Biomedical subjects

Angamuthu Selvapandiyan

Publications and source records attributed to Angamuthu Selvapandiyan.

7 recordsLinked to original sources

PubMed2006

Genetically modified live attenuated parasites as vaccines for leishmaniasis.

Leishmaniasis causes significant morbidity and mortality worldwide and is an important public health problem. Even though it is endemic in developing countries in tropical regions of the world,in recent years economic globalization and increased travel has extended its reach to people in developed countries. Leishmania is usually spread by the bite of the female sandfly. In addition, naïve populations can be exposed to Leishmania infection through transfusion of blood and blood products from infected asymptomatic individuals. There are several clinical forms of leishmaniasis caused by different species of the parasite. In some cases, the only possible cure for this disease is drug treatment. However, prolonged use of such drugs has led to parasite drug resistance. At present there are no effective vaccines against Leishmania. Many vaccine strategies have been pursued, including the use of whole cell lysate, killed, avirulent or irradiated parasites. Additionally, DNA vaccines and purified or recombinant parasite antigens have also been tested. Most of these strategies have shown some degree of effectiveness in animal models but little or no protection in humans. There is now a general consensus among Leishmania vaccine researchers that parasite persistence may be important for effective protective response and could be achieved by live attenuated parasite immunization. In this article we reviewed the efforts in developing genetically defined live attenuated Leishmania parasites as vaccine candidates with the goal of achieving a low level of parasite persistence without being virulent in the host and inducing protective immunity.

Animals↗

PubMed2005

Cloning and characterization of angiotensin converting enzyme related dipeptidylcarboxypeptidase from Leishmania donovani.

We report the first identification, gene cloning, recombinant expression and biochemical characterization of an angiotensin converting enzyme (ACE) related dipeptidylcarboxypeptidase (DCP) in a protozoan parasite. The mammalian counterpart of this enzyme, peptidyl dipeptidase A (a carboxyl dipeptidase) also known as ACE leads to the cleavage of angiotensin I to produce a potent vasopressor. The catalytic enzyme activity of its Escherichia coli DCP counter part can be inhibited by the antihypertensive drug captopril, suggesting that this class of enzymes constitutes a novel target for drugs and vaccines. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding a DCP enzyme for the kinetoplast protozoan Leishmania donovani (LdDCP) that was differentially expressed in promastigote and amastigote stages of the parasite life cycle. Both RNA and protein levels of LdDCP are higher in axenic amastigotes compared to promastigotes. Immuno-fluorescence analysis revealed the cytosolic expression of the protein. Primary structure analysis of LdDCP revealed the presence of an active Zn binding site. When expressed in E. coli, the recombinant enzyme showed carboxy-dipeptidase activity with synthetic substrates. Replacement of two histidine and one glutamic acid at positions 466, 470 and 467, respectively, with alanine residues in its active site resulted in loss of enzyme activity. Captopril, an ACE specific inhibitor was able both to reduce significantly LdDCP enzyme activity and to inhibit promastigote growth. Both its cytosolic location and close homology to DCPs from bacterial species suggests a role in parasite nutrition. Further, identification of LdDCP now provides an opportunity to investigate Leishmania peptidases for their potential as drug and vaccine targets.

Amino Acid Motifs↗

PubMed2005

A novel semiquantitative fluorescence-based multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood.

A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.

Animals↗

PubMed2005

A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.

Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.

Animals↗

PubMed2004

Centrin gene disruption impairs stage-specific basal body duplication and cell cycle progression in Leishmania.

Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes. To explore the role of centrin in the protozoan parasite Leishmania, we created Leishmania deficient in the centrin gene (LdCEN). Remarkably, centrin null mutants (LdCEN(-/-)) showed selective growth arrest as axenic amastigotes but not as promastigotes. Flow cytometry analysis confirmed that the mutant axenic amastigotes have a cell cycle arrest at the G(2)/M stage. The axenic amastigotes also showed failure of basal body duplication and failure of cytokinesis resulting in multinucleated "large" cells. Increased terminal deoxy uridine triphosphate nick end labeling positivity was observed in centrin mutant axenic amastigotes compared with wild type cells, suggesting the activation of a programmed cell death pathway. Growth of LdCEN(-/-) amastigotes in infected macrophages in vitro was inhibited and also resulted in large multinucleated parasites. Normal basal body duplication and cell division in the LdCEN knockout promastigote is unique and surprising. Further, this is the first report where disruption of a centrin gene displays stage-specific/cell type-specific failure in cell division in a eukaryote. The centrin null mutant defective in amastigote growth could be useful as a vaccine candidate against leishmaniasis.

Animals↗

PubMed2004

DNA polymorphism assay distinguishes isolates of Leishmania donovani that cause kala-azar from those that cause post-kala-azar dermal Leishmaniasis in humans.

Leishmania donovani in India causes visceral infection (kala-azar) and dermal infection (post-kala-azar dermal leishmaniasis). We report here the identification of polymorphism in a well-defined genetic locus among the Leishmania parasites causing the visceral and dermal manifestations, in a comparison of 15 post-kala-azar dermal leishmaniasis and 12 kala-azar patient isolates.

Animals↗

PubMed2003

Recombinantly expressed isoenzymic aminopeptidases from Helicoverpa armigera (American cotton bollworm) midgut display differential interaction with closely related Bacillus thuringiensis insecticidal proteins.

Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.

Amino Acid Sequence↗