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Biomedical subjects

Brian Ward

Publications and source records attributed to Brian Ward.

6 recordsLinked to original sources
PubMed2006

Development of a genetic assay to distinguish between Leishmania viannia species on the basis of isoenzyme differences.

BACKGROUND: Tegumentary leishmaniasis in Latin America is caused mainly by Leishmania viannia braziliensis complex parasites. L. braziliensis and Leishmania viannia peruviana are the 2 predominant Leishmania species in Peru. L. braziliensis is more virulent, because it can cause mucocutaneous leishmaniasis, known as espundia, that results in severe facial destruction. Early identification of the species that causes the initial cutaneous infection would greatly help to prevent mucocutaneous leishmaniasis, because it would allow more aggressive treatment and follow-up. However, because of the close genetic similarity of L. braziliensis and L. peruviana, there currently exists no simple assay to distinguish between these species. METHODS: We cloned the mannose phosphate isomerase gene from both L. braziliensis and L. peruviana. It is the only known isoenzyme capable of differentiating between L. braziliensis and L. peruviana in multilocus enzyme electrophoresis. Interestingly, only a single nucleotide polymorphism was found between the mannose phosphate isomerase genes from L. braziliensis and L. peruviana, resulting in an amino acid change from threonine to arginine at amino acid 361. A polymerase chain reaction assay was developed to distinguish the single nucleotide polymorphism of the mannose phosphate isomerase gene to allow for the specific identification of L. braziliensis or L. peruviana. RESULTS: This assay was validated with 31 reference strains that were previously typed by multilocus enzyme electrophoresis, successfully applied to patient biopsy samples, and adapted to a real-time polymerase chain reaction assay. CONCLUSIONS: This innovative approach combines new genetic knowledge with traditional biochemical fundamentals of multilocus enzyme electrophoresis to better manage leishmaniasis in Latin America.

Amino Acid Sequence↗
PubMed2002

HLA-G exhibits low level of polymorphism in indigenous East Africans.

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I antigen that is predominantly expressed on invasive cytotrophoblastic cells, and is postulated to be a mediator of maternal-fetal tolerance. Almost all studies in Caucasian and Asian populations have consistently reported that HLA-G exhibits low levels of allelic polymorphism unlike the classical class I genes. However, the concept that HLA-G is nonpolymorphic has recently been challenged in a single study of African-American subjects. We have examined the DNA sequences of the first seven HLA-G exons by single-strand conformational polymorphism (SSCP) and DNA direct sequencing procedures in 45 healthy individuals from an indigenous African population. Overall, we detected 14 sequence variations: 3 in the signal peptide (exon 1); 2 in the alpha-1 domain (exon 2); 5 in the alpha-2 domain (exon 3); 2 in alpha-3 domain (exon 4); 2 in transmembrane domain (exon 5); and none in the cytoplasmic tail (exons 6 and 7). Of these variants, only three result in amino acid substitutions at the protein level. Of particular interest, we identified a novel nucleotide substitution (C727T), 56 bp before the HLA-G gene transcription start site, located in the putative binding site for polyomavirus enhancer-binding protein 2 (PEBP2) transcriptor factor. These data confirm previous reports describing HLA-G exhibiting limited allelic polymorphism. Further studies are needed to determine the impact of the C727T polymorphism on the level or developmental regulation of HLA-G expression.

Africa, Eastern↗
PubMed2002

Exploring ligand-DNA space using type IIS restriction enzymes.

Investigating ligand-DNA interactions using type IIS restriction enzymes (IISRE) as footprinting reagents is reviewed and contemplated. Ligand binding at a IISRE's cleavage but not sequence recognition site protects DNA from strand scission. This spatial arrangement has been exploited in the development of qualitative (combinatorial) and quantitative ligand-DNA investigative methods collectively termed Type IIS Restriction Enzyme Footprinting (cIISREF and qIISREF respectively). In cIISREF, the consensus binding sequence of a ligand is sought by using a IISRE to segregate combinatorial library members that are bound by ligand from those that are not. A PCR is performed following the segregation step to enrich the library in ligand binding (i.e. uncut) sequences. It might be possible that diversities approaching 10(30) unique sequences could be simultaneously searched using this homogeneous and biologically relevant method. For qIISREF, a ligand-DNA equilibrium constant is measured by quantifying the amounts of target and control DNA IISRE cleavage products as a function of ligand concentration. The control sequence is engineered to not bind ligand. Along with illustrating these methods by reviewing published works, current concerns and future prospects for IISREF are discussed.

Base Sequence↗