Biomedical subjects

L Hoang

Publications and source records attributed to L Hoang.

9 recordsLinked to original sources

PubMed2001

Water magnetic relaxation dispersion in biological systems: the contribution of proton exchange and implications for the noninvasive detection of cartilage degradation.

Magnetic relaxation has been used extensively to study and characterize biological tissues. In particular, spin-lattice relaxation in the rotating frame (T(1rho)) of water in protein solutions has been demonstrated to be sensitive to macromolecular weight and composition. However, the nature of the contribution from low frequency processes to water relaxation remains unclear. We have examined this problem by studying the water T(1rho) dispersion in peptide solutions ((14)N- and (15)N-labeled), glycosaminoglycan solutions, and samples of bovine articular cartilage before and after proteoglycan degradation. We find in model systems and tissue that hydrogen exchange from NH and OH groups to water dominates the low frequency water T(1rho) dispersion, in the context of the model used to interpret the relaxation data. Further, low frequency dispersion changes are correlated with loss of proteoglycan from the extra-cellular matrix of articular cartilage. This finding has significance for the noninvasive detection of matrix degradation.

Amino Acid Sequence↗

PubMed2001

An amino acid code for protein folding.

Direct structural information obtained for many proteins supports the following conclusions. The amino acid sequences of proteins can stabilize not only the final native state but also a small set of discrete partially folded native-like intermediates. Intermediates are formed in steps that use as units the cooperative secondary structural elements of the native protein. Earlier intermediates guide the addition of subsequent units in a process of sequential stabilization mediated by native-like tertiary interactions. The resulting stepwise self-assembly process automatically constructs a folding pathway, whether linear or branched. These conclusions are drawn mainly from hydrogen exchange-based methods, which can depict the structure of infinitesimally populated folding intermediates at equilibrium and kinetic intermediates with subsecond lifetimes. Other kinetic studies show that the polypeptide chain enters the folding pathway after an initial free-energy-uphill conformational search. The search culminates by finding a native-like topology that can support forward (native-like) folding in a free-energy-downhill manner. This condition automatically defines an initial transition state, the search for which sets the maximum possible (two-state) folding rate. It also extends the sequential stabilization strategy, which depends on a native-like context, to the first step in the folding process. Thus the native structure naturally generates its own folding pathway. The same amino acid code that translates into the final equilibrium native structure-by virtue of propensities, patterning, secondary structural cueing, and tertiary context-also produces its kinetic accessibility.

Amino Acids↗

PubMed2000

Evaluation of molecular techniques to biotype Giardia duodenalis collected during an outbreak.

Twenty-seven Giardia duodenalis cyst-positive specimens (human, animal, or drinking water) were obtained from a waterborne outbreak in a community in British Columbia, western Canada. Parasite isolates were characterized using molecular techniques at 4 different steps of organism retrieval. None of the drinking water samples (n = 20) infected gerbils and none was successfully amplified using polymerase chain reaction (PCR). We were able to genotype 4 of 7 (human and animal) isolates by amplification of DNA from original specimens at the triosephosphate isomerase (tpi) gene locus using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Five of the original specimens inoculated into Mongolian gerbils (Meriones unguiculatus) were infective and genotyped at the tpi locus using parasite material collected from the gerbil (cysts and trophozoites). Pulsed field gel electrophoresis (PFGE) was used to biotype trophozoites collected from the gerbils as well as trophozoites from the 4 isolates that adapted to culture. Four of these 5 isolates displayed the same (designated outbreak) biotype at all parasite retrieval steps with all molecular techniques including the originally amplified isolates. PCR-RFLP identified an additional biotype group. The 4 isolates that adapted to in vitro culture were also characterized by isoenzyme electrophoresis (IE). Biotype groups identified in these axenized isolates were all the same with each molecular technique (PCR-RFLP, PFGE, IE) tested. Results of this study demonstrate a need for more sensitive molecular methods to detect and characterize Giardia in original host and environmental samples. Results are also consistent with evidence of biotype changes that occur during the presently used process of isolate retrieval.

Animals↗

PubMed1998

Bacterial endotoxin alters kinetics of BK channels in rat cerebrovascular smooth muscle cells.

Patch-clamp recordings were used to study the effects of Escherichia coli bacterial endotoxin (lipopolysaccharide, LPS) on the properties of large-conductance, Ca2+-dependent K+ channels (BK channels) in the membrane of enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs). LPS had negligible effects on the kinetic and conductance properties of BK channels when applied to the extracellular domain of these channels. However, acute application of LPS (10-100 microg/ml) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability of BK channels, leaving the conductance of these channels unaltered. The magnitude of this effect decreased as the concentration of free Ca2+ at the cytoplasmic membrane face was lowered, but was little affected by changes in membrane potential. Kinetic analysis showed that LPS accelerated reopening of BK channels while having little effect on mean channel open time. Detoxified E. coli LPS, from which the fatty acid chains of Lipid A were partially removed, showed slightly reduced activity when compared to the parent endotoxin molecule. A purified E. coli Lipid A had negligible effects on BK channel function. These results indicate that LPS activates BK channels in cerebrovascular smooth muscle cells when present at the cytoplasmic membrane face. This novel mechanism may provide insights into the regulation of BK channels by intracellular, membrane-associated elements.

Animals↗

PubMed1997

Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics.

The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.

Anaerobiosis↗

PubMed1996

PAH Mutation Analysis Consortium Database: a database for disease-producing and other allelic variation at the human PAH locus.

The PAH Mutation Analysis Consortium (81 investigators, 26 countries) is engaged in mutation detection at the human PAH locus. Ascertainment of probands occurs largely through newborn screening for hyperphenylalaninemia. A relational database records allelic variation (disease-producing and polymorphic) at the locus. Information is distributed by Newsletter, diskette (WINPAHDB software stand-alone executable on IBM compatible hardware), and at a 'real' site on the Worldwide Web (http://www.mcgill.ca/pahdb). The database presently records (Sept. 27, 1995) 248 alleles in 798 different associations (with polymorphic haplotype, geographic region and population) along with additional information. The database, as a record of human genetic diversity, at a particular locus, contributes to the study of human evolution and demic expansion; it also has medical relevance.

Alleles↗

PubMed1996

The phenylalanine hydroxylase locus: a marker for the history of phenylketonuria and human genetic diversity. PAH Mutation Analysis Consortium.

Disease-producing allelic variation describes one aspect of human genetic diversity. Phenylketonuria, the major type of hyperphenylalaninaemia and formerly a functional genetic lethal, has a 2% carrier frequency in temperate-zone populations. Newborn screening for hyperphenylalaninaemia (incidence of 1 in 10000) has made it one of the most widely ascertained human Mendelian traits; 99% of hyperphenylalaninaemia mutations map to the PAH (phenylalanine hydroxylase) gene on 12q24.1, and most cause phenylketonuria. The gene is well characterized. Analysis of 3986 mutant chromosomes by 81 investigators in 26 countries has identified 243 different mutations in 788 different associations (with polymorphic intragenic haplotypes [seven diallelic sites, one short tandem repeat, one variable number of tandem repeats], populations and regions). These data are compiled on a database accessible on the World-Wide Web or as a stand-alone software package. A few phenylketonuria alleles occur at high relative frequencies in particular populations on one or only a few haplotypes, suggesting positive selection in the past. Additional mechanisms (founder effect, drift and recurrent mutation) can explain frequencies and distributions of particular alleles. Allele stratification in Europeans and Orientals implies that mechanism(s) accounting for distribution and high frequencies of PAH alleles were acting before and during demic expansion in Europe and after the European and Oriental radiations.

Genetic Markers↗

PubMed1996

G protein alpha subunit multigene family in the Japanese puffer fish Fugu rubripes: PCR from a compact vertebrate genome.

We compare the complexity and organization of the G protein alpha subunit multigene family in the vertebrate genomes of mammals and the Japanese puffer fish Fugu rubripes. Fourteen Fugu G alpha genes were identified of the 16 genes characterized previously in mammals, including Fugu genes from the four classes of alpha subunits Gs, Gi, Gq, and G12. Fugu and mammalian G alpha coding sequences are highly homologous, and the intron/exon structure of the fish and mammalian orthologs is identical throughout the coding regions. A novel G alpha gene, G alpha p1, was also identified in Fugu rubripes and two other species of puffer fish. The complete sequence of Gnaz and the tandemly duplicated genes Gnai2 and Gnat1 were obtained from a Fugu genomic cosmid library. Introns in the puffer fish G alpha genes lacked repeat DNA sequences, other than simple sequence length repeats, and most introns were significantly shorter in Fugu than in mammalian orthologs. The compact genome of puffer fish provides a unique vertebrate model for characterizing multigene families and identifying novel genes directly from genomic DNA by PCR amplification with degenerate primers. The fact that Fugu encodes most, if not all, of the G protein alpha subunits identified in mammals strongly supports Fugu as a model organism for vertebrate genome research.

Amino Acid Sequence↗

PubMed1983

ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism.

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.

Kinetics↗