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Biomedical subjects

T Y Wu

Publications and source records attributed to T Y Wu.

At least 19 recordsLinked to original sources

Solid-phase synthesis of 2,3,5-trisubstituted indoles.

2,3,5-Trisubstituted indoles are synthesized in three steps starting from resin-bound aniline 2. R1 is introduced by a palladium-mediated coupling of the aryl iodide with terminal alkynes followed by intramolecular cyclization to form the indole core. Acylation at C-3 with an acid chloride in the presence of AlCl(3) catalyst introduces R2. The indole C-5 position is then diversified either by Sonagashira or Suzuki couplings with the aryl bromide. Finally, indole N-1 can be modified by post-cleavage methylation. [reaction: see text]

Journal Article↗

Sequence polymorphism in the coding region of mitochondrial genome encompassing position 8389-8865.

Analysis of the polymorphic sequences in mitochondrial DNA (mtDNA) has been widely applied to forensic tests and anthropology studies. However, these polymorphic data in human have thus far been derived from the displacement-loop and intergenic regions only. Here, we report the identification of clustered polymorphic sites in the mitochondria coding region encompassing position 8389-8865. The DNA sequences of 119 unrelated Chinese were determined by PCR amplification and direct sequencing. The results showed that heteroplasmy was found in five individuals, 39 sites were noted in this 477 bp region, and 41 haplotypes were identified. The probability of identity and allelic diversity were estimated as 0.1265 and 0.8809, respectively. The results suggest that sequence polymorphism from position 8389-8865 in human mtDNA can be used as a marker for identity investigation.

DNA, Mitochondrial↗

Tubulointerstitial nephritis associated with a novel mitochondrial point mutation.

BACKGROUND: Nephropathy caused by mitochondrial disorders is a relatively newly recognized disease. Only a few cases have been reported in the literature, and most of them are proximal tubulopathy-presenting Fanconi syndrome. Here we report on a novel mutation in two familial cases of tubulointerstitial nephropathy associated with concentrating defect. METHODS: Renal biopsy specimens were examined by light microscopy and electron microscopy. Mitochondrial genomic DNA isolated from renal biopsy specimens was amplified by polymerase chain reaction (PCR) and sequenced in its entirety. The DNA sequences were analyzed by (1) comparing with the Anderson et al's mitochondrial sequences; (2) comparing with DNA sequences obtained from 97 human controls, including both healthy individuals and patients with renal diseases; and (3) comparing with the counterparts in 90 different species. RESULTS: Dismorphic mitochondria with occasional intramitochondrial inclusions were found in the renal tubular epithelial cells. A novel mitochondrial point mutation was identified at the position 608, that is, the distal end of the anticodon stem of the tRNA(Phe) molecule. The A to G substitution at this position was not observed in 97 human controls and was found to be highly conserved in evolution. CONCLUSIONS: We have identified an A608G mutation of mitochondrial genome in two cases whose presentation include tubulointerstitial nephritis and stroke.

Animals↗

Disseminated cutaneous infection with Mycobacterium kansasii: genotyping versus phenotyping.

Mycobacterium kansasii is a common and virulent pathogen. However, primary cutaneous M kansasii infection has rarely been reported, totaling to only 34 cases since 1965. This low incidence has not been explained, but probably is due to difficulty in culture or phenotyping. We describe the first reported case of cutaneous M kansasii diagnosed by genotyping, which is based on the DNA extracted from the formalin-fixed and paraffin-embedded tissue.

Adult↗

Murine B16 melanoma vaccination-induced tumor immunity: identification of specific immune cells and functions involved.

Vaccination using inactivated B16 melanoma cells that have been treated in vitro for > 2 weeks with interferon-alpha (IFN-alpha) (B16alpha cells) has been shown to elicit a protective host antitumor immunity. In these studies, vaccination with B16alpha cells has been shown to provide protection against primary B16 tumor challenge, established B16 tumors, and metastatic B16 tumors. Specific immune cells and factors that might mediate this tumor immunity have now been evaluated. Macrophage depletion studies suggest that macrophage function is required for expression of tumor immunity either for processing of antigen or for cytokine production but that macrophage function is not involved in direct cytotoxicity against the B16 challenge tumor. CD8(+) T cell depletion studies show that cytotoxic T cell function is required for expression of tumor immunity. Syngeneic knockout mouse experiments offer further insights into the immune cells and factors that mediate the development and expression of tumor immunity. First, interleukin-12 (IL-12) knockout mouse experiments identify IL-12 as an important cytokine in mediating the development of tumor immunity. Second, specific knockout mouse experiments show that tumor immunity requires the function of CD4(+) T cells, CD8(+) T cells, and natural killer (NK) cells. Third, specific knockout mouse experiments show that tumor immunity does not require the function of B cells. The results suggest that vaccination with inactivated B16alpha cells induces an active, cell-mediated immunity to B16 melanoma cells. The tumor vaccination protocol with B16alpha cell vaccinations establishes a potent tumor immunity against B16 melanoma tumors in mice and may serve as a model for induction of tumor immunity against primary or secondary melanoma tumors in humans.

Animals↗

Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system.

A modified tetracycline-responsive expression system (TRES) for use in insect cells was developed. The TRES contains two components: one encodes a tetracycline-controllable transactivator (tTA) and the other contains a tet operator DNA sequence to drive the luciferase gene. Our results show that the human cytomegalovirus (CMV) promoter, an essential part for strong tTA expression in mammalian system, was not functional in insect cells. Thus further modifications were required. Functional tTA was efficiently expressed in Sf9, Sf21, and TN368 cells by the p10 promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in plasmid form with virus co-infection. An increase of up to 258-fold of luciferase activity was detected in these cells when both components in modified TRES were co-transfected. In order to further simplify the experiment, tTA, which is driven by the p10 promoter, was inserted into AcMNPV. Luciferase activity was also strongly stimulated by the infection of this tTA expression-recombinant virus with the transfection of a plasmid containing the second TRES component expressing luciferase. The luciferase expressions in these systems, either in plasmids or the tTA gene in virus and luciferase in plasmid, were significantly suppressed by tetracycline. The time course kinetics of tetracycline action to the TRES were further studied. Within a time span of 50 h, the luciferase activities could be fully suppressed or activated, respectively, corresponding to the addition or removal of tetracycline. These experiments have established a well-regulated gene expression system for further broad applications of molecular biological studies in insect cells.

Animals↗

Enhancement of radiosensitivity in human glioblastoma U138MG cells by tetrandrine.

Tetrandrine, a natural lipid-soluble alkaloid with a low molecular weight, has both anti-tumor activity and a tissue-protective effect, as demonstrated in our previous studies. We studied tetrandrine to determine whether or not it enhanced radiosensitivity in human glioblastoma U138MG cells. Tetrandrine at concentration of 5.0 and 7.5 eg/ml dramatically enhanced the growth-inhibiting effect of radiation. This effect is dose-dependent on the dose of both tetrandrine and radiation. Moreover, we found that tetrandrine eliminated the cell cycle perturbation induced by radiation. Cells treated with tetrandrine alone as well as with a combination of tetrandrine and radiation became rounded, floated up and grew sparsely under observation under an inverted light microscope, DNA fragmentation was also noted by gel electrophoresis. These findings suggest that tetrandrine has potential as an adjunct to radiotherapy for glioblastoma.

Alkaloids↗

Orally administered IFN-alpha acts alone and in synergistic combination with intraperitoneally administered IFN-gamma to exert an antitumor effect against B16 melanoma in mice.

Administration of interferons (IFN) via the intranasal route recently has been shown to exert an antitumor activity against a variety of tumors in mice, including B16 melanoma inoculated intravenously. This study confirms the antitumor activity of orally administered IFN-alpha against B16 melanoma challenge using another route of tumor inoculation, the intraperitoneal route. It further demonstrates that orally administered IFN-alpha can synergistically interact with intraperitoneally administered IFN-gamma but not with intraperitoneally administered IFN-alpha. The results support the interpretation that the oral route may provide an effective alternative or supplement to current methods of IFN administration for the control of malignancies.

Administration, Oral↗

Efficacy of B16 melanoma cells exposed in vitro to long-term IFN-alpha treatment (B16alpha cells) as a tumor vaccine in mice.

B16 melanoma cells exposed to >2 weeks of in vitro interferon-alpha (IFN-alpha) treatment (B16alpha cells) were UV inactivated and used for vaccination. This vaccination was efficacious against challenge with parental B16 cells in the absence of adjuvant therapy. Vaccinations based on parental cells and B16 cells exposed to short-term in vitro IFN-alpha treatment were not effective. The efficacy of B16alpha vaccination was evaluated using three B16 tumor models. Using intraperitoneal (i.p.) tumor challenge given after vaccination, vaccination efficacy depended on the concentration of IFN-alpha to which B16alpha cells were exposed, the number of inactivated B16alpha cells inoculated, the number of inoculations administered, and the amount of tumor burden. A significant fraction (30%) of vaccinated mice surviving initial challenge had durable immunity against a second parental tumor challenge. This immunity increased to 92% with administration of a single booster vaccination. Using metastatic tumor challenge given after vaccination, vaccination reduced lung metastases by approximately 67%. Using vaccination begun 3 days after subcutaneous (s.c.) tumor challenge, regression of established tumor occurred when vaccination was given i.p. (39%) or contralaterally s.c. (53%). Taken together, the results suggest that vaccination with inactivated B16alpha cells may serve as a model for induction of host tumor immunity against primary or secondary tumors.

Animals↗

Induction of apoptosis in human leukemic U937 cells by tetrandrine.

Tetrandrine, a calcium channel antagonist, is a plant alkaloid possessing various pharmacological activities including anti-tumor activity. We studied tetrandrine to determine whether or not this anti-tumor effect occurs through induction of apoptosis. Tetrandrine inhibited both proliferation and clonogenicity of human leukemic U937 cells at an optimal concentration of 2.5 microg/ml. This growth inhibition was dose and time dependent, and accompanied with evidence of apoptotic changes. The characteristic morphological changes of apoptosis were observed in U937 cells under light microscopy and DNA fragmentation was noted by gel electrophoresis. Moreover, flow cytometric detection of surface phosphatidyl serine expression of U937 cells after treatment with tetrandrine confirmed the induction of apoptosis in these cells. The induction of apoptosis by tetrandrine would appear to proceed via non-Ca2+-dependent pathways.

Alkaloids↗

A 2.9-kilobase noncoding nuclear RNA functions in the establishment of persistent Hz-1 viral infection.

Differential viral gene expression during both productive and persistent infections of Hz-1 virus in insect cells was elucidated. Despite more than 100 viral transcripts being expressed during productive viral infection, massive viral gene shutoff was observed during viral persistency, leaving the 2.9-kb persistence-associated transcript 1 (PAT1) as the only detectable viral RNA. Persistence-associated gene 1 (pag1), which encodes PAT1, was cloned and found to contain no significant open reading frames. PAT1 is not associated with the cellular translation machinery and is located exclusively in the nucleus. Further experiments showed that PAT1 is functional in the establishment of persistent Hz-1 viral infection in the cells. All the evidence collectively indicates that PAT1 is a novel nuclear transcript of viral origin. Our results showed that although PAT1 and XIST RNA, a mammalian X-inactive specific transcript, are transcribed by different genes, they have interesting similarities.

Animals↗

Use of sodium polystyrene sulfonate for reduction of plasma lithium concentrations after chronic lithium dosing in mice.

OBJECTIVES: Previous studies have shown that oral sodium polystyrene sulfonate lowers plasma lithium concentrations after acutely administered oral doses of lithium chloride. However, a significant proportion of lithium overdose cases resulting in morbidity and mortality are those in which exposure to lithium is chronic. This study was designed to determine whether multiple oral doses of sodium polystyrene sulfonate are effective in reducing plasma lithium concentrations after chronic dosing. DESIGN: Placebo-controlled animal study. INTERVENTIONS: One hundred thirty mice were given 75 mM lithium chloride in their drinking water for a period of 14 days. At the end of that period, half of the animals were given orogastric sodium polystyrene sulfonate at 5 g/kg/dose 0, 60, 120, 180, and 360 minutes after the cessation of lithium chloride; the remaining half received orogastric water at equivalent times. Subgroups of each group were sacrificed at 90, 150, 330, 480, 1440, and 2880 minutes after lithium chloride cessation and plasma analyzed for lithium content. Lithium concentrations were compared by analysis of variance and single degree of freedom contrasts. Significance was set at an alpha level of 0.05. RESULTS: Lithium concentration was lower overall in the animals treated with sodium polystyrene sulfonate (p < .0001) and specifically at 150, 330, and 480 minutes after lithium chloride cessation (p < .05). CONCLUSIONS: Repetitive oral doses of sodium polystyrene sulfonate effectively lowered plasma lithium concentrations. Further study may ultimately define a role for the use of sodium polystyrene sulfonate in the treatment of patients with chronic lithium toxicity.

Administration, Oral↗

B16 melanoma cells exposed in vitro to long-term IFN-alpha treatment (B16 alpha cells) as activators of tumor immunity in mice.

Mice inoculated with B16 melanoma cells exposed to long-term in vitro IFN-alpha treatment (> or = 14 days, B16 alpha cells) but not short-term in vitro IFN-alpha treatment (24 h) exhibited an enhanced survival time. Enhanced survival time also occurred when inactivated B16 alpha cells were inoculated at the same time as live B16 cells. Further, an even greater improvement in survival time was observed when the inactivated B16 alpha cells were inoculated before live B16 cell challenge. No enhancement in survival time was observed when mice were inoculated with inactivated, untreated B16 cells. Enhancement of survival time by B16 alpha cells was unrelated to retrovirus surface antigen expression. Long-lasting protective immunity to B16 cells was observed in mice that survived B16 alpha cell, but not normal B16 cell, challenge and subsequent IFN treatment. It is evident that inoculation with inactivated B16 alpha cells, but not with inactivated untreated B16 cells, was able to prolong significantly the survival time of mice either simultaneously or subsequently challenged with live B16 cells. Additionally, survival of B16 alpha-inoculated but not B16-inoculated mice was accompanied by a durable immunity. Inoculation of inactivated B16 alpha cells may serve as a model for the induction of host immunity to a parental primary or secondary tumor.

Animals↗

Exercise performance of Tibetan and Han adolescents at altitudes of 3,417 and 4,300 m.

The difference was studied between O2 transport in lifelong Tibetan adolescents and in newcomer Han adolescents acclimatized to high altitude. We measured minute ventilation, maximal O2 uptake, maximal cardiac output, and arterial O2 saturation during maximal exercise, using the incremental exercise technique, at altitudes of 3,417 and 4,300 m. The groups were well matched for age, height, and nutritional status. The Tibetans had been living at the altitudes for a longer period than the Hans (14.5 +/- 0.2 vs. 7.8 +/- 0.8 yr at 3,417 m, P < 0.01; and 14.7 +/- 0.3 vs. 5.3 +/- 0.7 yr at 4,300 m, P < 0.01, respectively). At rest, Tibetans had significantly greater vital capacity and maximal voluntary ventilation than the Hans at both altitudes. At maximal exercise, Tibetans compared with Hans had higher maximal O2 uptake (42.2 +/- 1.7 vs. 36.7 +/- 1.2 ml . min-1 . kg-1 at 3,417 m, P < 0.01; and 36.8 +/- 1.9 vs. 30.0 +/- 1. 4 ml . min-1 . kg-1 at 4,300 m, P < 0.01, respectively) and greater maximal cardiac output (12.8 +/- 0.3 vs. 11.4 +/- 0.2 l/min at 3,417 m, P < 0.01; 11.5 +/- 0.5 vs. 10.0 +/- 0.5 l/min at 4,300 m, P < 0. 05, respectively). Although the differences in arterial O2 saturation between Tibetans and Hans were not significant at rest and during mild exercise, the differences became greater with increases in exercise workload at both altitudes. We concluded that exposure to high altitude from birth to adolescence resulted in an efficient O2 transport and a greater aerobic exercise performance that may reflect a successful adaptation to life at high altitude.

Acclimatization↗

A study of the oligomeric state of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain.

The number of the subunits in an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking regents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits.

Animals↗

Purification and biochemical characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors of pig brain.

Two preparations of glutamate receptors were purified from the synaptic junctions of pig brain by a combination of detergent solubilization, anion-exchange chromatography, wheat-germ agglutinin affinity chromatography and sedimentation through sucrose gradients. These preparations were enriched in specific L-[3H]glutamate binding activity (> 5000 pmol of glutamate binding sites/mg of protein), and the rank order of ligand affinity for binding to these preparations was: quisqualate > 6-cyano-7- nitroquinoxaline-2,3-dione > alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) > L-glutamate > kainate > > N-methyl-D-aspartate approximately L-2-amino-4-phosphonobutyrate. SDS/PAGE analysis revealed that more than 80% of the protein in either of these preparations appeared as a single protein band of 106 kDa. Two-dimensional gel electrophoresis further revealed that these 106 kDa proteins consisted of a series of acidic proteins which were recognized by antibodies against rat AMPA receptor subunits. These 106 kDa proteins were also recognized by wheatgerm agglutinin and concanavalin A; in addition, peptide N-glycosidase F treatment of these preparations decreased their size to 99 kDa. Our results suggest that the putative glutamate receptors isolated here are likely to belong to the AMPA subtype of glutamate receptors in pig brain. Using the purification procedure reported here, 5 micrograms of AMPA receptor proteins can be isolated from 250 g of pig brain tissue.

Animals↗