Biomedical subjects

D N Rao

Publications and source records attributed to D N Rao.

At least 19 recordsLinked to original sources

PubMed2000

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence.

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.

2-Aminopurine↗

PubMed2000

Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias.

Free radicals are highly reactive species that have been implicated in the pathogenesis of many diseases. Reactive oxygen species can initiate lipid peroxidation and DNA damage leading to mutagenesis, carcinogenesis and cell death, if the antioxidant system is impaired. This study was undertaken to examine the prevalence of oxidative stress and the role of antioxidant defence in untreated leukemia patients. The generation of superoxide anion and hydrogen peroxide by leukocytes, plasma malondialdehyde levels, red cell copper zinc superoxide dismutase (Cu-Zn SOD) and glutathione peroxidase (GSH-PX) activities were determined in 30 patients with different types of leukemias prior to therapy. The superoxide anion generation by polymorphonuclear leukocytes was found to be significantly increased in leukemia patients especially those with acute lymphocytic and nonlymphocytic leukemias, while the hydrogen peroxide levels were comparable to the control values. Plasma lipid peroxidation products in untreated leukemia patients were in the normal range. Red cell Cu-Zn SOD and GSH-PX activities were significantly increased and showed no correlation with the hemoglobin content. Although superoxide generation was high, lipid peroxide levels were normal in these patients. This might be due to the increased activities of the antioxidant enzymes (SOD, GSH-PX) which counteract lipid peroxidation. Increased free radical generation, especially superoxide anion in leukemia patients and increased antioxidant defence enzymes, which is an adaptive protective response, are indicative of mild oxidative stress. There were no significant differences for the parameters cited above between different types of leukemias, suggesting that the changes are not specific to the type of leukemia.

Adult↗

PubMed2000

ATP-dependent restriction enzymes.

The phenomenon of restriction and modification (R-M) was first observed in the course of studies on bacteriophages in the early 1950s. It was only in the 1960s that work of Arber and colleagues provided a molecular explanation for the host specificity. DNA restriction and modification enzymes are responsible for the host-specific barriers to interstrain and interspecies transfer of genetic information that have been observed in a variety of bacterial cell types. R-M systems comprise an endonuclease and a methyltransferase activity. They serve to protect bacterial cells against bacteriophage infection, because incoming foreign DNA is specifically cleaved by the restriction enzyme if it contains the recognition sequence of the endonuclease. The DNA is protected from cleavage by a specific methylation within the recognition sequence, which is introduced by the methyltransferase. Classic R-M systems are now divided into three types on the basis of enzyme complexity, cofactor requirements, and position of DNA cleavage, although new systems are being discovered that do not fit readily into this classification. This review concentrates on multisubunit, multifunctional ATP-dependent restriction enzymes. A growing number of these enzymes are being subjected to biochemical and genetic studies that, when combined with ongoing structural analyses, promise to provide detailed models for mechanisms of DNA recognition and catalysis. It is now clear that DNA cleavage by these enzymes involves highly unusual modes of interaction between the enzymes and their substrates. These unique features of mechanism pose exciting questions and in addition have led to the suggestion that these enzymes may have biological functions beyond that of restriction and modification. The purpose of this review is to describe the exciting developments in our understanding of how the ATP-dependent restriction enzymes recognize specific DNA sequences and cleave or modify DNA.

Adenosine Triphosphate↗

PubMed2000

Humoral and cell mediated immune response to cadmium in mice.

The effect of 30, 100 and 300 ppm of cadmium chloride (CdCl2) exposure for 35 days on humoral and cell mediated immune response was examined in Swiss Albino mice. Body burden of cadmium in kidney, spleen and liver was determined and histopathology of these organs was also done. Cadmium chloride in doses of 100 and 300 ppm when fed in drinking water caused significant decrease in IgM and IgG titre against sheep red blood cells (SRBC) and a significant decrease in IgG titre against bovine serum albumin (BSA). The delayed type hypersensitivity response to SRBC and splenic T cell proliferation to BSA was also significantly decreased following 100 amd 300 ppm cadmium exposure. Cadmium accumulation in the spleen, liver and kidney was associated with degeneration and inflammatory changes. It is concluded that cadmium causes significant suppression of humoral and cell mediated immune response in mice which could be due to its cytotoxic action on liver, kidney and immune cells.

Animals↗

PubMed2000

Identification of immunodominant epitope of F1 antigen of Yersinia pestis.

The F1 antigen of Yersinia pestis has been identified as one of the major protective antigens of this bacterium. The present study aims to delineate major and minor antigenic sites of F1 antigen. Using algorithmic predictions, five peptide sequences (P1, P2, P3, P4 and P5) spanning the C-terminal region were identified and synthesized. Antibodies were generated in mice against the peptides, native F1 protein and polymerized F1 antigen using liposomes as mode of immunization. Cross-reactivity between F1 antigen and peptides was tested using both solid and solution phase assays. Similar assays were done with rabbit anti-F1 sera. Competitive inhibition assays using a different combination of antisera and competing antigen identified P2 peptide FFVRSIGSKGGKLAAGKYTDAVTV (142-165) as the immunodominant sequence. The results indicate that this sequence appears to be exposed on the surface of F1 molecule. In a solid phase binding assay, P2 peptide was recognized even at high F1 antisera dilution. However, when antisera raised to different peptides were tested for binding to F1 antigen, antisera to P4 peptide showed maximal immunoreactivity. This implies more accessibility of this region during immobilization on solid surface. There was consistency in the results obtained for different strains of mice as well as for the rabbit antisera. Such a sequence of F1 antigen, which is recognized widely in animals of different genetic background, would be useful for diagnosis and subunit vaccine.

Amino Acid Sequence↗

PubMed1999

Bioactive fragment of human interleukin-1beta (163-171) modulates the immune response to synthetic peptides of RESA of Plasmodium falciparum.

In this study a bioactive fragment, residues 163-171 of human interleukin-1beta (hu IL-1beta), that mimics the functions of the whole IL-1 molecule was chemically linked to two peptide sequences of the ring erythrocyte surface antigen (RESA), an asexual blood stage antigen of Plasmodium falciparum. The immunogenicity of different formulations was studied in different haplotypes of inbred mice. The peptide-IL-1beta conjugates delivered in liposomes showed the maximal antibody production. Antigen entrapped in liposomes at a dose of 5 microg showed antibody levels comparable to that of peptide conjugate in alum. The IgG subclass profile with different RESA peptides and its conjugates at various doses in liposomes induced predominantly IgG2a/2b isotypes while alum-delivered formulations showed both IgG1 and IgG2a/2b isotypes. In vitro studies of merozoite reinvasion showed varying degree (50-85%) of inhibition, maximum being with the peptide conjugates in liposomes (80-87%). Thus, this approach suggests that linking of hu IL-1beta is instrumental in augmenting the immune response against otherwise poorly immunogenic synthetic peptides.

Animals↗

PubMed1999

Enzymatic and nonenzymatic production of free radicals from the carcinogens 4-nitroquinoline N-oxide and 4-hydroxylaminoquinoline N-oxide.

The anion radicals of 4-nitroquinoline N-oxide (4-NQO) and 4-nitrosoquinoline N-oxide (4-NOQO) carcinogens were detected and characterized by electron spin resonance (ESR) spectroscopy. The structures of the radical intermediates were examined by density functional theory (DFT) at the level of hybrid unrestricted uBecke3LYP. The formation of superoxide anion radical catalyzed by flavin-containing enzymes such as cytochrome P450 reductase or xanthine oxidase in the presence of 4-NQO or 4-nitroquinoline N-oxide was studied by spin-trapping experiments. In this case, the ESR signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-superoxide radical adduct was observed, and its formation was inhibited by superoxide dismutase (SOD). No ESR signal was detected when the two-electron-transferring flavoenzyme DT-diaphorase (NADPH-quinone oxidoreductase) was used. The above is consistent with a one-electron reduction in the metabolism of these nitro compounds to anion free radicals by various flavoenzyme reductases.

4-Hydroxyaminoquinoline-1-oxide↗

PubMed1999

Alcohol as an additional risk factor in laryngopharyngeal cancer in Mumbai--a case-control study.

A retrospective case-control study of 1698 male pharyngeal and laryngeal cancers seen at the Tata Memorial Hospital, Mumbai from 1980 to 1984 was undertaken to assess the association between the cancers and chewing, smoking, and alcohol habits. Male controls were chosen from persons who attended the hospital during the same period and who were diagnosed as free from cancer, benign tumor, and infectious disease. Statistical analysis was based on unconditional logistic regression method. Bidi smoking and alcohol drinking emerged as significant factors for pharyngeal and laryngeal cancers. Illiterates had 50 to 60% excess risk for pharyngeal cancer only. Nonvegetarian diet did not emerge as significant factor in our study.

Adult↗

PubMed1999

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.

AIDS Vaccines↗

PubMed1999

Correlation of T-cell response and lymphokine profile with RESA peptides of Plasmodium falciparum containing a universal T-cell epitope and an immunopotentiator, polytuftsin.

Two RESA repeat sequences, (EENVEHDA)2 and (DDEHVEEPTVA)2, were chemically linked to a universal T-cell epitope, CS.T3 and polytuftsin, and a natural immunopotentiator, was physically mixed with these conjugates. The immunogens were studied for in vitro antigen-induced T-cell proliferation, and cytokine levels were measured in the culture supernatants. The RESA peptide(s)-CS.T3 conjugate containing polytuftsin showed the highest stimulation index (SI) as compared to the RESA peptide-CS.T3 conjugates or RESA peptides alone. Spleen cells from mice primed with either RESA peptide(s)-CS.T3 conjugate or RESA peptide-CS.T3 conjugate containing polytuftsin, when pulsed in vitro with the respective RESA peptide, showed a higher proliferation index as compared to spleen cells primed and pulsed in vitro with the respective RESA peptides. This observation has an important relevance during natural reinfection for boosting the immune response. The culture supernatants from the cells primed and pulsed in vitro with RESA peptide-CS.T3 conjugate and RESA peptide-CS.T3 conjugate containing polytuftsin showed higher IL-2 and IFN-gamma levels as compared to the RESA peptides alone. Very low IL-4 levels were detected with the above formulations. The cytokine profile is suggestive of a CD4+ TH1 type of immune response, which is ideal for the killing of intracellular pathogens like the malarial parasite.

Adjuvants, Immunologic↗

PubMed1999

Potentiation of immune response against the RESA peptides of Plasmodium falciparum by incorporating a universal T-cell epitope (CS.T3) and an immunomodulator (polytuftsin), and delivery through liposomes.

Synthetic peptides representing repeat sequences of ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum have shown poor immunogenicity and protection. In this study, the RESA peptides [(EENVEHDA)2 and (DDEHVEEPTVA)2] were chemically linked to a universal T-cell determinant, CS.T3, derived from the CS protein of P. falciparum. Polytuftsin (TKPR)40, a polymer of naturally occurring immunomodulator "tuftsin," was physically mixed with these conjugates. These preparations in alum and liposomes were immunized in four inbred strains of mice with different genetic backgrounds to study the humoral response. In the case of liposome-entrapped preparations, a 10 microg dose of antigen showed the optimum antibody response. Mice immunized with liposome containing RESA peptide(s)-CS.T3 conjugate along with polytuftsin showed the highest antibody levels in all the strains, whereas the RESA peptide(s) alone, adsorbed on alum or entrapped in liposomes, showed either poor or moderate antibody levels. The antibodies raised against liposome-entrapped preparations in both high-responder strain (SJL/J H-2s) and low-responder strain (FVB/J H-2q) showed 2 4-fold lower Kd values as compared to the alum adsorbed preparations, suggestive of high affinity antibodies. All the antigen preparations predominantly induced IgG2a and IgG2b isotype response, suggesting that the T-helper response involved is of the CD4 Thl type. The in vitro merozoite reinvasion inhibition assay showed 50-92% inhibition with sera raised against different antigen formulations. The highest percentage inhibition was observed with the RESA peptide-CS.T3 conjugate containing polytuftsin in liposomes. Thus, the incorporation of peptide antigens inside liposomes not only reduced the antigen dose by 5-fold but also elicited a high titre with high affinity antibodies and the inhibition of merozoites to RBC in vitro. Therefore, we conclude that the incorporation of these synthetic constructs in liposomes could be a useful strategy for the development of a subunit immunogen against malaria.

Adjuvants, Immunologic↗

PubMed1999

Estimation & significance of serum & synovial fluid malondialdehyde levels in rheumatoid arthritis.

Serum and synovial fluid (SF) levels of malondialdehyde (MDA), a marker of free radical induced lipid peroxidation, were estimated in patients of rheumatoid arthritis (RA) and compared with healthy controls and patients of osteoarthritis (OA). While serum MDA levels were similar in healthy controls (0.24 +/- 0.10 nmol/ml) and OA (0.28 +/- 0.11 nmol/ml), the serum levels in RA (0.47 +/- 0.19 nmol/ml) were significantly higher as compared to both healthy controls and OA patients; and correlated with synovial fluid (SF) MDA levels. No difference was observed in SF-MDA levels in RA (0.17 +/- 0.07 nmol/ml) and OA (0.16 +/- 0.09). MDA levels did not correlate with markers of disease activity in RA like joint counts, duration of morning stiffness, erythrocyte sedimentation rate etc. Increased serum MDA levels in RA suggest the role of free radicals in the pathogenesis of this inflammatory arthropathy and support the need for further studies assessing the therapeutic role of free radical scavengers in RA.

Arthritis, Rheumatoid↗

PubMed1998

Probing the role of cysteine residues in the EcoP15I DNA methyltransferase.

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.

Amino Acid Substitution↗

PubMed1998

Influence of immunoadjuvants and a promiscous T-cell determinant on the immunogenicity of RESA peptide antigen of P. falciparum.

Synthetic peptide antigens representing the repeat sequences of malarial antigens showed poor immunogenicity and protection in clinical trials. In the present study, RESA, an asexual blood stage antigen, containing (EENVEHDA)2 and (DDEHVEEPTVA)2 sequences were chemically linked to a promiscous T-cell determinant (CS.T3) of the circumsporozoite protein of P. falciparum. The synthetic constructs either alone or coentrapped with immunoadjuvants (nor muramyl dipeptide/lauroyl tetrapeptide) were administered in liposomes to mice of varying genetic background and the immunogenicity of different formulations were compared under identical experimental conditions. The RESA peptide formulations containing the T-cell determinant and the adjuvants generated high titre and affinity antibodies in all the strains, as compared to peptide(s) alone. The booster immunization induced a strong anamnestic response in each group. Though the major IgG isotype is of IgG1 and IgG2b interestingly, formulations containing CS.T3 have a higher proportion of cytophilic IgG2b isotype. There was a significant fall in the levels of IgG2b isotype while IgG1 levels were maintained same in the third bleed (day 60, without booster immunization). The mixed peptide group preparation containing the adjuvant is found to be a better immunogen than that of respective peptides itself. The in vitro merozoite reinvasion inhibition assay showed 76-96% inhibition with the formulations containing RESA peptide(s)-CS.T3 and the adjuvant, while with peptides alone the inhibition was 50-56%. This study highlights the importance of an alternative approach for developing peptide based immunogen against malaria.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1998

Survival analysis of 5595 head and neck cancers--results of conventional treatment in a high-risk population.

This is a study of 5595 head and neck cancer patients treated during 1987-89 at TMH, Mumbai. The study included 1970 oral cancers (ICD 140-145), 1495 oropharyngeal cancers (ICD 1410, 1453, 146), 1255 hypopharyngeal cancers (ICD 148), 125 nasopharyngeal cancers (ICD 147) and 750 laryngeal cancers (ICD 161). The clinical extent of disease at presentation was based on TNM group staging (UICC 1978). For the majority of sites, patients attended the hospital during stage III and stage IV of the disease; the only exception was for cancers of the lower lip, anterior tongue and vocal cord when between 46.2% and 56.5% of patients with localized cancer (stage I and II) were seen. Generally, surgery either alone or with radiation has been administered for oral cancer patients whereas radiation either alone or in combination with chemotherapy was administered for other head and neck sites. The overall 5-year survival rate was in the range of 20-43% for oral cancer, 8-25% for pharyngeal cancers and 25-62% for laryngeal cancer. The 5-year relative survival rates were more or less in agreement with the results published by the Eurocare study for head and neck cancers. The importance of primary prevention in head and neck cancer is stressed.

Combined Modality Therapy↗

PubMed1998

Influence of iron on growth and extracellular products of Acinetobacter baumannii.

Iron is an important nutrient required by bacteria for optimal growth. Acquisition of iron from the host where iron is restricted is an important mediator of bacterial pathogenesis. In iron deplete chemically defined medium (CDM-Fe) growth of Acinetobacter baumannii was restricted as compared to iron replete medium (CDM + Fe). Bacteria developed four high molecular weight outer membrane proteins (OMPs) of 88, 84, 80 and 77 kDa in CDM-Fe medium which were absent in CDM + Fe medium, and are known iron regulated outer membrane proteins (IROMPs). A. baumannii secreted siderophores extracellularly into the medium which act as iron chelators which had been demonstrated in the supernatants of CDM-Fe media. The siderophore was of catechol type. This shows that A. baumannii under iron restricted conditions express IROMPs along with production of catechol type siderophore in order to acquire iron from the external milieu.

Acinetobacter↗

PubMed1998

Preparation & standardization of the goat lung surfactant extract.

Exogenous goat lung surfactant extract (GLSE) was obtained by chloroform-methanol extraction of the centrifuged lung lavage. Four lungs provided around 250 mg of surfactant. GLSE has a phospholipid content of 50 percent, cholesterol of 1.5 percent and protein content under 1 percent. The preparation was bacteriologically sterile and had a shelf life of at least one month at 4 degrees C. Endotracheal administration of surfactant (50 mg/kg) in a rat lung model rendered surfactant deficient by saline lavage, restored the compliance characteristics to normal, thus documenting biological activity of the preparation. The cost of a course of 2 doses of surfactant replacement therapy was estimated to be as low as Rs 500/-. The study opens up the possibility of surfactant replacement therapy for the management of neonatal respiratory distress syndrome in India by an indigenous product at an affordable cost.

Animals↗

PubMed1998

Estimate of cancer incidence in India in 1991.

Cancer incidence and eighteen site-specific age standardised rates in India were estimated for the year 1991. With the establishment of National Cancer Registry Programme, incidence rates per 100,000 are available from six metropolitan registries and one rural registry. Using population census data for India in 1991, about 609,000 new cancer cases were estimated to have been diagnosed in the country in 1991. The estimated age standardised rates per 100,000 were 96.4 for males and 88.2 for females. The five most common cancers were lung (10.6%) pharynx (9.1%), oesophagus (6.7%), tongue (6.6%) and stomach (5.7%) among males and cervix (23.5%), breast (19.3%), ovary (5.5%) oesophagus (4.4%), and mouth (3.9%) among females. A comparison of estimated ASRs for two two largest countries in Asia (China and India) showed differences in the pattern of cancer.

Adolescent↗