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J C Shih

Publications and source records attributed to J C Shih.

At least 91 records · Page 5Linked to original sources
PubMed1996

Characterization and partial purification of human monoamine oxidase-B expressed in Escherichia coli.

Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endogenous and dietary monoamines. A full-length cDNA of the B-type of MAO, isolated from a human liver cDNA library, was cloned into a prokaryotic expression vector (pET11c). Escherichia coli which was transfected with the recombinant plasmid expressed an insoluble protein product with the expected molecular weight (65 kDa). However, in the inclusion body fraction, where most of the recombinant protein was present, no MAO activity was observed. In contrast, the membrane fraction of the bacterial lysates expressed catalytic activity as estimated by oxidative deamination of beta-phenylethylamine and tyramine. The active enzyme protein was solubilized with Triton X-100 and partly purified (80-fold) on a DEAE-Sepharose column. This enzyme activity showed properties very similar to those of human brain and platelet MAO-B. Moreover, a single band of the expected molecular size was observed on an immunoblot. The peak fraction from the DEAE-Sepharose separation was further purified on a tyramine-Sepharose column, yielding a highly purified enzyme (190-fold), visible as a band on a sodium dodecyl sulfate-containing polyacrylamide gel.

Blood Platelets↗
PubMed1996

Monoamine oxidase gene transcription in human cell lines: treatment with psychoactive drugs and ethanol.

In the present study transcriptional activities has been measured with different fragments of the 5'-flanking sequence of the human monoamine oxidase (MAO) genes linked to human growth hormone which was used as a reporter gene. SH-SY5Y neuroblastoma cells and 1242 MG glioma cells were compared under basal conditions as well as after treatments with different drugs. Under basal conditions, the relative reporter activities of the different promoter fragments were similar for both cell lines. No changes in promoter activities, were observed when cells were treated with L-deprenyl, lithium chloride or raclopride. In contrast, increases (2-3-fold) in both reporter gene expression and enzyme activity were observed after ethanol treatment of cells transfected with MAO-B fragments. Gel retardation analysis showed that ethanol caused changes in transcription factor binding to the MAO-B core promoter in both the SH-SY5Y and 1242 MG cell lines in a cell-type specific fashion.

Alcohol Drinking↗
PubMed1996

Systematic screening for mutations in the human serotonin-2A (5-HT2A) receptor gene: identification of two naturally occurring receptor variants and association analysis in schizophrenia.

A statistically significant association between a silent mutation (102T/C) in the serotonin-2A (5-HT2A) receptor gene and schizophrenia has recently been reported in a sample of Japanese patients and healthy controls. This finding suggests that genetic predisposition to schizophrenia may be affected by a functional 5-HT2A receptor variant that is in linkage disequilibrium with 102T/C. In the present study, we have sought to identify genetic variation in the 5-HT2A receptor gene by screening genomic DNA samples from 91 unrelated subjects comprising 45 patients with schizophrenia and 46 healthy controls by using single-strand conformation analysis. We have identified four nucleotide sequence variants. Two sequence changes would result in protein alterations: a substitution of threonine by asparagine at position 25 (Thr25Asn), and a substitution of histidine by tyrosine at position 452 (His452Tyr). In order to test for a possible contribution to the development of schizophrenia, we have determined allele frequencies in extended samples of unrelated patients and healthy controls. The two amino acid substitutions are found with similar frequencies in patients and controls, indicating that the presence of these variants is not causally related to the development of schizophrenia. However, the reported association of the non-coding polymorphism 102T/C with the disease has also been detected in our sample (P=0.041, odds ratio=1.28, 95% confidence interval 1.012-1.623).

Alleles↗
PubMed1996

Identification of a region important for human monoamine oxidase B substrate and inhibitor selectivity.

Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161-375 with the corresponding region of MAO-B, termed AB(161-375)A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants (k(cat)/K(m))for the oxidation of beta-phenylethylamine (PEA) (1.6 x 10(5) s-1 M-1) and benzylamine (2.4 x 10(4) s-1 M-1) by AB (161-375)A were similar to wild-type MAO-B (PEA, 8 x 10(5)s(-1) M(-1); benzylamine, 4.9 x 10(4) s(-1) M(-1). Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB(161-375)A or wild-type MAO-B. Furthermore, (AB161-375)A was more sensitive to the MAO-B specific inhibitor deprenyl (IC50 2.7 +/- 0.4 x 10(-8) M) than to the MAO-A specific inhibitor clorgyline (IC50 5.4 +/- 0.8 x 10(-7) M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed ++(+BA152-366B), lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152-366 contain a domain(s) that confers substrate and inhibitor selectivity.

Base Sequence↗
PubMed1996

Determination of transcription initiation sites and promoter activity of the human 5-HT2A receptor gene.

The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes. Previous data addressing the regulation of this receptor are extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, we have identified the promoter region of the human 5-HT2AR gene. Anchored PCR has mapped a cluster of transcription initiation sites at nucleotides -1157, -1137, -1127 (numbered sequentially as sites 1, 2 and 3). An additional initiation site (site 4) was detected at -496, 631 bp downstream of site 3. Promoter activity was defined by transfection studies. Several 5' flanking fragments linked to the human growth hormone reporter gene were transfected into two human cell lines, SHSY-5Y (neuroblastoma) and HeLa (cervix carcinoma) which express 5-HT2AR. A 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites 1 to 3 and 5' of the downstream initiation site 4, exhibited significant promoter activity in both cell lines. Inclusion of additional sequences upstream (the 1.6 kb PvuII/PvuII fragment) had little effect on the promoter activity, but the extension of the 0.74 kb fragment downstream to include a 0.45 kb PvuII/SmaI fragment drastically decreased the promoter activity. These results suggest that the promoter activity for human 5-HT2AR gene resides in the 0.74 kb HaeIII/PvuII fragment and the 0.45 kb PvuII/SmaI fragment may contain a silencer for the gene expression.

Base Sequence↗
PubMed1996

Intact 5-HT2A receptor exons and the adjoining intron regions in schizophrenia.

Genes that regulate serotonergic (5-HT) systems may underlie the etiology of schizophrenia. In this study the gene encoding the 5-HT2A receptor in schizophrenics and healthy controls was examined. First, we sequenced all exons and the flanking introns of the 5-HT2A receptor gene in 10 schizophrenics and 10 controls. The substitution of C for T at position 102 in exon, which had been reported by Warren et al. (1993), was confirmed. Restriction fragment length polymorphism (RFLP) analysis revealed no association between polymorphism and schizophrenia. There was no association between the polymorphism and subdiagnosis, family history, age of onset, amounts of antipsychotics, or positive and negative symptoms before or after medication. Other polymorphisms in the gene were screened in 100 schizophrenics by the single-strand conformation polymorphism method, but none was found. Our results suggest that an abnormality in the 5-HT2A receptor gene in schizophrenia is unlikely.

Adult↗
PubMed1996

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry.

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinson's and Alzheimer's diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

Aged↗
PubMed1996

Influence of C terminus on monoamine oxidase A and B catalytic activity.

Monoamine oxidase (MAO) A and B play important roles in the metabolism of neurotransmitters and dietary amines. The domains important for enzyme specificities were studied by construction of chimeric MAOA/B enzymes. Exchange of the N-terminal 45 amino acids of MAOA with the N-terminal 36 residues of MAOB (chimeric enzymes B36A and A45B) resulted in the same substrate and inhibitor sensitivities as the wild-type MAOA or B. Thus, the N terminus may not be responsible for MAOA or B enzyme specificities. When MAOB C-terminal residues 393-520 were replaced with MAOA C-terminal residues 402-527 (chimeric B393A) catalytic activity was not detectable. Chimeric B393A consists of eight residues with different charges, three less proline residues (458, 476, and 490), and one additional proline at 518 compared with wild-type MAOB. These differences may have induced conformational changes and affected MAOB catalytic activity. Thus, the C terminus of MAOB is critical for maintaining MAOB in an active form. It is interesting that when the C terminus of MAOA was switched with MAOB (chimeric A402B), little effect was observed on MAOA catalytic activity. This new information is valuable for further studies of the structure and function relationship of this important enzyme.

Amino Acid Sequence↗
PubMed1995

Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA.

Deficiency in monoamine oxidase A (MAOA), an enzyme that degrades serotonin and norepinephrine, has recently been shown to be associated with aggressive behavior in men of a Dutch family. A line of transgenic mice was isolated in which transgene integration caused a deletion in the gene encoding MAOA, providing an animal model of MAOA deficiency. In pup brains, serotonin concentrations were increased up to ninefold, and serotonin-like immunoreactivity was present in catecholaminergic neurons. In pup and adult brains, norepinephrine concentrations were increased up to twofold, and cytoarchitectural changes were observed in the somatosensory cortex. Pup behavioral alterations, including trembling, difficulty in righting, and fearfulness were reversed by the serotonin synthesis inhibitor parachlorophenylalanine. Adults manifested a distinct behavioral syndrome, including enhanced aggression in males.

Aggression↗
PubMed1995

Possible role of viruses in atherosclerosis.

Relative to the role of viruses in the pathogenesis of atherosclerosis a number of questions remain to be answered. Being ubiquitous, are the herpesvirus infections the norm or a diseased state in humans and animals? In all cases studied, including MDV induced atherosclerosis, direct isolation of viruses from arterial tissues has not been successful. Are herpesviruses always latent or dormant in vascular tissues? What is the relationship between provirus and disease development? Are they setting the stage for the pathogenic process triggered by certain environmental factors? Is hypercholesterolemia to trigger the development of atherosclerosis in the presence of proviruses? Is the reactivated and infectious virus the injuring agent that initiates atherogenesis? Or, are the proviral genes activated to transform arterial cells? In the latter case, are these proviral genes equivalent to "proto-atherogenes"? Hopefully, further study on Japanese quail will help clarify many of these questions.

Animals↗
PubMed1995

Dietary nitroprusside alleviates atherosclerosis in hypercholesterolemic Japanese quail (Coturnix coturnix japonica).

Male Japanese quail (Coturnix coturnix japonica), susceptible to cholesterol-induced atherosclerosis, were rendered hypercholesterolemic by feeding a diet containing 0.5% cholesterol in two experiments. Half the animals also received a dietary supplement of sodium nitroprusside ranging in concentration from 0.005% to 0.015%. After 10 weeks on the diets, serum was obtained for cholesterol analysis, the animals were killed, and the aortae removed and examined for the presence of atherosclerotic lesions. The number of animals having lesions and the severity of the lesions was reduced in a dose dependent manner among those animals receiving nitroprusside. Serum cholesterol was also reduced in response to increasing levels of dietary nitroprusside. These findings indicate that, in this model, dietary nitroprusside, a source of nitric oxide, can reduce the appearance and severity of atherosclerotic lesions in the aorta.

Animals↗
PubMed1995

Extrauterine twin-twin transfusion syndrome after birth in conjoined twins.

Cross-circulation of conjoined twins in utero has been demonstrated and the extent of its severity has varied. Significant 'extrauterine' twin-twin transfusion syndrome after birth, however, caused by an unbalanced shunting within the circulation has not been reported. In a case of omphalopagus conjoined twins complicated by hypovolemia in one twin and hypervolemia in the other, we demonstrated by color Doppler imaging an aberrant vessel from the hepatic artery of one twin to the hepatic vein of the other. Unbalanced shunting through this aberrant vessel was suspected and urgent separation of the conjoined twins was undertaken. Circulatory changes after birth leading to 'extrauterine twin-twin transfusion syndrome' are discussed. A possible explanation of the pathophysiology of intrauterine twin-twin transfusion syndrome is also given.

Adult↗
PubMed1995

Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1.

Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.

Amino Acid Sequence↗
PubMed1995

Characterization of the human 5-HT2A receptor gene promoter.

The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes and has been shown to be extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, it is important to characterize its promoter. In this report, the 5' end of the human 5-HT2AR gene was cloned and characterized. Anchored PCR mapped multiple transcription initiation sites at nucleotides -1157, -1137, -1127, and -496. Transfection of chimeric growth hormone plasmids containing various DNA fragments into 5-HT2AR-positive human cell lines (SHSY-5Y, neuroblastoma; HeLa, cervix carcinoma) showed that the 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites between -1157 and -1127 and 5' of the downstream initiation site (at -496), exhibited significant promoter activity. This promoter activity was not affected by the sequence upstream of the 0.74 kb fragment. The sequence downstream (the 0.45 kb PvuII/SmaI fragment) strongly repressed this promoter activity, suggesting the presence of a silencer. Sequence analysis combined with gel retardation and Dnase 1 footprinting assay identified multiple cis and trans elements for this fragment, including Sp1, PEA3, cyclic AMP response element (CRE)-like sequence, and E-boxes. Two novel transcription factors have been detected by gel retardation and DNase 1 footprinting assay; one of them may be specific for human. The transcription factors and promoter activities were low in the negative cell line NCI-H460 (human lung large cell carcinoma). Interestingly, the 0.39 kb fragment, isolated from the 3' end of the 0.74 kb fragment, exhibited the highest promoter activity. The possibility that this 0.39 kb fragment may be an alternative promoter is discussed. These new data are essential for further study of the regulation of 5-HT2AR gene expression.

Base Sequence↗
PubMed1994

Culture characterization and viral infection of aortic smooth muscle cells from Japanese quail susceptible to atherosclerosis.

Proliferation of aortic smooth muscle cells (SMCs), an important feature of atherosclerosis, occurs in the early stage of the disease in Japanese quail when fed an atherogenic diet. Quail aortic SMCs were isolated from a total of 32 quail and cultured in Eagle's minimum essential medium supplemented with 10% bovine fetal serum. Studies were carried out to characterize the cells in vitro. They grew actively in the early passages, but began to develop cytopathic changes after 3-4 passages and eventually cell death. The pathobiology had similarities to virus infected cells. Whole-mount electron microscopic examination detected virus-like particles in the cultured cells. All cell lines tested were positive for viral infection as screened by DNA dot blot hybridization using a genomic library of Marek's disease herpesvirus as a probe. However, the attempt to isolate the virus was unsuccessful. In summary, these cell lines are believed to be infected by a putative quail herpesvirus. Results of this in vitro cellular study support previous findings that the atherosclerosis susceptible quail are latently infected in vivo. Future studies will be needed to determine whether the virus plays a role in the pathogenesis of atherosclerosis in this animal model.

Animals↗
PubMed1994

Identification of human monoamine oxidase (MAO) A and B gene promoters.

The promoter of human monoamine oxidase (MAO) A and B genes have been identified. The core promoter region of MAO A is comprised of two 90 bp repeats each of which contains two Sp1 elements and lacks a TATA box. The MAO B core promoter region contains two sets of overlapping Sp1 sites which flank a CACCC element all upstream of a TATA box. The different organization of the MAO A and B promoters may underlie their different cell and tissue specific expression.

Base Sequence↗
PubMed1994

Does the loop electrosurgical excision procedure adversely affect the histopathological interpretation of cervical conization specimens?

OBJECTIVE: To assess the suitability of conized specimens obtained by loop electrosurgical excision procedure for histopathological interpretation. METHODS: We evaluated the histological pictures of 215 tissue sections obtained by loop electrosurgical excision procedure. These sections came from 32 cases of patients with various degrees of cervical intraepithelial neoplasia. All women included in our study had a satisfactory colposcopy and no cytological or colposcopic evidence of invasive cancer. We quantified the thickness of thermal damage in the tissue sections using a stage-mounted, calibrated grid microscope. RESULTS: At its greatest extent, thermal damage occurring next to incision lines ranged from 160 to 520 mu (mean 262, SE 14 mu). Two different zones of thermal damage were produced by LEEP: the carbonization and coagulation zones. The carbonization zone was located at the outermost layer and was very thin, measuring from 10 to 30 mu in depth. The coagulation zone was adjacent to the carbonization zone, was eosinophilic, and was significantly deeper than the carbonization zone at its points of greatest thickness (150 to 500 mu; p < 0.0001, Student's t-test). The depth of the coagulation zone correlated significantly with the depth of the carbonization zone (p = 0.041, least linear correlation). Tissue distortion was present in 53% (17/32) of the cases, and appeared only in the coagulation zone. CONCLUSION: Tissue structure from the diseased portions of the conized specimens was generally well preserved. The area of thermal damage was limited and thus did not result in diagnostic problems. We conclude that LEEP is a reliable method for obtaining tissue samples for histopathological examination.

Adult↗