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Biomedical subjects

W Tan

Publications and source records attributed to W Tan.

At least 91 records · Page 5Linked to original sources
PubMed1997

AU-rich mRNA instability elements on human papillomavirus type 1 late mRNAs and c-fos mRNAs interact with the same cellular factors.

Expression levels of human papillomavirus type 1 late genes are determined in part by an AU-rich inhibitory sequence in the 3' untranslated region on the late mRNAs. Fine mapping of the AU rich inhibitory sequence revealed that it mapped to a 57 nucleotide sequence, consisting of an AU-rich region containing two AUUUA motifs and a U-rich region containing three UUUUU motifs. An internal deletion showed that the U-rich region was required for inhibition. Point mutations in the AUUUA- and UUUUU-motifs inactivated the inhibitory sequence. Analysis of the stability of mRNAs containing the AU-rich sequence in sense or anti-sense orientation showed that mRNAs containing the AU-rich sequence in sense orientation had reduced half life. Analysis of RNA-protein interactions revealed that binding to the inhibitory sequence of three poly(U) binding proteins (38, 44 and 46 kDa) correlated with inhibition and that the same proteins bind to the AU-rich mRNA instability element in the 3' untranslated region on the human c-fos mRNA. We speculate that the human papillomavirus late mRNAs, produced from several hundred copies of the virus genome present in infected cells, compete with the c-fos mRNAs for destabilizing cellular factors and that this may lead to elevated Fos protein levels in human papillomavirus infected cells.

Adenine↗
PubMed1997

Neurofilament heavy chain-like messenger RNA and protein are present in benign prostate and down-regulated in prostatic carcinoma.

Differences in gene expression between benign and malignant human prostate specimens were investigated using the differential display technique. RNA samples from paired benign and malignant areas microdissected from opposite sides of the same prostate gland were used for reverse transcription PCR. A 477-bp band was identified that was consistently present in benign prostate but absent or diminished in intensity in malignant tissue. This band was cloned, and the sequence demonstrated 99% identity with a region in the fourth exon of the human neurofilament heavy chain gene (NF-H). Northern blotting with a cDNA probe derived from this band confirmed the presence of a similarly sized message of approximately 3.9 kb in both prostate and brain, and reverse transcription PCR using primers specific to an upstream region of exon 4 confirmed NF-H-like mRNA expression in benign prostatic tissue. Immunostaining with a monoclonal antibody to NF-H showed a positive reaction in benign prostatic epithelial cells but complete absence of staining in prostatic cancer cells. These data demonstrate the presence of a NF-H-like gene product in normal prostatic epithelial cells that is down-regulated or absent in prostatic carcinomas.

Base Sequence↗
PubMed1997

[Effects of gallbladder ruptured under laparoscopic cholecystectomy on pulmonary function].

To assess effects of gallbladder ruptured and bile running out under laparoscopic cholecystectomy on pulmonary function, we preprospectively studied 20 patients without gallbladder rupture (group A), 10 with gallbladder rupture (group B), and 10 open cholecystectomy (group C). Significant differences were found in the group B and C to all pulmonary function values compared with preoperative values until the 5th to 7th postoperative day (P < 0.05). The group A showed only a significant difference on the first day after operation (P < 0.05). There are no significant differences between groups B and C. Comparing the group A with the group B and C respectively showed a significant differences from the 1st to 7th day (P < 0.05). This study confirms that the group A is superior to the groups B and C. Postoperative peritonitis is also an important cause of pulmonary dysfunction to reduce small airway function. Performing LC, we should try to avoid rupture of gall bladder or hemorrhage to fasten the recovery of patients' pulmonary function and reduce the chance of pulmonary complications.

Cholecystectomy↗
PubMed1997

[Expression of hepatitis C viral structure protein by recombinant vaccinia virus].

The hepatitis C viral (HCV) DNA fragment coding for the core and two glycoproteins E1 + E2/NS1 were inserted into vaccinia virus vector pJSA1175, under the control of the promoter 7.5K. 143TK- cells transfected with the HCV recombinant plasmid pJSA1175CE in the presence of infectious TK+ vaccinia virus (Tiantan stain) yielded recombinant vaccinia virus and expressed an approximately 90 kDa polyprotein. Southern blot hybridization showed that the inserted HCV cDNA was located at the vaccinia virus TK+ gene, J fragment. One polypeptide band between 106 and 80 kDa was revealed in SDS-PAGE which reacted with anti-core murine serum. The polypeptide could be secreted into the medium, the total amount was about the same as in the cells.

Gene Expression↗
PubMed1997

[Core protein of hepatitis C virus expressed in transgenic mice is associated with the expression of Fas molecule].

Hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide but the pathogenesis of liver cell injury and the molecular mechanism of persistent infection of HCV are not well understood. The aim of this work is to establish the animal model of HCV infection for studying the biosynthesis and pathogenesis of HCV structural protein in transgenic mice. The structural genes (C+ E1 + E2) and 5'-uncoding region gene (5'UCR) of HCV isolated from China was inserted into pcDNA3 vector. The transgenic mice were produced by microinfecting this plasmid into mouse (C57BL/6 x ICR) embryos and the integrated mice were identified. The expression of core protein of HCV and mFas molecule were confirmed by immunohistochemical analysis using monoclonal antibody. The results revealed that the expression of core protein was detected in many mouses' tissues with nuclear staining, while the heart tissue was with cytoplasmic staining. The expression level in tissues was indicated as: heart>lung>kidney>liver. At meantime, Fas molecule was detected in heart, kidney and liver tissues, and the expression of Fas was parallel to that of core protein. Transgenic mouse containing HCV structural gene was established and the expression of Fas molecule may be associated with the expression of core protein of HCV. The Fas system or other signaling pathway to the programmed cell death may play important roles in HCV infection. Our transgenic mouse will provide a useful animal model to critically address issues of immune pathogenesis, direct cytopathic potential and carcinogenesis of the structural protein of HCV.

Animals↗
PubMed1997

Expression in Escherichia coli and purification of human thrombopoietin.

Human thrombopoietin (TPO) has been successfully overexpressed in Escherichia coli, with an expression level of about 12% of total cellular protein. The full-length TPO gene was subcloned into the prokaryotic expression vector pKK233-2 under the control of the inducible tac promoter. The recombinant protein was produced mainly in the form of inclusion body. By efficient renaturation and one-step purification, the recombinant protein was purified to homogeneity. The specific activity and yield of recombinant TPO can reach 2 x 10(4) units/mg and 2 mg/g of wet E. coli cells respectively.

Chromatography, Liquid↗
PubMed1997

Use of an automated fluorescent microsphere method to measure regional blood flow in the fetal lamb.

We have developed a method for measuring regional blood flow by means of fluorescent microspheres in all organs and tissues of the fetal lamb, including brain, heart, lung, liver, gut, spleen, kidney, adrenal, brown fat, skin, muscle, bone, and placenta. Five different fluorescent-labeled microspheres were used: blue (B), yellow-green (Y), orange (O), red (R), and crimson (C). An automated, 96-well microplate fluorescent reader (bottom reading) was chosen for the assay because of the rapidity and high throughput that it offers. Tissue samples were digested by 4 M ethanolic KOH. The sedimentation method and dye extraction with Cellosolve acetate, as previously reported by others, were used for the sample processing. The bones were crushed and allowed to directly soak in Cellosolve acetate to extract the dye. The relationship between microsphere number and fluorescent intensity was linear over a broad range of microsphere numbers (80-20,000/mL). The coefficients of variation of within-run and between-run precision were 3.39 +/- 1.10% and 4.54 +/- 1.10%, respectively. Recovery of microspheres from tissues and blood averaged 94.3 +/- 2.5% and was not dependent on microsphere number. The spillover of the fluorescent signals into adjacent colors was 4.0 +/- 0.1% for O to Y, 8.1 +/- 0.4% for O to R, and 9.1 +/- 0.5% for R to C, and these values were constant over a wide range in concentrations of the microsphere pairs. No evidence was obtained for quenching of the emission of one fluorophore via photon absorption by another fluorophore. The measurements of regional blood flow obtained with fluorescent microspheres in three chronically instrumented fetal lambs at approximately 140 days gestation were similar to the flow estimates obtained using radioactive microspheres in four other fetal lambs at the same gestational age. The fluorescent method is thus a viable alternative to the radioactive technique for the measurement of regional blood flow to all fetal organs and tissues, particularly when an automated fluorescent microplate reader is employed to reduce analysis time.

Animals↗
PubMed1996

Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site.

Base Sequence↗
PubMed1996

Identification of nuclear and cytoplasmic proteins that interact specifically with an AU-rich, cis-acting inhibitory sequence in the 3' untranslated region of human papillomavirus type 1 late mRNAs.

Expression of human papillomavirus late genes encoding L1 and L2 capsid proteins is restricted to terminally differentiated epithelial cells. We have previously identified and characterized an AU-rich, cis-acting negative regulatory element in the 3' untranslated region of human papillomavirus type 1 late mRNAs. This element acts posttranscriptionally to reduce mRNA levels and the translation efficiency of mRNAs. The experiments reported here are a continuation of our previous work. We have used RNA gel shifts and UV cross-linking assays to identify cellular proteins that interact with the inhibitory RNA sequence of human papillomavirus type 1. RNA gel shift assays established that cellular proteins interact with the AU-rich sequence. The binding of nuclear proteins was inhibited by competition with poly(U), whereas the binding of cytoplasmic proteins was inhibited by competition with poly(U) and also by competition with poly(A) and poly(G). Two nuclear proteins and two cytoplasmic proteins that bind specifically to the AU-rich RNA sequence were identified by UV cross-linking. These proteins did not bind to the 3' untranslated region of human papillomavirus type 1 early mRNAs, which does not show inhibitory activity. The cellular proteins identified in our experiments may therefore be involved in the inhibition of human papillomavirus type 1 late gene expression in nondifferentiated epithelial cells.

Base Sequence↗
PubMed1996

Pharmacokinetics and tissue disposition of a chimeric oligodeoxynucleoside phosphorothioate in rats after intravenous administration.

Antisense oligonucleotides represent a novel therapeutic principle for designing drugs against various diseases. Oligonucleotides can be chemically modified to improve their pharmacokinetics and in vivo stability, and it is important to understand the effect of these modifications. In the present study, the pharmacokinetics of a 25-mer phosphorothioate oligonucleotide containing four contiguous, internucleotide, methylphosphonate linkages at the 3'- and 5'-ends (chimeric oligonucleotide) were determined in rats after i.v. administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. Plasma disappearance of the oligonucleotide could be described by a two-compartment model, with half-lives of 0.38 and 52.9 hr. The intact chimeric oligonucleotide was detected in plasma up to 6 hr after dosing. Urinary excretion represented the major elimination pathway, with approximately 21% of the administered dose being excreted within 24 hr and 35% being excreted over a 240-hr period after dosing. The majority of the radioactivity in urine was associated with the intact oligonucleotide within 6 hr after dosing and with increasing degradation products thereafter. Fecal excretion was a minor elimination pathway. The oligonucleotide was widely distributed in tissues, with the majority of the radioactivity in most tissues being intact up to 48 hr after dosing. Compared with oligodeoxynucleotide phosphorothioates, the chimeric oligonucleotide was significantly more stable in vivo. The presence of intact oligonucleotide in plasma and tissues even 12 hr after dosing is a significant advantage over an "all"-phosphorothioate analog. Thus, the chimeric oligonucleotide could provide a longer duration of action as an antisense agent after its administration.

Animals↗
PubMed1995

Double-stranded damage of DNA.RNA hybrids by neocarzinostatin chromophore: selective C-1' chemistry on the RNA strand.

Glutathione-activated neocarzinostatin chromophore generates bistranded lesions in the hybrid formed by yeast tRNA(phe) and DNA complementary to its 31-mer 3' terminus. To elucidate the chemistry of the RNA cleavage reaction and to show that the lesions are double-stranded (ds), a series of shorter oligoribonucleotides containing the target sequence r(AGAAUUC).(GAATTCT) (underlining indicates major attack site) was studied as substrates. In addition to cleavage at both U residues, major damage was produced in the form of an abasic site at the U residues. Evidence for abasic site formation on the RNA strand was obtained from sequencing-gel analysis and measurement of uracil base release. Initial evidence for the ds nature of the damage came from experiments in which 2'-O-methyluridine was substituted for uridine in the RNA at one or both of the target sites. The site containing the substitution was not a target for cleavage or abasic site formation, and the particular T residue, staggered two nucleotides in the 3' direction on the complementary DNA strand, was cleaved significantly less. These studies were valuable in identifying the DNA ds partner of the RNA attack site. Direct evidence for ds lesions came from analysis of the products from a hairpin oligonucleotide construct in which the RNA and DNA strands were linked by four T residues and contained an internal 32P label at the 3' end of the RNA strand. Substitution of deuterium for hydrogen at the C-1' position of the U residues led to a substantial isotope effect (k1H/k2H = 3) upon the formation of the RNA abasic lesion and the RNA cleavage products, providing conclusive evidence for selective 1' chemistry. On the other hand, cleavage at the T residues on the complementary DNA strand involved C-5' hydrogen abstraction, as was also true for the T residue in an oligodeoxynucleotide analogue of the RNA strand. Chemical mechanisms to account for the RNA cleavage and abasic site formation via C-1' hydrogen abstraction are proposed.

Antibiotics, Antineoplastic↗
PubMed1995

Neurotransmitter imaging in living cells based on native fluorescence detection.

A UV laser-based optical microscope and CCD detection system with high sensitivity has been developed to image neurotransmitters in living cells. We demonstrate the detection of serotonin that has been taken up into individual living glial cells (astrocytes) based on its native fluorescence. We found that the fluorescence intensity of astrocytes increased by up to 10 times after serotonin uptake. The temporal resolution of this detection system at 10(-4) M serotonin is as fast as 50 ms, and the spatial resolution is diffraction limited. This UV laser microscope imaging system shows promise for studies of spatial-temporal dynamics of neurotransmitter levels in living neurons and glia.

Animals↗
PubMed1995

Simultaneous determination of enzyme activity and enzyme quantity in single human erythrocytes.

An integrated laser-based fluorescence enzyme assay and particle-counting immunoassay system with zeptomole sensitivity has been developed for simultaneous determinations of activity and concentration of enzymes in single erythrocytes. The product NADPH of the reaction between glucose-6-phosphate and NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) is monitored. Simultaneously, the agglutination of antibody-coated particles in the presence of G6PDH produces large particles which are counted by light scattering. The correlation between the two parameters indicates that on average only about 35% of the enzyme in individual cells is active. There is more than a 10-fold cell-to-cell variation in the ratios of the two parameters. This indicates that there are multiple mechanisms for the degradation of intracellular enzymes as a function of cell age.

Adult↗
PubMed1995

Rhabdomyolysis and myoglobinuric acute renal failure associated with classic heat stroke.

Classic heat stroke is a disorder of thermal regulation that predominantly affects elderly patients during heat waves. In contrast to exertional heat stroke, rhabdomyolysis and myoglobinuric acute renal failure are considered to be unusual manifestations of classic heat stroke. We retrospectively reviewed the charts of seven patients admitted to Maimonides Medical Center with classic heat stroke over a 3-day period during a heat wave in July 1993. Three of these patients with classic heat stroke had rhabdomyolysis, but no renal failure; two completely recovered; and one had an ataxic gait disturbance. Three additional patients had rhabdomyolysis and myoglobinuric acute renal failure; one of them completely recovered, one survived with quadriplegia, and one died. Our findings suggest that rhabdomyolysis and myoglobinuric acute renal failure are common manifestations of classic heat stroke. Recognition of this complication warrants rigorous hydration and alkalinization of the urine to prevent or attenuate myoglobinuric acute renal failure.

Acute Kidney Injury↗