PubMed Health⌕ Search

Biomedical subjects

R Madhubala

Publications and source records attributed to R Madhubala.

At least 19 recordsLinked to original sources
PubMed2001

The Leishmania genome project: new insights into gene organization and function.

The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Animals↗
PubMed2001

Effect of Leishmania donovani lipophosphoglycan on ornithine decarboxylase activity in macrophages.

Lipophosphoglycan (LPG), a major surface molecule from Leishmania donovani, stimulated ornithine decarboxylase (ODC) activity in macrophages in a dose- and time-dependent manner. LPG stimulated the rapid increase in ODC activity within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. However, LPG-induced ODC activity was a transient event because 3 hr after exposure to LPG, no stimulation of ODC activity was detectable. ODC activity appeared to be coupled to the activation of protein kinase C (PKC) in macrophages, as activators of PKC caused a rapid increase in the ODC activity. Macrophages pretreated with LPG for 1 hr became unresponsive to subsequent stimulation by the PKC activators 1-oleoyl-2-acetyl-glycerol and the calcium ionophore A23187. In contrast, the ability of macrophages to express ODC activity in response to the cyclic AMP analogue dibutyryl cyclic AMP was not impaired by LPG.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2001

Detection of Leishmania causing visceral leishmaniasis in the Old and New Worlds by a polymerase chain reaction assay based on telomeric sequences.

We present a new polymerase chain reaction assay based on telomeric sequences of Leishmania donovani. When this assay was used in dilutions of purified L. donovani DNA, a strong amplification signal was observed with 1 fg of DNA. In a specificity test that used purified DNA from Old World and New World Leishmania, the assay recognized all parasites isolated from patients with visceral leishmaniasis, except for 2 isolates of Leishmania colombiensis from Venezuela and 1 isolate from Brazil. All Leishmania major and Leishmania tropica isolates tested were negative, except for one isolate in each species. We also used the assay on fresh and archive bone marrow samples recovered from Giemsa-stained slides and from dried blood stains.

Adult↗
PubMed2000

Immunization with recombinant LD1 antigens protects against experimental leishmaniasis.

The genes, ORFF and BT1 (previously ORFG), are part of the multigenic LD1 locus on chromosome 35 which is frequently amplified in Leishmania. BT1 encodes a biopterin transporter, while the function of the ORFF gene product is unknown, but it is localized to the nucleus. We show here that immunization of mice with recombinant ORFF and BT1 proteins, individually, or in combination, conferred partial protection against challenge with Leishmania donovani. Protection correlated with the production of antigen-specific antibodies and in vitro splenocyte proliferation. Thus, these antigens can be potential vaccine candidates against visceral leishmaniasis.

Animals↗
PubMed2000

In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase.

The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins. We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region. The predicted molecular mass of the entire transcript is 166 kDa. The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331. The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P. falciparum. SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain. The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P. falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs.

Adenosylmethionine Decarboxylase↗
PubMed2000

Genomic organization and gene function in Leishmania.

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion ( approximately 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, i. e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Animals↗
PubMed2000

Inhibition of glutathione synthesis as a chemotherapeutic strategy for leishmaniasis.

This study focuses on the use of buthionine sulphoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, on Leishmania donovani growth. The effect of BSO on amastigote multiplication within macrophages showed that 5 mM BSO decreased infectivity by about 50% and the mean number of amastigotes per 100 infected macrophages by 21%. The mechanism may be that BSO resulted in enhanced nitric oxide (NO) levels within macrophages, probably due to inhibition of GSH content since GSH (10 mm) given after BSO treatment led to a decrease in NO compared to macrophages treated with BSO alone which were preexposed to the Leishmania surface molecule lipophosphoglycan.

Animals↗
PubMed2000

Isolation and characterization of human breast adenocarcinoma cells made resistant to alpha-difluoromethylornithine.

Human breast adenocarcinoma cells MCF-7 were selected for resistance to ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine (DFMO). Stepwise increments of the concentration of DFMO resulted in selection of MCF-7 cells that were capable of growing in the presence of 1.0 mM DFMO. This capacity was associated with a 10-fold increase in ODC activity and marked enhancement in the synthesis rate of ODC protein as verified by a 2-hr [35S]methionine labeling of cellular proteins followed by immunoprecipitation and SDS-PAGE. The resistant cells had much higher concentration of putrescine, spermidine, and spermine than the control cells. A 25-fold increase in ED50 (effective dose causing 50% inhibition) for the antiproliferative action of DFMO in these resistant cells was observed. The susceptibility of wild-type and resistant cell lines to other inhibitors of the polyamine biosynthetic pathway and adriamycin is also reported.

Adenocarcinoma↗
PubMed2000

Plasmodium falciparum: detection and strain identification of Indian isolates by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed for detection and strain identification of P. falciparum in a comparative field study of Indian isolates. The primers were selected from highly conserved regions flanking the variable, tandemly repeated regions of highly polymorphic cell surface antigens, major merozoite surface antigen-1 (MSP-1), major surface antigen-2 (MSP-2), circumsporozoite surface antigen (CSP) and ring-infected erythrocyte surface antigen (RESA). Out of the 52 microscopically positive P. falciparum infected field samples, 47 samples were positive by PCR. Variation in the size of the amplified products was observed using MSP-1, MSP-2 specific primers respectively in different field isolates of P. falciparum, but CSP and RESA did not exhibit any variation in size of the amplified product. The multiplex PCR results demonstrated that amplified products from these surface antigens vary in size and there is a specific pattern for each strain and this could be utilized to identify a particular field isolate. One P. falciparum infected field sample detected by the above PCR method was found to be a mixed infection by two different strains. Five microscopically positive P. vivax infeced samples were also analyzed by PCR method using P. falciparum cell surface antigen (MSP-2) specific primers. PCR results showed one P. vivax infected sample was positive when P. falciparum specific primers were used, this could be due to inaccurate and reduced limit of detection of Plasmodial species by microscopic examination.

Animals↗
PubMed1999

The Leishmania donovani LD1 locus gene ORFG encodes a biopterin transporter (BT1).

We have previously described two genes, ORFF and ORFG, from the LD1 locus near one telomere of chromosome 35, which are frequently amplified in Leishmania isolates. In Leishmania donovani LSB-51.1, gene conversion of the rRNA gene locus on chromosome 27 with these two genes resulted in their over-expression, because of their transcription by the RNA polymerase I-mediated rRNA promoter. The predicted ORFG protein has substantial sequence homology to the ESAG10 gene product from the Trypanosoma brucei VSG expression site and both are putative membrane proteins. Using successive rounds of gene replacement of the three ORFG genes in L. donovani LSB-51.1, ORFG null mutants were obtained. These mutant cell lines show a direct relationship between ORFG mRNA, protein expression levels and active transport of biopterin into the cells. Transformation of the null mutant with a plasmid containing ORFG restores biopterin transport activity. In addition, the null mutants are unable to grow in the absence of supplemental biopterin. Thus, ORFG encodes a biopterin transporter and has been renamed BTI.

Animals↗
PubMed1999

Effect of the microtubule stabilising agent taxol on leishmanial protozoan parasites in vitro.

Taxol, a mitotic spindle toxin, was found to selectively inhibit the proliferation of Leishmania donovani in vitro at nanomolar concentrations with an IC50 of 35 nM. Concentrations of taxol as high as 50 nM, however, did not affect J774A.1 murine macrophages. Taxol (30 nM) also inhibited amastigote multiplication within a J774A.1 macrophage cell line when used in a 10-day experiment. It resulted in the in vitro assembly of L. donovani microtubules in a dose-dependent manner. When promastigotes were exposed to different concentrations of taxol for 24 h, cells were largely blocked in the G2-M phase of the cell cycle and there was a marked reduction in the percentage of cells in the S phase. The selective nature of taxol action against the parasite and its effectiveness in controlling amastigote multiplication emphasise its use as a promising chemotherapeutic against kala-azar.

Animals↗
PubMed1999

Isolation of a taxol-resistant Leishmania donovani promastigote mutant that exhibits a multidrug-resistant phenotype.

We raised a strain of Leishmania donovani in the laboratory that was resistant to 500 nM taxol. The IC50 of the wild-type strain for taxol was 35 nM and that of the taxol-resistant strain (T-500) was 1 microM. The T-500 strain exhibited a Mdr phenotype; it was also resistant to other unrelated drugs like vinblastine, adriamycin and the commonly used antimonial drugs pentostam and glucantime. Verapamil (20 nM), a calcium channel blocker, was found to reverse the resistance of T-500 to taxol. Acquired resistance to taxol has been reported to be mediated by alterations involving tubulin in cancer cells. Thus polymerisation assays with tubulin fractions in wild-type versus taxol-resistant cells (T-500) were performed in vitro. The tubulin fraction from T-500 was more resistant to in vitro polymerisation than the tubulin isolated from the wild-type, suggesting that this is one means by which the parasite may acquire resistance to taxol.

Animals↗
PubMed1999

Leishmania donovani: characterization and expression of ORFF, a gene amplified from the LDI locus.

The LD1 locus is a 27.5-kb region of chromosome 35 that is conserved among all species of Leishmania and is amplified in several different isolates. Here, we report the genomic distribution of ORFF, a gene from the LD1 region, and its expression at the RNA and protein levels in two Indian isolates of Leishmania donovani. In both of these isolates, ORFF was present as a single copy on chromosome 35. Densitometric analysis of ORFF mRNA abundance revealed relative abundance of 0.2 and 1.0 in AG83 and S-Lal, respectively. Antiserum against recombinant ORFF protein detected a protein of the predicted size ( approximately 34 kDa) in both strains. The protein is most abundant in mid-log-phase promastigotes and has a nuclear localization. The ORFF protein is preferentially expressed in L. donovani amastigotes but, in contrast, is expressed at higher levels in L. major promastigotes.

Animals↗
PubMed1999

Serodiagnosis of leishmaniasis with recombinant ORFF antigen.

The serodiagnostic potential of recombinant ORFF protein (rORFF) from Leishmania infantum was assessed by ELISA. Of 49 sera from confirmed cases of visceral leishmaniasis (VL), all were seropositive using 5 ng of rORFF and serum diluted 1:20, while only 38 were positive with 500 ng of soluble antigen (SA) and 44 were positive by a direct agglutination test. There was also a positive correlation between spleen size and level of seropositivity with rORFF or SA. The reciprocal endpoint titer with rORFF was 1,280 for sera from VL patients, but < 20 with sera from malaria, filariasis, and tuberculosis patients, as well as with sera from healthy individuals from endemic and non-endemic areas. Sera from 10 confirmed cutaneous leishmaniasis cases from Turkey were negative or only weakly positive with rORFF although 9 were positive with SA. Thus, rORFF protein appears useful as a sensitive reagent for the differential diagnosis of VL caused by the Leishmania donovani complex.

Animals↗
PubMed1998

Chemoprevention of DMBA-induced transplacental and translactational carcinogenesis in mice by oil from mustard seeds (Brassica spp.).

The present study reports the chemopreventive potential of the oil from mustard seed on 7,12-dimethylbenz[a]anthracene-induced transplacental and translactational carcinogenesis in Swiss albino mice. Gestating females were treated with mustard oil at dose levels of 0.05 and 0.10 ml per day from days 13 to 19 of gestation. In addition, they were given DMBA (3 mg/animal) on days 15-17 of gestation. The percentage of tumour incidence in the F1 progeny was reduced significantly at both dose levels from 65% in the control group to 29% and 16%, respectively, in the experimental groups. The mean number of tumours per effective F1 progeny was reduced from 1.56 in the control group to 0.93 and 0.41 in the animals treated with lower and higher doses of mustard oil, respectively. When lactating mothers were given the mustard oil at dose levels of 0.05 and 0.10 ml per day for the first 15 days of lactation in addition to DMBA given on days 3, 6, 9, 12 and 15 of lactation, the multiple site tumour incidence was brought down significantly from a control value of 70% to 32% and 18%, respectively, in lower and higher dose groups. The mean number of tumours in the F1 mouse was reduced from a control value of 1.71 to 0.96 at the lower dose level and to 0.34 at the higher dose level. From earlier studies done in our laboratory, it appears that mustard oil exerts its effect by inducing the enzymes of drug detoxification and also by changing the profile of the antioxidant defence system. The quantitative and qualitative nature of the active principles and their passage into the F1 progeny remains to be seen.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Difluoromethylornithine antagonizes taxol cytotoxicity in MCF-7 human breast cancer cells.

Taxol is a naturally occurring anticancer agent. We studied the combined effects of taxol with 0.1 mM of the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) in the MCF-7 human breast adenocarcinoma cell line. The effects of taxol on MCF-7 cells were evident at 0.05-1 microM and the half-maximum inhibition was calculated to be 0.05 microM. Although the cells in the control group continued to proliferate during an 8-day growth period, cells in the taxol-treated group showed approximately 78% inhibition on day 6 and approximately 92% inhibition on day 8. The combined effects of different concentrations of taxol with 0.1 mM DFMO for 48 h showed that DFMO reversed the cytotoxicity of taxol. The combined effects of 0.5 microM taxol and 0.1 mM DFMO over an 8-day period resulted in the reversal of taxol cytotoxicity by 74% on the sixth day of culture. Pretreatment and posttreatment with 0.1 mM DFMO protected the MCF-7 human breast adenocarcinoma cells from the cytotoxic effect of taxol. Polyamine levels were inhibited in cells treated with DFMO for 24 h. In a separate experiment, we verified that the addition of exogenous putrescine along with taxol and DFMO to cultures for 48 h restored the cytotoxic effects of taxol. Following exposure to 0.5 microM taxol, over 59% of MCF-7 cells were in G2/M phase. DFMO (0.1 mM) showed only a slight increase in the G1 phase of the cell cycle. However, in cells treated with taxol and DFMO, there was no change in the percent of cells in the G2/M phase compared to taxol-treated cells. Therefore, depletion of cellular polyamines may not interfere with cell cycle changes induced by taxol. Treatment of MCF-7 cells with 0.5 microM taxol resulted in the fragmentation of genomic DNA, indicating apoptosis, whereas the combined effects of taxol with DFMO inhibited DNA fragmentation.

Adenocarcinoma↗
PubMed1996

Antileishmanial effect of a potent S-adenosylmethionine decarboxylase inhibitor: CGP 40215A.

CGP 40215A, specific S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor was found to inhibit the growth of Leishmania donovani promastigotes (strain UR6) in a dose-dependent manner with an IC50 of 18 microM. The growth inhibition was reversed with 100 microM of spermidine and spermine. The growth inhibition in vitro by this inhibitor was accompanied by a significant decrease in AdoMetDC activity and spermidine levels. CGP 40215A was more potent than other AdoMetDC inhibitors, Berenil or methyl glyoxal (bis) guanyl hydrazone. The combination of CGP 40215A with other polyamine biosynthetic inhibitors like DL-alpha-difluoromethylornithine or the bis (benzyl) polyamine analogue (MDL 27695) shows an accentuated inhibitory effect on leishmanial growth.

Adenosylmethionine Decarboxylase↗