relative power, power asymmetry, and coherence. All the results of two-session comparisons were transformed into z score values. Our preliminary study revealed that there exists a specific multivariate profile of QEEG for specific disease entity. And all the patients with z score less than or equal to 3.0 turned out to be free from any significant sequelate.

Adult↗
PubMed1990

Demarcation and localization of primary sensor and motor areas in human cortex by cortical somatosensory. Evoked potential (Co-SEP) during operation in surgery for epilepsy and intracranial tumor.

In neurosurgical operation, when the focus is close to the central fissure, precise delineation of the precentral motor and postcentral somatosensory areas is cardinal in defining the extent of the surgical resection. Eight ASA II-III patients scheduled for neurosurgery for intractable complex partial seizure (n = 5) and intracranial tumor (n = 3) were enrolled in the study. Subdural grids of electrodes were used for recording cortical evoked somatosensory potentials elicited by stimulating the median nerves to locate the primary somatosensory cortex and to delineate the central fissure through demonstration of phase-reversal pattern across the rolandic fissure. In all cases under general anesthesia, we were able to determine precisely the location of the central fissure. At the same time, we detected the evoked potential from SII (second sensory area) in one patient with intracerebral tumor. In conclusion, Co-SEP under general anesthesia is feasible and recommended to be performed as a routine intraoperatively for intractable epilepsy and tumor originating close to the primary motor or sensory strip in order to avoid iatrogenic damage to the vital cerebral cortex.

Brain Neoplasms↗
PubMed1989

Expression of Yb1 glutathione S-transferase using a baculovirus expression system.

A full-length cDNA clone was isolated for rat liver Yb1 glutathione S-transferase (EC 2.5.1.18). The coding sequence of Yb1 cDNA was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The enzymatically active recombinant Yb1 glutathione S-transferase protein has a native molecular weight of 42,000 daltons (by molecular sieve chromatography), a subunit molecular weight of 26,500 daltons (by SDS-polyacrylamide gel electrophoresis), a pI of 8.4 and an extinction coefficient E1%280 of 5.6 +/- 0.4.

Animals↗
PubMed1988

Hydrolysis of p-nitrophenyl phenylphosphonate catalysed by bovine pancreatic deoxyribonuclease.

The ability of bovine pancreatic DNAase to hydrolyse the synthetic substrate p-nitrophenyl phenylphosphonate (NPPP) is intrinsic and is not due to the contamination of the DNAase preparation by nonspecific phosphodiesterases because the activities of DNA and NPPP hydrolysis are co-eluted from a DEAE-cellulose column with use of the Ca2+-affinity elution method and because the two activities are decreased simultaneously when the purified enzyme is treated with Cu2+/iodoacetate, an active-site-labelling agent for DNAase. NPPP hydrolysis is facilitated by the metal ion-DNAase. At relatively high Na+ concentrations, where the metal ion-DNA interaction is weak, DNA hydrolysis is also facilitated by the metal ion-DNAase. With NPPP as substrate the Michaelis constants are Km 3.7 mM for Mn2+ and Km 49 mM for Mg2+ in 0.2 M-Tris/HCl buffer, pH 7.2. Ca2+ competes with Mn2+, with Ki 64 mM. Free Cu2+ ions non-competitively inhibit DNAase-catalysed DNA or NPPP hydrolysis in the presence of Mn2+ or Mg2+ and the inhibition is not relieved by Ca2+. The affinity of Cu2+ for free DNAase is higher than that for Mn2+-DNAase. Mn2+ is not bound to DNAase via a simple ionic interaction, as Mn2+ remains bound in the presence of relatively high Na+ concentrations and induces a near-u.v. difference absorption spectrum. The kinetics of NPPP hydrolysis catalysed by Mn2+-DNAase are sigmoidal. From the Hill equation, h = 2.0 is obtained, suggesting that more than two NPPP molecules are bound per molecule of DNAase with a certain amount of co-operativity. Because DNAase in solution is a monomer with a single catalytic site, the multiple NPPP molecules on a single protein molecule are probably in one location, resulting in a co-operative interaction that may resemble that in the stacked base-pairs of double-helical DNA.

Animals↗
PubMed1988

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing.

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.

Amino Acid Sequence↗