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Biomedical subjects

T Bui

Publications and source records attributed to T Bui.

At least 19 recordsLinked to original sources

Effect of nonlinear summation of synaptic currents on the input-output properties of spinal motoneurons.

A single spinal motoneuron receives tens of thousands of synapses. The neurotransmitters released by many of these synapses act on iontotropic receptors and alter the driving potential of neighboring synapses. This interaction introduces an intrinsic nonlinearity in motoneuron input-output properties where the response to two simultaneous inputs is less than the linear sum of the responses to each input alone. Our goal was to determine the impact of this nonlinearity on the current delivered to the soma during activation of predetermined numbers and distributions of excitatory and inhibitory synapses. To accomplish this goal we constructed compartmental models constrained by detailed measurements of the geometry of the dendritic trees of three feline motoneurons. The current "lost" as a result of local changes in driving potential was substantial and resulted in a highly nonlinear relationship between the number of active synapses and the current reaching the soma. Background synaptic activity consisting of a balanced activation of excitatory and inhibitory synapses further decreased the current delivered to the soma, but reduced the nonlinearity with respect to the total number of active excitatory synapses. Unexpectedly, simulations that mimicked experimental measures of nonlinear summation, activation of two sets of excitatory synapses, resulted in nearly linear summation. This result suggests that nonlinear summation can be difficult to detect, despite the substantial "loss" of current arising from nonlinear summation. The magnitude of this "loss" appears to limit motoneuron activity, based solely on activation of iontotropic receptors, to levels that are inadequate to generate functionally meaningful muscle forces.

Animals↗

Undermethylated DNA as a source of microsatellites from a conifer genome.

Developing microsatellites from the large, highly duplicated conifer genome requires special tools. To improve the efficiency of developing Pinus taeda L. microsatellites, undermethylated (UM) DNA fragments were used to construct a microsatellite-enriched copy library. A methylation-sensitive restriction enzyme, McrBC, was used to enrich for UM DNA before library construction. Digested DNA fragments larger than 9 kb were then excised and digested with RsaI and used to construct nine dinucleotide and trinucleotide libraries. A total of 1016 microsatellite-positive clones were detected among 11 904 clones and 620 of these were unique. Of 245 primer sets that produced a PCR product, 113 could be developed as UM microsatellite markers and 70 were polymorphic. Inheritance and marker informativeness were tested for a random sample of 36 polymorphic markers using a three-generation outbred pedigree. Thirty-one microsatellites (86%) had single-locus inheritance despite the highly duplicated nature of the P. taeda genome. Nineteen UM microsatellites had highly informative intercross mating type configurations. Allele number and frequency were estimated for eleven UM microsatellites using a population survey. Allele numbers for these UM microsatellites ranged from 3 to 12 with an average of 5.7 alleles/locus. Frequencies for the 63 alleles were mostly in the low-common range; only 14 of the 63 were in the rare allele (q < 0.05) class. Enriching for UM DNA was an efficient method for developing polymorphic microsatellites from a large plant genome.

DNA Methylation↗

Semi-quantitative RT-PCR method to estimate full-length mRNA levels of the multidrug resistance gene.

Expression levels of P-glycoprotein (P-gp), the transporter encoded by the human multidrug resistance gene (MDR1), may play an important role in drug disposition. The ability to quantitate full-length MDR1 mRNA levels may be predictive of P-gp expression and function. Therefore, a semi-quantitative RT-PCR assay was developed to assess full-length MDR1 mRNA levels. Levels offull-length 3.8-kb MDR1 mRNA were estimated by comparing PCR amplification of the RNA extract with that of an internal standard, deltaMDR1. The 2.9-kb deltaMDR1 competitor RNA standard was constructed by deleting 965 bpfrom the interior of MDR1 mRNA. The full-length MDR1 and deltaMDR1 share identical 5' and 3'primer binding sequences, allowing for their simultaneous amplification in the same RT-PCR. With this approach, MDR1 mRNA levels can be sensitively and reliably estimated with a detection limit of 2000 copies. Full-length MDR1 mRNA levels in various human cell lines and lymphocytes from leukemia patients varied over 100-fold, ranging from 0.3 to 36.5 x 10(5) copies/microg total RNA. The semi-quantitative full-length RT-PCR assay may be useful in estimating MDR1 mRNA levels to assess P-gp expression, which may be important in studying the role of P-gp in drug disposition and cancer chemotherapy efficacy.

Cell Line↗

Dendritic cells from nonobese diabetic mice exhibit a defect in NF-kappa B regulation due to a hyperactive I kappa B kinase.

Insulin-dependent diabetes mellitus (IDDM) is characterized by the T cell-mediated destruction of insulin-producing beta cells. Accordingly, APCs, such as macrophage, have also been shown to be important in the disease process. However, the role(s) of dendritic cells (DCs) that exhibit potent APC function remains undefined in IDDM. Here we demonstrate that DCs derived from nonobese diabetic (NOD) mice, a model for IDDM, are more sensitive to various forms of stimulation compared with those from C57BL/6 and BALB/c mice, resulting in increased IL-12 secretion. This property is a consequence of hyperactivation of NF-kappaB, a transcription factor known to regulate IL-12 gene expression. Specifically, NOD DCs exhibit persistent hyperactivation of both IkappaB kinase and NF-kappaB in response to stimuli, in addition to selective degradation of IkappaBepsilon. Transfection of NOD DCs with a modified form of IkappaBalpha significantly reduced IL-12 secretion, suggesting that hyperactivation of NF-kappaB was in part responsible for increased IL-12 production. An enhanced capacity of NOD DCs to secrete IL-12 would be expected to contribute to the development of pathogenic Th1 (Tc1) cells during the diabetogenic response.

Active Transport, Cell Nucleus↗

Amino acid catalyzed direct asymmetric aldol reactions: a bioorganic approach to catalytic asymmetric carbon-carbon bond-forming reactions.

Direct asymmetric catalytic aldol reactions have been successfully performed using aldehydes and unmodified ketones together with commercially available chiral cyclic secondary amines as catalysts. Structure-based catalyst screening identified L-proline and 5,5-dimethyl thiazolidinium-4-carboxylate (DMTC) as the most powerful amino acid catalysts for the reaction of both acyclic and cyclic ketones as aldol donors with aromatic and aliphatic aldehydes to afford the corresponding aldol products with high regio-, diastereo-, and enantioselectivities. Reactions employing hydroxyacetone as an aldol donor provide anti-1,2-diols as the major product with ee values up to >99%. The reactions are assumed to proceed via a metal-free Zimmerman-Traxler-type transition state and involve an enamine intermediate. The observed stereochemistry of the products is in accordance with the proposed transition state. Further supporting evidence is provided by the lack of nonlinear effects. The reactions tolerate a small amount of water (<4 vol %), do not require inert reaction conditions and preformed enolate equivalents, and can be conveniently performed at room temperature in various solvents. In addition, reaction conditions that facilitate catalyst recovery as well as immobilization are described. Finally, mechanistically related addition reactions such as ketone additions to imines (Mannich-type reactions) and to nitro-olefins and alpha,beta-unsaturated diesters (Michael-type reactions) have also been developed.

Acetone↗

Antigen-specific mediated suppression of beta cell autoimmunity by plasmid DNA vaccination.

In this study, we have investigated the use of plasmid DNA (pDNA) vaccination to elicit Th2 effector cell function in an Ag-specific manner and in turn prevent insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. pDNA recombinants were engineered encoding a secreted fusion protein consisting of a fragment of glutamic acid decarboxylase 65 (GAD65) linked to IgGFc, and IL-4. Intramuscular injection of pDNA encoding GAD65-IgGFc and IL-4 effectively prevented diabetes in NOD mice treated at early or late preclinical stages of IDDM. This protection was GAD65-specific since NOD mice immunized with pDNA encoding hen egg lysozyme-IgGFc and IL-4 continued to develop diabetes. Furthermore, disease prevention correlated with suppression of insulitis and induction of GAD65-specific regulatory Th2 cells. Importantly, GAD65-specific immune deviation was dependent on pDNA-encoded IL-4. In fact, GAD65-specific Th1 cell reactivity was significantly enhanced in animals immunized with pDNA encoding only GAD65-IgGFc. Finally, NOD.IL4(null) mice treated with pDNA encoding GAD65-IgGFc and IL-4 continued to develop diabetes, indicating that endogenous IL-4 was also required for disease prevention. These results demonstrate that pDNA vaccination is an effective strategy to elicit beta cell-specific Th2 regulatory cell function for the purpose of preventing IDDM even at a late stage of disease development.

Animals↗

Suppression of maternal virus load with zidovudine, didanosine, and indinavir combination therapy prevents mother-to-fetus HIV transmission in macaques.

Recently, we developed a maternal-fetal macaque model using a highly pathogenic HIV-2 strain, HIV-2287, to study the time course of HIV transmission in utero. Most pregnant macaques (Macaca nemestrina) infected with HIV-2287 (10-103 infective doses) transmitted HIV to their fetuses, as verified by positive identification of virus-infected mononuclear cells and free viral RNA in fetal blood. To determine whether an antiretroviral drug combination therapy composed of two dideoxynucleosides, azidothymidine (15 mg/kg) and dideoxyinosine (15 mg/kg), and a protease inhibitor, indinavir (25 mg/kg), could completely inhibit mother-to-fetus HIV transmission, we administered these drugs orally through gastric catheters to five pregnant macaques infected with 10 infective doses of HIV-2287. Beginning 30 minutes after HIV inoculation, the dams were given the combination antiviral therapy three times daily until delivery by cesarean section. Drug treatment reduced the maternal virus load to a minimally detectable level but did not prevent primary HIV-2287 infection. All fetal and infant blood samples were virus negative by internally controlled RNA polymerase chain reaction (QC-RNA-PCR) and virus coculture assays. Fetal and infant CD4+ T-cell levels remained normal throughout the experiment. These findings strongly suggest that combination chemotherapy with azidothymidine, dideoxyinosine, and indinavir can suppress maternal viral load enough to prevent mother-to-fetus transmission of HIV.

Animals↗

An efficient benchtop system for multigram-scale kinetic resolutions using aldolase antibodies.

The preparative scale kinetic resolution of racemic aldols 1-4 using aldolase antibodies 38C2 (Aldrich no. 47995-0) and 84G3 (Aldrich no. 52785-8) is described. These reactions use a biphasic aqueous/organic solvent system that allows the catalyst to be reused. Reaction scales range from miligrams to grams, with 0.0086 to 0.12 mol% of antibody binding sites. Because antibodies 38C2 and 84G3 have opposite enantioselectivities, both aldol product enantiomers are accessible by kinetic resolution.

Aldehydes↗

Inhibition of RNA polymerase I transcription in differentiated myeloid leukemia cells by inactivation of selectivity factor 1.

Transcription by RNA polymerase I (pol I) regulates the rate of ribosome biogenesis and the biosynthetic potential of the cell; therefore, it plays an important role in the control of cell growth. Differentiation of the human promyelocytic leukemic cell line U937 is accompanied by drastic decreases in pol I transcriptional activity. We have used cell-free extracts prepared from undifferentiated and differentiated U937 cells to investigate the molecular mechanisms responsible for this inhibitory process. Our analysis indicates that the activity of the TATA binding protein (TBP)/TBP-associated factor (TAF) complex selectivity factor 1 (SL1), one of the factors required for accurate and promoter-specific transcription by RNA pol I, is severely repressed in differentiated U937 cells. Moreover, the reduction in SL1 activity is not a consequence of a decrease in SL1, because there is no detectable difference in the abundance of TBP or TAFs before and after U937 cell differentiation. In conclusion, our results indicate that the selectivity factor SL1 is an important target for the regulation of pol I transcription during cell differentiation.

Cell Differentiation↗

Molecular typing of pathogenic fungi.

In this Round Table, the application of several methods of molecular typing were discussed in reference to four important pathogenic fungi: Coccidioides immitis, Histoplasma capsulatum, Candida albicans and Paracoccidioides brasiliensis. Among the different methods the following were discussed: restriction fragment length polymorphisms (RFLP), single nucleotide polymorphisms, random amplified polymorphic DNA (RAPD), polymerase chain reaction (PCR)-RFLP and microsatellites. By means of these methods, several important biological questions related to speciation, mode of reproduction and population genetics could be approached. The basic information obtained from this approach has implications in the understanding of these pathogenic fungi in relation to their behavior and the development of pathogenic features, such as resistance to antimicrobials and virulence factors used for colonization of mammalian hosts. The knowledge obtained from these studies could also be used for the development of innovative diagnostic methods, as well as for novel therapeutic approaches and production of vaccines.

Genetic Techniques↗

Formulation of HIV-envelope protein with lipid vesicles expressing ganglioside GM1 associated to cholera toxin B enhances mucosal immune responses.

Taking advantage of the ability of pentameric cholera toxin B subunit (CTB) to bind selectively to GM1, we developed recently a CTB-mediated GM1 lipid vesicle delivery system to target drugs and proteins to mucosal tissues [1]. In this report, we present the use of such a strategy to deliver an HIV envelope protein (HIV-env) to mucosal tissues via intranasal route. Intranasal administration of a recombinant HIV envelope protein formulated in CTB-associated GM1 lipid vesicles enhanced mucosal IgA antibody responses detected in the nasal and gut tissues, compared to that of control animals immunized with antigen formulated in GM1-free vesicles with CTB or formulated in alum-associated vesicles with CTB. We found a nearly 2- to 3-fold enhancement in IgA antibody titers detected both in nasal and gut tissues using the CTB-GM1 lipid vesicle delivery system, compared to using the GM1-free lipid vesicle system. Intranasal administration of HIV-env formulated in the CTB-associated GM1 vesicles also induced a significant level of serum IgG and cellular immune responses against HIV-env. IgG isotype analysis indicates that CTB in GM1 vesicle delivery system enhanced both IgG1 and IgG2a while CTB in alum formulation enhanced only IgG1. However, IgA and IgG antibody responses against CTB were similar for GM1 vesicles regardless of whether HIV-env was present in the vaccine formulation. Collectively, these data indicate that delivery of HIV-env to mucosal epithelial cells with CTB-associated GM1 lipid vesicles enhanced mucosal and systemic immune responses against the HIV-envelope protein. It is possible that both the CTB-mediated targeted delivery of antigen-loaded GM1 lipid vesicles and mucosal adjuvanticity of CTB may be involved in enhancing the immune responses.

Adjuvants, Immunologic↗

Association with oleic acid vesicles enhances manganese III mesoporphyrin absorption and biliary elimination in rats.

OBJECTIVE: To determine whether the association of manganese III mesoporphyrin (MnMeso), an oral MR contrast agent, to oleic acid (OA) vesicles will improve absorption and delivery of MnMeso to the liver. METHODS: MnMeso or MnMeso-OA vesicle suspension was intraduodenally administered to rats and the time course of MnMeso concentration in plasma, bile, and intestinal solution was determined. Tissue concentrations of MnMeso in the liver were also determined. RESULTS: Association of MnMeso to OA reduced the time it took to reach one-half absorption maximum, TC50, and enhanced the rate and the extent of biliary elimination of this contrast agent. In addition, the results obtained from dose-titration studies indicate that MnMeso-OA vesicle complex may enhance MnMeso accumulation in the liver, observed as a lower dose required to reach equivalent tissue concentration. Taken together, complexion to OA vesicles may provide rapid absorption, enhanced liver accumulation, and efficient elimination of MnMeso. CONCLUSIONS: Association with OA vesicles may enhance the rate of MnMeso absorption and biliary excretion while promoting the extent of liver accumulation of MnMeso in rats. This strategy may provide a means to provide safe and effective use of MnMeso as an MR contrast medium.

Animals↗

Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia.

Cryptococcus neoformans var. gattii lives in association with certain species of eucalyptus trees and is a causative agent of cryptococcosis. It exists as two mating types, MATalpha and MATa, which is determined by a single-locus, two-allele system. In the closely related C. neoformans var. neoformans, the alpha mating type has been found to outnumber its a counterpart by at least 30:1, but there have been very limited data on the proportions of each mating type in C. neoformans var. gattii. In the present study, specific PCR primers were designed to amplify two separate alpha-mating-type genes from C. neoformans var. gattii strains. These were used to survey for the presence of the two mating types in clinical and environmental collections of C. neoformans var. gattii strains from Australia. Sixty-eight of 69 clinical isolates produced both alpha mating type-specific bands and were assumed to be of the alpha mating type. The majority of environmental isolates were also of the alpha mating type, but the a mating type was located in two separate areas. In one area, the a mating type outnumbered the alpha mating type by 27:2, but in the second area, the ratio of the two mating types was close to the 50:50 ratio expected for sexual recombination.

Animals↗

Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity.

Short term exposure of the testis to heat causes degeneration of germ cells. However, the mechanisms underlying this process are poorly understood. The major objectives of this study were to determine whether the heat-induced loss of germ cells in the adult rat occurs via apoptosis, to document its stage-specific and cell-specific distribution, and to examine whether intratesticular testosterone (T) plays any role in the stage specificity of heat-induced germ cell death. Testes of adult male Sprague-Dawley rats were exposed to 22 C (control) or 43 C for 15 min. Animals were killed on days 1, 2, 9, and 56 after heat exposure. Germ cell apoptosis was characterized by DNA gel electrophoresis and in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling assay. The incidence of germ cell apoptosis [apoptotic index (AI)] was quite low in control rats (AI = 0.04-0.1). Mild hyperthermia within 1 or 2 days resulted in a marked activation (AI = 4.7-5.6) of germ cell apoptosis predominantly at early (I-IV) and late (XII-XIV) stages. Stages V-VI and VII-VIII were relatively protected from heat-induced apoptosis. Spermatocytes, including pachytenes at stages I-IV and IX-XII, diplotene and dividing spermatocytes at stages XIII-XIV, and early (steps 1-4) spermatids, were most susceptible to heat. On day 9, the majority of the tubules were severely damaged and displayed only a few remaining apoptotic germ cells. By day 56, spermatogenesis was completely recovered, and the incidence of germ cell apoptosis was compatible with the control levels. To determine whether intratesticular T plays a role in protecting germ cells at stages VII-VIII against heat-induced cell death, adult rats were exposed to local testicular heating on day 2 or were given a daily sc injection of GnRH antagonist (GnRH-A) for 4 days with and without a single exposure of testes to heat applied on day 2. By day 4, the incidence of increased germ cell apoptosis at stages other than VII-VIII were not different between heat-treated and GnRH-A- plus heat-treated groups, whereas the control group and the group treated with GnRH-A alone showed minimal apoptosis. GnRH-A addition to heat resulted in a further increase in apoptosis (by 3.2-fold) at stages VII-VIII over the values measured in the heat-treated group, and it became comparable to that at all other stages. Collectively, these results provide evidence that 1) heat induces germ cell apoptosis in a stage-specific and cell-specific fashion; and 2) intratesticular T plays a pivotal role in protecting germ cells at stages VII-VIII against heat-induced cell death. However, the possible involvement of various other factors, including growth factors, thermoprotectants, cytokines, and various death-related proteins, in protecting germ cells against heat-induced apoptosis cannot be ruled out.

Animals↗

Effects of cyclopentenone prostaglandins and related compounds on insulin-like growth factor-I and Waf1 gene expression.

The molecular pathways by which the cyclopentenone prostaglandins (PGA and PGJ series) inhibit cell growth and tumorigenicity are poorly understood. These cellular responses may be caused by specific regulation of growth-related and stress-induced genes. A variety of prostaglandins were tested for their ability to regulate insulin-like growth factor-I (IGF-I) and Waf1 gene expression in C6 rat glioma cells. The prostaglandins (in order of potency) PGJ2 > PGA1 > PGA2, approximately PGD2 >> PGE2 all significantly repressed IGF-I gene expression. With the exception of PGE2, the same prostaglandins that repressed IGF-I also induced Waf1 gene expression. However, the order of potency for Waf1 induction was different than for IGF-I repression: PGA2 > PGA1 approximately PGJ2 > PGD2. The different order of potency of the prostaglandins in regulating IGF-I and Waf1 gene expression suggests that different intracellular signals may be involved in regulating the two genes. Augmentation of glutathione levels by pretreatment of cells with N-acetyl-L-cysteine attenuated the effect of PGA2 on IGF-I and Waf1 gene expression. conversely, depletion of the intracellular glutathione pool by pretreatment with buthionine sulfoximine potentiated the effect of PGA2 on the expression of both genes. These results suggest that conjugation with glutathione prevents the regulation of gene expression by PGA2. We also tested the effect of several simpler compounds that contain a five-membered ring system on IGF-I and Waf1 gene expression. 2-Cyclopenten-1-one, but not cyclopentene or cyclopentene, repressed IGF-I and induced Waf1 gene expression, demonstrating the requirement for an alpha, beta-unsaturated carbonyl for regulation of the two genes. The dione compound 4-cyclopentene-1,3-dione, which has two potentially reactive carbons rather than one, was considerably more potent than 2-cyclopentene-1-one in repressing IGF-I gene expression (IC50 = 30 microM for 4-cyclopentene-1,3-dione as compared with 167 microM for 2-cyclopentene-1-one). Additional results indicated that diethyl maleate, which has two alpha,beta-unsaturated carbonyls in a non-cyclic configuration, also repressed IGF-I gene expression (IC50 = 214 microM) and induced Waf1 gene expression, indicating that the cyclic structure is not required for either effect.

Animals↗

Cauda equina syndrome after incidental total spinal anesthesia with 2% lidocaine.

A 77 year-old male presented for a right popliteal distal vein bypass graft procedure with continuous epidural anesthesia and light general endotracheal anesthesia. After the uneventful placement of the epidural needle and catheter at the L2-L3 interspace, fentanyl 150 microg and a total of 72 ml of preservative-free 2% lidocaine with epinephrine was continually injected through the epidural catheter for the duration of the more than 5 hour procedure. At the end of the procedure, it was noted that the patient had developed total spinal anesthesia, and his pupils were fixed and dilated. Further examination confirmed that the catheter tip was placed in the subarachnoid space. The patient was unable to turn or sit up by himself for over 1 month. Over a 12-month period, he improved to walking with a quad cane, but he required self-catheterization. Although numerous factors were considered, neurotoxicity of 2% lidocaine solution has been discussed as the potential cause. We were unable to find any other factors that could have caused the cauda equina syndrome.

Aged↗

Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53.

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.

Adenoviridae↗

Fourier transform infrared spectrometry: a versatile technique for real world samples.

The versatility of FTIR spectrometry was explored by considering a variety of samples drawn from industrial applications, materials science and biomedical research. These samples included polymeric insulators, bauxite ore, clay, human hair and human skin. A range of sampling techniques suitable for these samples is discussed, in particular FTIR microscopy, FTIR emission spectroscopy, attenuated total reflectance and photoacoustic FTIR spectrometry. The power of modern data processing techniques, particularly multivariate analysis, to extract useful information from spectral data is also illustrated.

Hair↗