Biomedical subjects

A P Tsou

Publications and source records attributed to A P Tsou.

9 recordsLinked to original sources

PubMed2000

Clinical experience in using polymerase chain reaction for rapid diagnosis of pulmonary tuberculosis.

BACKGROUND: Polymerase chain reaction (PCR) techniques have revolutionized the diagnosis of tuberculosis (TB). PCR has significantly improved the sensitivity and specificity of existing diagnostic methods. In this study, we report our experience using a modified IS6110-based nested PCR assay for rapid diagnosis of pulmonary TB. METHODS: A total of 327 respiratory specimens from 275 patients suspected of having pulmonary TB at Taipei Veterans General Hospital were tested using the nested PCR assay, acid-fast smear and culture for the presence of Mycobacterium tuberculosis complex (MTB). Nested PCR was performed with IS6110-based primers specific for MTB. We reviewed the medical records of patients and analyzed the clinical features. The PCR results were compared with the final clinical diagnosis. RESULTS: We identified MTB in 167 of 327 samples by the nested PCR assay. No non-tuberculous Mycobacterium (NTM) was identified among the clinical samples. Diagnosis by PCR took about 6 hours in this study. The sensitivity and specificity compared with culture were 94.7% and 100%, respectively for the smear-positive, culture-positive samples, and 76.7% and 98.6% for the smear-negative, culture-positive samples. The overall sensitivity, specificity, positive and negative predictive values, compared with culture results, were 91.7%, 98.6%, 98.8% and 90.6%, respectively. Two specimens positive by PCR and negative by culture were taken from patients on anti-TB drug therapy. These specimens were culture-positive before anti-TB drug therapy. After resolution of the discrepancies by studying the patients' clinical data, both specificity and positive predictive value reached 100%. CONCLUSIONS: The results indicated that this in-house nested PCR assay is a rapid and sensitive method for diagnosing pulmonary TB. It is also good for excluding infections caused by NTM.

Humans↗

PubMed1998

Parallel hybridization analysis of multiple protein kinase genes: identification of gene expression patterns characteristic of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of human cancer deaths worldwide. To identify alterations of the genetic program associated with human HCC, we designed a new protocol based on the high-density replica method to analyze protein kinase gene expression in normal liver, HCC, and HCC-derived cell lines. RNA was prepared for reverse transcription and cDNA was used for PCR amplification of the conserved catalytic domain of protein kinase genes. Initially, from a pair of HCC and the adjacent noncancerous tissues, we sequenced 228 samples and identified 26 genes that represent different tyrosine kinase subfamilies. High-density grid filters were then prepared to assist the identification, by hybridization, of genes that are differentially expressed in normal vs HCC samples. Eleven tyrosine kinase genes were tested, and positive signals were reliably scored by doubly offset duplicates and by two independent gene-specific probes. Of the 11 genes tested, PDGF receptor-beta, MEKK-3, axl, and FGFR-4 are preferentially expressed in tumor samples. Additionally, we analyzed protein kinase gene expression in five HCC cell lines and identified distinct kinase gene expression patterns in different cell lines. Our results suggest that multiple kinases are activated in different tumors and confirm that there is molecular heterogeneity in the mechanisms sustaining autonomous cell growth in liver tumor formation.

Blotting, Northern↗

PubMed1998

Cloning, expression and chromosomal localization of human Ca2+/calmodulin-dependent protein kinase kinase.

A human cDNA clone encoding the calcium/calmodulin-dependent protein kinase kinase (CaMKK) was isolated by RT-PCR amplification of the fragment corresponding to the conserved kinase catalytic domain followed by rapid amplification of cDNA ends and cDNA library screening. Compilation of nucleotide sequencing data yielded a consensus cDNA sequence of 1.9 kb with an open reading frame of 1,251 nucleotides in length which translates to a polypeptide of 417 amino acids (47 kd). It showed significant homology to the rat brain CaMKK isozymes. The human CaMKK, which was expressed as a Flag-tagged protein in human non-small cell lung cancer H- 1299 cells followed by immunoprecipitation with anti-Flag antibody, was shown to phosphorylate recombinant human CaMK I in a calcium/CaM-dependent fashion. Northern blot analysis revealed that human CaMKK is ubiquitously expressed, with brain showing the highest level of expression. The CaMKK gene is localized to human chromosome 12. The presence of cDNA clones with divergent 3' terminal sequences suggests a family of CaMKK variants which may arise from alternative splicing.

Alternative Splicing↗

PubMed1997

Cloned and native guinea pig 5-ht7 receptors. Characterization using an integrated approach.

Structural criteria, i.e., primary sequence homology, indicates a unique 5-HT subtype. Operational criteria suggest that this is also true, although no selective agonist or antagonist is available to fully define the receptor, and thus its function in vivo. Transductional data provide perhaps the weakest criterion to define the receptor, since at least two other subtypes (5-HT4 and 5-ht6) signal via the same second messenger. These criteria, taken together, suggest that the cloned sequence represents an endogenously expressed 5-HT receptor and should be referred to as "5-HT7" receptors, rather than "5-ht7".

Amino Acid Sequence↗

PubMed1994

Cloning and expression of a 5-hydroxytryptamine7 receptor positively coupled to adenylyl cyclase.

A cDNA clone (designated as GP2-7) encoding a novel 5-hydroxytryptamine (5-HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5-HT receptor subtypes (34-48%). The sequence of GP2-7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5-HT7. mRNA for GP2-7 was detected in cortical and limbic brain regions. Transiently expressed GP2-7 showed high-affinity binding to [3H]5-HT (pKi = 9.0) with the following rank order of affinities: 5-carboxyamidotryptamine (5-CT) > 5-HT = 5-methoxytryptamine (5-MeOT) > methiothepin > 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) > spiperone >> sumatriptan. Adenylyl cyclase activity in CHO-K1 cells transiently transfected with GP2-7 was stimulated by several analogues of 5-HT with the following order of potency: 5-CT > 5-HT = 5-MeOT > dipropyl-5-CT > 8-OH-DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2-7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5-CT.

Adenylyl Cyclases↗

PubMed1990

1B1075: a brain- and pituitary-specific mRNA that encodes a novel chromogranin/secretogranin-like component of intracellular vesicles.

The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role.

Aging↗

PubMed1989

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression.

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody Specificity↗

PubMed1988

Glucocorticoids selectively inhibit the transcription of the interleukin 1 beta gene and decrease the stability of interleukin 1 beta mRNA.

Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1 beta mRNA. Glucocorticoids markedly decreased IL-1 beta mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1 beta mRNA, without affecting the stability of beta-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.

Cycloheximide↗

PubMed1986

Structural characterization of a heterogeneous family of rat brain mRNAs.

A large heterogeneous family of RNAs derived from a single rat gene contains members that differ from each other at one or more of three positions. Their 5' ends are nested and transcription can begin at 22 or more sites covering 265 nucleotides. Many of the 5' ends are detectable only in brain RNAs, and even 5' ends common with other tissues appear with different absolute and relative abundances in brain RNA. The central portions of the RNAs are of two forms, differing only by the presence or absence of 17 nucleotides; these forms are probably produced by alternative splicing. Polyadenylation occurs at either of two sites. This complicated family of 88 RNAs encodes two novel putative proteins that differ at their C termini.

Animals↗