Biomedical subjects

S V Nguyen

Publications and source records attributed to S V Nguyen.

At least 19 recordsLinked to original sources

PubMed2001

Large-scale analysis of the Alu Ya5 and Yb8 subfamilies and their contribution to human genomic diversity.

We have utilized computational biology to screen GenBank for the presence of recently integrated Ya5 and Yb8 Alu family members. Our analysis identified 2640 Ya5 Alu family members and 1852 Yb8 Alu family members from the draft sequence of the human genome. We selected a set of 475 of these elements for detailed analyses. Analysis of the DNA sequences from the individual Alu elements revealed a low level of random mutations within both subfamilies consistent with the recent origin of these elements within the human genome. Polymerase chain reaction assays were used to determine the phylogenetic distribution and human genomic variation associated with each Alu repeat. Over 99 % of the Ya5 and Yb8 Alu family members were restricted to the human genome and absent from orthologous positions within the genomes of several non-human primates, confirming the recent origin of these Alu subfamilies in the human genome. Approximately 1 % of the analyzed Ya5 and Yb8 Alu family members had integrated into previously undefined repeated regions of the human genome. Analysis of mosaic Yb8 elements suggests gene conversion played an important role in generating sequence diversity among these elements. Of the 475 evaluated elements, a total of 106 of the Ya5 and Yb8 Alu family members were polymorphic for insertion presence/absence within the genomes of a diverse array of human populations. The newly identified Alu insertion polymorphisms will be useful tools for the study of human genomic diversity.

Alu Elements↗

PubMed2001

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR.

Proper cellular response to genotoxic insult often requires the activity of one or more members of a family of high-molecular weight protein kinases referred to as phosphatidylinositol-3 kinase (PIK)-like proteins. While catalytic activity is an indispensable part of PIK-like protein function, little is currently known about factors that control their activity and/or functions. This deficiency stems, in large part, from our lack of knowledge concerning functionally significant subdomains within the large non-catalytic domain of these proteins. We have determined that the transcript encoding the PIK-like protein ATR undergoes alternate splicing within the region of the mRNA encoding its non-catalytic domain. This conclusion is based on the sequencing of a human expressed sequence tag clone encoding a portion of the ATR cDNA, and is supported by the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays conducted on total and polyA+ RNA, as well as sequencing of cloned RT-PCR products. Cloning and sequencing of a segment of human genomic DNA indicated that this event arises from splicing of a single 192 bp exon within the ATR gene. Analysis of several human tissues indicated that alternate ATR transcripts are differentially expressed, suggesting that this region of the ATR protein may be of functional importance.

Alternative Splicing↗

PubMed2001

Patterns of ancestral human diversity: an analysis of Alu-insertion and restriction-site polymorphisms.

We have analyzed 35 widely distributed, polymorphic Alu loci in 715 individuals from 31 world populations. The average frequency of Alu insertions (the derived state) is lowest in Africa (.42) but is higher and similar in India (.55), Europe (.56), and Asia (.57). A comparison with 30 restriction-site polymorphisms (RSPs) for which the ancestral state has been determined shows that the frequency of derived RSP alleles is also lower in Africa (.35) than it is in Asia (.45) and in Europe (.46). Neighbor-joining networks based on Alu insertions or RSPs are rooted in Africa and show African populations as separate from other populations, with high statistical support. Correlations between genetic distances based on Alu and nuclear RSPs, short tandem-repeat polymorphisms, and mtDNA, in the same individuals, are high and significant. For the 35 loci, Alu gene diversity and the diversity attributable to population subdivision is highest in Africa but is lower and similar in Europe and Asia. The distribution of ancestral alleles is consistent with an origin of early modern human populations in sub-Saharan Africa, the isolation and preservation of ancestral alleles within Africa, and an expansion out of Africa into Eurasia. This expansion is characterized by increasing frequencies of Alu inserts and by derived RSP alleles with reduced genetic diversity in non-African populations.

Africa↗

PubMed2001

Phylogenetic analysis of the Friedreich ataxia GAA trinucleotide repeat.

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome.

Alleles↗

PubMed2001

Alu insertion polymorphisms for the study of human genomic diversity.

Genomic database mining has been a very useful aid in the identification and retrieval of recently integrated Alu elements from the human genome. We analyzed Alu elements retrieved from the GenBank database and identified two new Alu subfamilies, Alu Yb9 and Alu Yc2, and further characterized Yc1 subfamily members. Some members of each of the three subfamilies have inserted in the human genome so recently that about a one-third of the analyzed elements are polymorphic for the presence/absence of the Alu repeat in diverse human populations. These newly identified Alu insertion polymorphisms will serve as identical-by-descent genetic markers for the study of human evolution and forensics. Three previously classified Alu Y elements linked with disease belong to the Yc1 subfamily, supporting the retroposition potential of this subfamily and demonstrating that the Alu Y subfamily currently has a very low amplification rate in the human genome.

Alu Elements↗

PubMed2001

Genetic evidence on the origins of Indian caste populations.

The origins and affinities of the approximately 1 billion people living on the subcontinent of India have long been contested. This is owing, in part, to the many different waves of immigrants that have influenced the genetic structure of India. In the most recent of these waves, Indo-European-speaking people from West Eurasia entered India from the Northwest and diffused throughout the subcontinent. They purportedly admixed with or displaced indigenous Dravidic-speaking populations. Subsequently they may have established the Hindu caste system and placed themselves primarily in castes of higher rank. To explore the impact of West Eurasians on contemporary Indian caste populations, we compared mtDNA (400 bp of hypervariable region 1 and 14 restriction site polymorphisms) and Y-chromosome (20 biallelic polymorphisms and 5 short tandem repeats) variation in approximately 265 males from eight castes of different rank to approximately 750 Africans, Asians, Europeans, and other Indians. For maternally inherited mtDNA, each caste is most similar to Asians. However, 20%-30% of Indian mtDNA haplotypes belong to West Eurasian haplogroups, and the frequency of these haplotypes is proportional to caste rank, the highest frequency of West Eurasian haplotypes being found in the upper castes. In contrast, for paternally inherited Y-chromosome variation each caste is more similar to Europeans than to Asians. Moreover, the affinity to Europeans is proportionate to caste rank, the upper castes being most similar to Europeans, particularly East Europeans. These findings are consistent with greater West Eurasian male admixture with castes of higher rank. Nevertheless, the mitochondrial genome and the Y chromosome each represents only a single haploid locus and is more susceptible to large stochastic variation, bottlenecks, and selective sweeps. Thus, to increase the power of our analysis, we assayed 40 independent, biparentally inherited autosomal loci (1 LINE-1 and 39 Alu elements) in all of the caste and continental populations (approximately 600 individuals). Analysis of these data demonstrated that the upper castes have a higher affinity to Europeans than to Asians, and the upper castes are significantly more similar to Europeans than are the lower castes. Collectively, all five datasets show a trend toward upper castes being more similar to Europeans, whereas lower castes are more similar to Asians. We conclude that Indian castes are most likely to be of proto-Asian origin with West Eurasian admixture resulting in rank-related and sex-specific differences in the genetic affinities of castes to Asians and Europeans.

Adult↗

PubMed2000

Potential gene conversion and source genes for recently integrated Alu elements.

Alu elements comprise >10% of the human genome. We have used a computational biology approach to analyze the human genomic DNA sequence databases to determine the impact of gene conversion on the sequence diversity of recently integrated Alu elements and to identify Alu elements that were potentially retroposition competent. We analyzed 269 Alu Ya5 elements and identified 23 members of a new Alu subfamily termed Ya5a2 with an estimated copy number of 35 members, including the de novo Alu insertion in the NF1 gene. Our analysis of Alu elements containing one to four (Ya1-Ya4) of the Ya5 subfamily-specific mutations suggests that gene conversion contributed as much as 10%-20% of the variation between recently integrated Alu elements. In addition, analysis of the middle A-rich region of the different Alu Ya5 members indicates a tendency toward expansion of this region and subsequent generation of simple sequence repeats. Mining the databases for putative retroposition-competent elements that share 100% nucleotide identity to the previously reported de novo Alu insertions linked to human diseases resulted in the retrieval of 13 exact matches to the NF1 Alu repeat, three to the Alu element in BRCA2, and one to the Alu element in FGFR2 (Apert syndrome). Transient transfections of the potential source gene for the Apert's Alu with its endogenous flanking genomic sequences demonstrated the transcriptional and presumptive transpositional competency of the element.

Alleles↗

PubMed1999

Hypoxia regulates the expression of the adrenomedullin and HIF-1 genes in cultured HL-1 cardiomyocytes.

Adrenomedullin (AM) is a hypotensive protein expressed in a variety of cells and tissues. We observed previously that the expression of the adrenomedullin gene increases substantially in the developing rat heart and in cultured adult rat ventricular cardiac myocytes in response to hypoxia as a function of time. An adrenomedullin promoter-luciferase reporter construct was used to show that this increase in adrenomedullin mRNA resulted from increased transcription in response to hypoxia. We report here additional evidence documenting that this hypoxia-induced transcription of the adrenomedullin gene is regulated by the hypoxia-inducible factor-1 (HIF-1) transcription factor. We used Northern blot analysis to show an increase in the levels of AM and HIF-1alpha mRNA but not HIF-1beta mRNA in the HL-1 cardiac myocyte cell line in response to hypoxia. Furthermore, Western blot analysis revealed that the levels of both HIF-1alpha and HIF-1beta protein increased under hypoxic conditions. Data from electrophoretic mobility shift assays indicate that the heterodimeric HIF-1 complex binds to the HIF-1-responsive elements. Combined data from these studies demonstrate that the AM gene is regulated by hypoxia-responsive elements localized in the AM promoter region.

Adrenomedullin↗

PubMed1999

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene.

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.

Animals↗

PubMed1999

Characterization of the Coxiella burnetti sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase.

The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a lambda EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced. The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N-terminal region was only 42%. The gene was expressed in E. coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS-PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever. The study results suggest that the C. burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever.

Acyltransferases↗

PubMed1999

Sequence comparison of the VP2 variable region of infectious bursal disease virus isolates from Vietnam.

The variable region in the VP2 gene of twenty-three infectious bursal disease virus (IBDV) isolates, collected in Vietnam in 1997 and 1998, was amplified as cDNA by using the reverse transcription-polymerase chain reaction and sequenced. Analysis of amino acid substitutions and phylogenetic relationships of the deduced amino acid sequences (residues 206-350) showed that the nineteen Vietnamese vv IBDVs clustered with the European vv IBDVs, Japanese vv IBDVs and Chinese vv strains, and that the four vietnamese virulent strains were closely related to European virulent strain 52/70. These results suggest that Vietnamese vv IBDVs, European vv IBDVs, Japanese vv IBDVs and Chinese vv strains have the same origin.

Amino Acid Sequence↗

PubMed1999

Recently integrated human Alu repeats: finding needles in the haystack.

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution.

Animals↗

PubMed1998

Adrenomedullin gene expression is developmentally regulated and induced by hypoxia in rat ventricular cardiac myocytes.

Adrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at -1095 and -770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O2) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5'-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at -1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter.

Adrenomedullin↗

PubMed1998

Antigenic characteristics of polypeptides of Coxiella burnetii isolates.

Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti-C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.

Animals↗

PubMed1998

Clinical evaluation of a new PCR assay for detection of Coxiella burnetii in human serum samples.

A nested PCR method was developed for the detection of Coxiella burnetii in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the com1 gene encoding a 27-kDa outer membrane protein of C. burnetii. The primers amplified the predicted fragments of 21 various strains of C. burnetii but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes SspI and SalI. The utility of the nested PCR was evaluated by testing human serum samples. The com1 gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the com1 gene appeared to be a sensitive, specific, and useful method for the detection of C. burnetii in serum samples.

Antibodies, Bacterial↗

PubMed1997

Evaluation of the high-density agglutination test for Coxiella burnetii antibodies in animals.

The usefulness of the high-density particle agglutination (HDPA) test as a potential tool for the detection of anti-Coxiella burnetii antibodies in animal sera was studied by using 619 cow, 589 dog, and 150 cat serum samples and antisera from rabbits, guinea pigs, and mice. The sensitivity and specificity of the test versus those of the reference microimmunofluorescence test were determined at two different threshold titer values. At the cutoff value of 1:16, the sensitivities of the HDPA test for cow, dog, and cat sera were 94.3, 95, and 91.3%, respectively, and the specificities were 95.5, 95.3, and 91.3%, respectively. At the cutoff value of 1:32, the sensitivities were 86.7, 88.3, and 82.6%, respectively, and the specificities were 99, 99.2, and 98.4%, respectively. For the group of immune laboratory animals all samples from rabbits, guinea pigs, and mice were positive by the HDPA test. The erythrocyte-sensitizing substance from phase II C. burnetii was found to contain protein and carbohydrate, and both fractions are immunoreactive. The study results show that the HDPA test is a useful tool in the epizootiological survey of Coxiella infection in animals.

Agglutination Tests↗

PubMed1996

Rapid method for detection of Coxiella burnetii antibodies using high-density particle agglutination.

A high-density particle agglutination test, using erythrocyte-sensitizing substance from phase II Coxiella burnetii adsorbed to high-density composite particles, was developed for rapid serodiagnosis of Q fever. The test was compared with the microimmunofluorescence test for sensitivity and specificity by using 3,036 human serum samples collected in Gifu Prefecture, Japan. An excellent agreement was found between the two tests for the acute-phase group and paired serum samples, but some discordant results were observed in the single-sample group. The sensitivity and specificity of the high-density particle agglutination test were both 100% in the former group and 81.6 and 99.9%, respectively, in the latter group. The test is a very promising tool for routine serodiagnosis of Q fever because of its simplicity, sensitivity, and specificity.

Agglutination Tests↗

PubMed1996

Q fever pneumonia in children in Japan.

The prevalence of Q fever pneumonia among children with atypical pneumonia from whom only an acute-phase serum sample was available was traced by using an indirect immunofluorescence (IF) test, nested PCR, and isolation. Twenty (34.5%) of 58 sera were found to have both polyvalent and immunoglobulin M antibodies to the phase II antigen of Coxiella burnetii by the IF test. Q fever pneumonia was present in 23 (39.7%) of 58 patients as determined by both the nested PCR and isolation and in 20 patients as determined by the IF test. The sensitivities for nested PCR and isolation were 100%, and that for the IF test was 87%. Our results indicate that nested PCR was faster and more sensitive than isolation and the IF test in the diagnosis of acute Q fever when a single acute-phase serum was available. These findings suggest that C. burnetii is an important cause of atypical pneumonia in children in Japan.

Antibodies, Bacterial↗