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Y D Sharma

Publications and source records attributed to Y D Sharma.

At least 19 recordsLinked to original sources
PubMed2005

Genetic alteration in drug resistance markers of Plasmodium falciparum.

Plasmodium falciparum shows plasticity in its genome. For its survival it can delete certain genes (or portions) if not needed for its growth and has the capability to regulate its genes under various stages of its life cycle as well as under unfavourable environmental conditions. Parasite shows enormous amount of antigenic variation under immune pressure leading to the emergence of vaccine resistant strains. Similarly, under drug pressure it allows mutations to settle in the target genes. It is becoming more and more clear that with the continuous exposure to a drug, the parasite accumulates more and more number of mutations in these genes. By measuring the number of these point mutations among field isolates one can predict the efficacy of a particular drug. Therefore, these markers are useful tools at epidemiological level. This molecular surveillance can also help in slowing down the drug resistance if supported by a careful drug usage policy. Further studies are required to develop molecular markers for rest of the antimalarial drugs as well as the improvement on the existing molecular tools for accurate and rapid detection of drug resistant malaria.

Animals↗
PubMed2003

Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparum among chloroquine responders in India.

Chloroquine-resistant Plasmodium falciparum needs to be monitored in the field for effective malaria control strategies. A point mutation K76T in the P. falciparum chloroquine resistance transporter (Pfcrt) protein has recently been proposed as a molecular marker for the faster detection of chloroquine-resistant falciparum malaria in field. We describe here the evaluation of this marker in Indian P. falciparum isolates. A total of 274 Indian P. falciparum isolates were analyzed for the K76T mutation. This mutation was detected in all the clinical isolates obtained from the in vivo chloroquine non-responders. But majority of the clinical isolates from chloroquine responders (71 of 74 patients, i.e. 96%) also harbored this mutation. The K76T mutation was indeed highly prevalent (91%) among 213 clinical isolates. There was a significant association between K76T mutation and the in vitro chloroquine response (P<0.05) but six isolates showed discordant results. In conclusion, the K76T mutation fails to differentiate majority of the chloroquine responders from that of the non-responders and thus will be of limited use in the field in India.

Adolescent↗
PubMed2001

Antibodies detected against Plasmodium falciparum haemozoin with inhibitory properties to cytokine production.

Haemozoin, the malaria pigment, regulates the synthesis of several host cytokines and has been found to be associated with the disease severity. Here we describe that malarial patients produce a significant amount of anti-haemozoin IgM antibodies. Levels of these antibodies were higher among the complicated Plasmodium falciparum cases compared to the non-complicated P. falciparum group and Plasmodium vivax patients. The P. falciparum haemozoin also induced the synthesis of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) by the monocytes of the healthy individuals, but the production of these cytokines by the monocytes was inhibited in the presence of the anti-haemozoin IgM antibodies. Therefore, it seems that the host produces these antibodies (mainly IgM types) during malarial infection that can influence the progression of the disease by inhibiting the production of cytokines.

Animals↗
PubMed2001

Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among Indian isolates of Plasmodium falciparum.

The malaria parasite contains a nuclear genome with 14 chromosomes and two extrachromosomal DNA molecules of 6 kb and 35 kb in size. The smallest genome, known as the 6 kb element or mitochondrial DNA, has been sequenced from several Plasmodium falciparum isolates because this is a potential drug target. Here we describe the complete nucleotide sequence of this element from an Indian isolate of P. falciparum. It is 5967 bp in size and shows 99.6% homology with the 6 kb element of other isolates. The element contains three open reading frames for mitochondrial proteins-cytochrome oxidase subunit I (CoI), subunit III (CoIII) and cytochrome b (Cyb) which were found to be expressed during blood stages of the parasite. We have also sequenced the entire cyb gene from several Indian isolates of P. falciparum. The rate of mutation in this gene was very low since 12 of 14 isolates showed the identical sequence. Only one isolate showed a maximum change in five amino acids whereas the other isolate showed only one amino acid change. However, none of the Indian isolates showed any change in those amino acids of cyb which are associated with resistance to various drugs as these drugs are not yet commonly used in India.

Amino Acid Sequence↗
PubMed2001

Allelic variation in the cg2 gene does not correlate with chloroquine resistance among Indian Plasmodium falciparum isolates.

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR amplification and sequencing of all the four repeat regions (kappa (kappa), gamma (gamma), psi (psi) and omega (omega)) of this gene, from Indian isolates. There were variant forms for each of these repeat regions (two for kappa and gamma, and three for psi and omega) among the 123 Indian isolates of P. falciparum. Among these isolates certain forms of psi and omega repeats were uniquely present while some of the reported forms of the kappa and omega repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11 different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the gamma and omega domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine resistance.

Alleles↗
PubMed2001

Computer modeling of small heat-shock metalloprotease of the human malaria parasite Plasmodium vivax.

We present here computer generated model of N-terminal fragment, amino acids (aa) 36-245, of a Plasmodium vivax heat shock metalloprotease called PVHSP28, whose gene was cloned and characterised earlier. The fragment showed homology with HSPs from many organisms, including Escherichia coli and Haemophilus influenzae. PVHSP28 had the signature sequence 'HEXXH' and 'EXXXD' of Zinc metalloproteases. Being the first malarial HSP possessing metalloprotease activity, PVHSP28 is an ideal target for the design of new anti-malarial drugs. However, except for a small region (aa 62-132) which had 24.6% sequence similarity with 1TAQ (a DNA polymerase), it did not show sequence similarity with any published structures in protein data bank. Hence it could not be modelled using any automated modeling programs. We modelled 36-245 aa of PVHSP28 using predicted secondary structure as well as experimentally determined and predicted properties of the protein on the basis of its amino acid sequence, using various Internet tools and in-house package MODEL. The model was energy minimised using Sander's module of AMBER 5.0, working on a Silicon Graphics machine, with all atom force field.

Amino Acid Sequence↗
PubMed2001

Epidemiologic investigations of a malaria outbreak in northern Delhi area.

Epidemiologic investigations revealed a 56.7 and 13.32% slide positivity rate in febrile and afebrile malaria cases, respectively. In both cases, Plasmodium falciparum was predominant. Anopheles culicifacies resistant to dichlorodiphenyltrichloroethane and benzene hexachloride (hexachlorocyclohexane) was found breeding profusely in pools and ponds created by excavation of earth around brick kiln in the region. Furthermore, children were not found to be producing significant levels of antibodies and a large percentage of patients harbored chloroquine-resistant parasites. Also, more than 1 P. falciparum strain was present in the population. We detected 2 strains, VI and VII, of which type VI was predominant.

Adolescent↗
PubMed2000

Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro.

PURPOSE: To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses. METHODS: Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed. RESULTS: At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium. CONCLUSIONS: Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.

Animals↗
PubMed2000

Metalloprotease activity in a small heat shock protein of the human malaria parasite Plasmodium vivax.

The malaria parasite affects millions of people each year, lives and multiplies in two different hosts, and synthesizes a large number of proteases and heat shock proteins (HSPs) for its survival. We describe here the characterization of a metalloprotease activity which resides in the small HSP (PVHSP28) of the common but noncultivable human malaria parasite Plasmodium vivax. The protein is expressed by erythrocytic stages of the parasite. It is expressed as a approximately 55-kDa polypeptide which is then processed to the 28-kDa mature protein. The latter was found to be an active protease in gelatin zymography. This protease showed its optimal activity at 37 degrees C (pH 7.6). It also retained its proteolytic activity at higher temperatures of up to 55 degrees C. The enzyme belongs to the metalloprotease class, as its proteolytic activity was most effectively blocked by 1,10-phenanthroline and was restored to a maximal level by the addition of zinc metal ions. Inhibitors for the cysteine, serine, and aspartate classes of proteases were ineffective against this enzyme. A homology search indicates that PVHSP28 probably belongs to a new class of HSPs which possess the metalloprotease signature sequence.

Amino Acid Sequence↗
PubMed1999

Genetic polymorphism of falciparum malaria vaccine candidate antigen genes among field isolates in India.

The present study was designed to investigate the genetic diversity of Plasmodium falciparum among field isolates from India. A total of 71 clinical isolates were analyzed by the polymerase chain reaction (PCR) for the amplification of repeat regions of malaria vaccine candidate antigen genes, i.e., merozoite surface antigen-1 (MSA-1), MSA-2, and circumsporozoite protein (CSP). All three genes showed variation; MSA-2 has the maximum number of 10 variant forms while MSA-1 and CSP had 8 and 6 variants, respectively. Some variant forms were more common than others among the clinical isolates. There were mixed alleles for each gene in several (27 of 71) cases. The MSA-2 gene showed the maximum number of cases with mixed alleles (22 of 65 [33.85%]) compared with MSA-1 (10 of 68 [14.7%]) and CSP (10 of 65 [15.38%]). Fifty-five (88.7%) of 62 clinical isolates of P. falciparum showed a different genotype. The malaria hyperendemic region (Orissa) not only showed the maximum number of variant forms of each gene but also the maximum number of cases with mixed alleles compared with the non-hyperendemic regions (Madhya Pradesh and Rajasthan). The presence of such large numbers of P. falciparum strains in India should be taken into account in future malaria vaccine programs.

Alleles↗
PubMed1998

Alu elements in a Plasmodium vivax antigen gene.

Plasmodium vivax is a very common human malaria parasite but it is poorly characterized at the molecular level. Here, we describe the isolation and characterization of an antigen coding gene of P. vivax which contains Alu elements. This gene, called Pv-Alu, is expressed during the erythrocytic phase of the parasite. The encoded 200 amino acid long polypeptide is highly hydrophobic, contains transmembrane domains, and is rich in leucine (19.4%), serine (15.9%), proline (15.4%) and phenylalanine (15.4%). The 5'-untranslated region and part of the 3'-end coding region of Pv-Alu show significant homology to different Alu families. The presence of Alu elements in the coding region of a parasite antigen gene is significant from a functional and evolutionary viewpoint.

Amino Acid Sequence↗
PubMed1998

Partial sequence analysis of a Plasmodium vivax cation transporting ATPase gene homologue & a putative pseudohomologue.

Molecular characterization of P. vivax is essential to develop suitable antimalarial drugs and vaccines. We describe here isolation and sequence analysis of a partial cDNA of a calcium ATPase as well as a putative pseudogene from this parasite. The immunoscreening of lambda gtll- P. vivax DNA library with patients serum has earlier resulted in the isolation of several seroreactive clones including Pv14. This clone contains a 299 bp insert having 18 amino acids (aa) reading frame fused with beta galactosidase. A larger fragment of approximately 15 kb was isolated from the EMBL3 library for Pv14 but it had only 2 extra aa in its reading frame. The far upstream region of Pv14 revealed a 101aa long putative open reading frame (ORF) showing homology to a variety of calcium ATPases in the M8 and M9 transmembrane region. But in the absence of a transcript in the parasite could indicate that it represents a pseudogene. However, the real gene for calcium ATPase in P. vivax was detected by RT-PCR using degenerate primers, designed from the conserved sequences of energy transduction and phosphorylation domains. The amplified cDNA-PCR product of 550 bp was cloned and sequenced which showed a significant aa homology to the calcium ATPase4 of P. falciparum. The present study, therefore, establishes the existence of calcium ion pumps in P. vivax which will be useful in drug development.

Amino Acid Sequence↗
PubMed1997

Variations in the C-terminal repeats of the knob-associated histidine-rich protein of Plasmodium falciparum.

The knob-associated histidine rich protein (KAHRP) of Plasmodium falciparum plays an important role in the pathophysiology of cerebral malaria. In the present study, the immunogenic C-terminal repeat domain of the KAHRP gene was amplified, cloned and sequenced from the Indian (RJ181) and Honduran (HB3) isolates of P. falciparum. Based on the number and types of repeats in the domain, we report here the presence of three unique variant forms of KAHRP among these isolates. The Indian isolate (RJ181) contained four units of the decapeptide repeats whereas the Honduran isolate (HB3) contained two forms i.e. one form containing four decapeptide repeats plus a tetrapeptide subunit and the other form containing three decapeptide repeats plus a tetrapeptide subunit. Thus, all together, the number of KAHRP variants is increased to five which includes previously described two variants, each containing either 3 or 5 decapeptide repeats. This high rate of variability in the antigenic domain of the KAHRP gene via deletion or addition of whole or part of the decapeptide units could be involved in the evasion of host immune system possibly by providing the speculative complementarity to the vargene product. The results of the present study will be useful in designing the suitable molecular therapeutic reagents for cerebral malaria.

Amino Acid Sequence↗
PubMed1997

Knob proteins in falciparum malaria.

Knob proteins play a significant role in the pathophysiology of cerebral malaria caused by Plasmodium falciparum. Most of these proteins are of parasite origin and can be divided into two major classes: (i) the cytoadherent proteins present at the surface of the knobs; and (ii) the submembranous structural proteins which are placed towards the cytoplasmic side in the knobs. Several surface proteins [viz., P. falciparum-infected erythrocyte membrane protein-1 (PFEMP-1), sequestrin, pfalhesin] and submembranous structural proteins [viz., knob-associated histidine-rich protein (KAHRP), PFEMP-2, PFEMP-3] of the knobs have been identified and characterized to a certain extent. The structural proteins interact with several host (e.g., spectrin, actin, band 4.1 etc.) as well as parasite (e.g., PFEMP-1) molecules to produce functional knobs. The surface proteins on the other hand interact with several adhesion molecules of the endothelial cell through receptor-ligand type of binding. Knob proteins are important from the point of view of malaria control since immunotherapeutic agents can be developed to block as well as reverse the cytoadherence phenomenon. The surface proteins are also good vaccine candidates except that they show a high rate of antigenic variation. Nevertheless, the use of ribozyme or antisense oligonucleotides to inhibit the expression of knob proteins (e.g., KAHRP alone or with surface protein) can be used as a molecular therapeutic agent.

Animals↗
PubMed1996

High prevalence of chloroquine resistant Plasmodium falciparum infection in Rajasthan epidemic.

Plasmodium falciparum is the main killer among all human malaria parasites. In 1994, there was a falciparum malaria epidemic in Rajasthan, India, with many deaths. We have investigated active falciparum malaria cases from this epidemic and found that most of the parasite isolates (95%) were resistant to chloroquine. Nevertheless, all the tested isolates from the epidemic, were sensitive to mefloquine and quinine and ninety percent were also susceptible to sulfadoxine/pyrimethamine. Most individuals had moderate levels of TNF-alpha (20-220 pg/ml) and anti-parasite IgM antibodies compared to IgG levels which were relatively lower. In conclusion, the high transmission rate of the chloroquine resistant P. falciparum parasite could be the probable cause of the disease epidemic in Rajasthan. The timely drug sensitivity test and availability of appropriate antimalarial drugs are, therefore, warranted.

Animals↗