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Biomedical subjects

A N Smith

Publications and source records attributed to A N Smith.

At least 19 recordsLinked to original sources
PubMed2006

A distinct molecular profile associated with mucinous epithelial ovarian cancer.

Mucinous epithelial ovarian cancers (MOC) are clinically and morphologically distinct from the other histological subtypes of ovarian cancer. To determine the genetic basis of MOC and to identify potential tumour markers, gene expression profiling of 49 primary ovarian cancers of different histological subtypes was performed using a customised oligonucleotide microarray containing >59 000 probesets. The results show that MOC express a genetic profile that both differs and overlaps with other subtypes of epithelial ovarian cancer. Concordant with its histological phenotype, MOC express genes characteristic of mucinous carcinomas of varying epithelial origin, including intestinal carcinomas. Differences in gene expression between MOC and other histological subtypes of ovarian cancer were confirmed by RT-PCR and/or immunohistochemistry. In particular, galectin 4 (LGALS4) was highly and specifically expressed in MOC, but expressed at lower levels in benign mucinous cysts and borderline (atypical proliferative) tumours, supporting a malignant progression model of MOC. Hence LGALS4 may have application as an early and differential diagnostic marker of MOC.

Adenocarcinoma, Mucinous↗
PubMed2005

Laboratory markers associated with progression of HIV infection.

Infection with HIV may develop to AIDS at different rates in different individuals, with a spectrum varying from rapid progression to long-term non-progression. The variable course of HIV-1 infection causes emotional trauma for the infected person and complicates the design and interpretation of therapeutic trials because of unrecognized differences in prognosis. Owing to the variable clinical expression of HIV infection, the use of non-clinical disease markers has become important to patient management. Thus, it is essential to have tests which can accurately assess the stage of infection in an individual, as well as predict its course and monitor its progression. These laboratory tests are valuable during the period of clinical latency and subsequently supplement various clinical parameters.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties.

In recent years, several inhibitors that prevent caspase activation and apoptosis have emerged. At high doses, however, these inhibitors can have nonspecific effects and/or become cytotoxic. In this study, we determined the effectiveness of broad spectrum caspase inhibitors to prevent apoptosis. A carboxy terminal phenoxy group conjugated to the amino acids valine and aspartate (Q-VD-OPh) potently inhibited apoptosis. Q-VD-OPh was significantly more effective in preventing apoptosis than the widely used inhibitors, ZVAD-fmk and Boc-D-fmk, and was also equally effective in preventing apoptosis mediated by the three major apoptotic pathways, caspase 9/3, caspase 8/10, and caspase 12. In addition to the increased effectiveness, Q-VD-OPh was not toxic to cells even at extremely high concentrations. Our data indicate that the specificity, effectiveness, and reduced toxicity of caspase inhibitors can be significantly enhanced using carboxyterminal o-phenoxy groups and may have important uses in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis.

Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initially cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögren's syndrome.

Animals↗
PubMed2003

The pathogenesis of membranoproliferative glomerulonephritis in KwaZulu-Natal, South Africa is unrelated to hepatitis C virus infection.

Idiopathic membranoproliferative glomerulonephritis (MPGN) is a well-defined clinicopathological entity with a poor prognosis, with 50% of patients progressing to end stage renal disease (ESRD) within 10 years. It was reported in about 36% of adult Black patients with nephrotic syndrome in our center previously [Seedat et al. 1988]. Hepatitis C virus (HCV) infection has been shown to be associated with cryoglobulinemic as well as non-cryoglobulinemic (or idiopathic glomerulonephritis). The aim of this study was to determine whether an association exists between HCV infection and idiopathic MPGN in a population with a relatively high prevalence of MPGN. We studied adult patients referred with glomerular disease over a two-year period, 104 patients had primary glomerulonephritis. All 23 (22%) patients with idiopathic MPGN were enrolled, as well as 32 age-matched patients presenting with other primary glomerular diseases. We examined serum from all 55 patients for evidence of HCV antibodies and HCV RNA. None of the 55 patients showed evidence of HCV infection. Chronic renal failure was present in 82.6% of the patients with idiopathic MPGN and it was advanced in 52,2%, who either were dialysis-requiring at presentation or progressed to ESRD soon thereafter; 30.4% had moderate chronic renal failure, while only 17.4% had normal renal function. HCV infection is not associated with idiopathic MPGN in our patients. Idiopathic MPGN remains an idiopathic disease, possibly with a poor prognosis in our population.

Adolescent↗
PubMed2002

Stage of the epidemic and viral phenotype should influence recommendations regarding mother-to-child transmission of HIV-1.

This article argues for a new approach to use of nevirapine in the prevention of vertical transmission of HIV-1. Existing antenatal surveillance should be strengthened to plan geographical allocation, and subsequent evaluation, of a "nevirapine plus" programme. As the epidemic evolves the programme should also and, ideally, care should be tailored to individual women. Underpinning this approach is evidence that a more virulent viral phenotype appears in many patients with advanced HIV-1 infection. This phenotype will become more common at the population level as the epidemic progresses. As efficacy of zidovudine correlates with viral phenotype, and use of the drug may alter phenotype, there is an urgent need for a replacement that is safe to use with nevirapine.

Anti-HIV Agents↗
PubMed2002

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss.

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

Acidosis, Renal Tubular↗
PubMed2001

Use of surgery and electron beam irradiation, with or without chemotherapy, for treatment of vaccine-associated sarcomas in cats: 78 cases (1996-2000).

OBJECTIVE: To evaluate responses of cats with vaccine-associated sarcomas to treatment with surgery and radiotherapy, with or without adjunctive chemotherapy. DESIGN: Retrospective study. ANIMALS: 76 cats (78 tumors). PROCEDURE: Medical records were reviewed. Factors potentially associated with survival time, time to recurrence, and time to development of metastases were evaluated. RESULTS: Following excision, electron beam radiation, and, in some cases, chemotherapy, 32 (41%) cats experienced recurrence, and 9 (12%) cats developed metastases. One- and 2-year survival rates were 86 and 44%, respectively. Median survival time from onset of disease was 730 days (range, 30 to 2,014 days). Median disease-free interval was 405 days (range, 30 to 925 days). Cats that underwent only 1 surgery prior to radiotherapy had a lower recurrence rate than did cats that underwent > 1 surgery and had a significantly longer disease-free interval. Survival time and disease-free interval decreased as time between surgery and the start of radiotherapy increased. Cats that developed metastases had significantly shorter survival times and disease-free intervals than did cats that did not develop metastases. Castrated male cats had a significantly shorter survival time than did spayed female cats. Cats with larger tumors prior to the first surgery had shorter survival times. Twenty-six cats received chemotherapy in addition to surgery and radiotherapy. Whether cats received chemotherapy was not associated with recurrence rate, metastasis rate, or survival time. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that excision followed by electron beam irradiation may be beneficial for treatment of cats with vaccine-associated sarcomas. Extent of excision prior to radiotherapy did not seem to be associated with recurrence rate.

Animals↗
PubMed2001

Molecular cloning and characterization of Atp6n1b: a novel fourth murine vacuolar H+-ATPase a-subunit gene.

The 116-kDa a-subunit of the vacuolar proton pump (H(+)-ATPase) exists as several isoforms encoded by different genes and with different patterns of tissue expression. Its function within the multisubunit pump complex has yet to be elucidated. To date, three isoforms have been identified in mouse (designated a1-a3). We now report the cloning and characterization of Atp6n1b, encoding a novel fourth murine isoform (a4). Murine a4 has 833 residues and shows 85% amino acid identity to the human kidney-specific ATP6N1B protein in which loss-of-function alterations cause autosomal recessive distal renal tubular acidosis. The human and murine genes have similar genomic organization; furthermore, Atp6n1b maps to a region of mouse chromosome 6 that is syntenic with the segment of human 7q33-34 containing ATP6N1B. Together these findings establish the two genes as orthologs. The mouse a4 protein is 61, 52, and 47% identical to a1, a2, and a3, respectively. Phylogenetic analysis confirms that among vertebrates there are four a-subunit families, with a4 most resembling a1. Northern blot analysis of Atp6n1b reveals a 3.7-kilobase a4 transcript in kidney but not other major organs, and a reverse transcription polymerase chain reaction in 12 mouse tissues detects expression in kidney alone. Immunofluorescence studies in murine kidney demonstrate high intensity a4 staining at the surface of intercalated cells, with additional expression in the proximal tubule (not previously reported in human kidney). Similar apical a4 immunostaining is also present in male genital tissue. Identification of this novel murine kidney-enriched 116-kDa a-subunit provides a molecular tool for investigation of the currently unknown role of this protein, which is essential for proper function of the apical renal vacuolar H(+)-ATPase in man.

Amino Acid Sequence↗
PubMed2001

Haplotype tagging for the identification of common disease genes.

Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.

Base Sequence↗
PubMed2001

Characterization of a TGFbeta-responsive human trophoblast-derived cell line.

The placenta is formed by developing trophoblast cells to facilitate fluid, gas and nutrient exchange with the mother. Inappropriate trophoblast responsiveness can lead to life threatening complications during pregnancy including intrauterine growth retardation, pre-eclampsia, spontaneous abortion and malignancy that could lead to fetal loss. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine required for embryonic development and is an important regulator of human trophoblast function. Although TGFbeta is critical for placental and embryonic development, there are currently no established TGFbeta-responsive human trophoblast-derived cell lines available to study the mechanisms by which TGFbeta regulates trophoblast function. Our studies have examined the transformed human trophoblast-derived cell line, ED27, to determine if it is responsive to TGFbeta. Our data indicate that TGFbeta dose responsively and reversibly inhibits cell growth in ED27 cells and induces classic TGFbeta response genes, fibronectin and plasminogen activator inhibitor 1 (PAI-1). TGFbeta also induces an inhibitor of trophoblast invasion, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in ED27 cells. Our studies have identified a human trophoblast-derived cell line that parallels isolated primary human trophoblasts in their responses to TGFbeta. This cell line may provide us with the opportunity to determine TGFbeta-mediated responses on human trophoblast functions not previously possible.

Apoptosis↗
PubMed2001

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.

Africa, Southern↗
PubMed2000

Disseminated infection with Phialemonium obovatum in a German shepherd dog.

A 4-year-old spayed female German Shepherd Dog was evaluated because of left forelimb lameness. A fungal granuloma on the distal portion of the radius was determined to be the cause of the lameness; the infecting organism was identified as Phialemonium obovatum. Despite aggressive treatment with amphotericin B, itraconazole, and ketoconazole and curettage of the local area, the dog developed systemic disease and was euthanatized 5 months after initial evaluation. Immune dysfunction may have played a role in development of disseminated disease, because although serum concentrations of total IgG, IgA, and IgM were within or greater than reference ranges, results of lymphocyte proliferation assays were abnormal, which indicated cellular immune dysfunction. Infection with Phialemonium obovatum should be considered as a differential diagnosis when branching fungal organisms are detected during histologic, cytologic, or microbiologic evaluation of tissue specimens.

Animals↗
PubMed2000

Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing.

The multi-subunit H+-ATPase pump is present at particularly high density on the apical (luminal) surface of -intercalated cells of the cortical collecting duct of the distal nephron, where vectorial proton transport is required for urinary acidification. The complete subunit composition of the apical ATPase, however, has not been fully agreed upon. Functional failure of -intercalated cells results in a group of disorders, the distal renal tubular acidoses (dRTA), whose features include metabolic acidosis accompanied by disturbances of potassium balance, urinary calcium solubility, bone physiology and growth. Mutations in the gene encoding the B-subunit of the apical pump (ATP6B1) cause dRTA accompanied by deafness. We previously localized a gene for dRTA with preserved hearing to 7q33-34 (ref. 4). We report here the identification of this gene, ATP6N1B, which encodes an 840 amino acid novel kidney-specific isoform of ATP6N1A, the 116-kD non-catalytic accessory subunit of the proton pump. Northern-blot analysis demonstrated ATP6N1B expression in kidney but not other main organs. Immunofluorescence studies in human kidney cortex revealed that ATP6N1B localizes almost exclusively to the apical surface of -intercalated cells. We screened nine dRTA kindreds with normal audiometry that linked to the ATP6N1B locus, and identified different homozygous mutations in ATP6N1B in eight. These include nonsense, deletion and splice-site changes, all of which will truncate the protein. Our findings identify a new kidney-specific proton pump 116-kD accessory subunit that is highly expressed in proton-secreting cells in the distal nephron, and illustrate its essential role in normal vectorial acid transport into the urine by the kidney.

Acidosis, Renal Tubular↗
PubMed2000

Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa.

Genotypic analysis of isoniazid (INH) resistance in 79 isolates of M. tuberculosis (MTB) was undertaken by PCR-single strand conformation polymorphism (SSCP), Msp1 restriction enzyme analysis and sequence analysis of specific regions of three genes (part of the coding sequence of katG, and promoter regions of the inhA operon and ahpC) in order to determine the particular allelic variants within these genes. The epidemiologic relatedness was determined using IS6110 and polymorphic G-C region (PGRS (MTB484(1)) based restriction fragment length polymorphism (RFLP). Mutations in katG, inhA locus and ahpC were identified in 77/79, 19/79 and 10/79 isolates respectively. The ability of PCR-SSCP to detect mutations associated with INH resistance in katG, inhA and ahpC genes was 100% (CI 91.2-99.7%), 98.7% (CI 74.0-99.9%), and 100% (CI 69.2-100%) respectively. Specificity was 100%. All isolates with mutations in the 209 bp fragment of the MTB katG gene containing the Ser315Thr codon were positive by PCR-RFLP using Msp1 enzyme restriction analysis. Sixteen of 19 isolates with alterations on the 3' end of the ribosome binding site upstream of mabA in inhA locus simultaneously harbored Ser315Thr mutations in KatG. In 9/10 isolates, mutations in the ahpC promoter region were located in the 105 bp oxyR-ahpC intergenic region. None of 17 INH drug susceptible isolates harbored mutations in any of the three genetic regions, although the katG1 allele (Arg 463 Leu) was present in one isolate. Characterization by IS6110/PGRS(MTB484(1))RFLP analysis revealed that a number of drug resistant clones are widespread in the community. We conclude that the frequency of the Ser315Thr katG mutation in the local strain population makes the PCR-RFLP MTB katG assay a reliable, rapid and useful method for detecting INH resistance.

Alleles↗
PubMed2000

Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.

Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.

Base Sequence↗