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Biomedical subjects

R Rajagopal

Publications and source records attributed to R Rajagopal.

At least 19 recordsLinked to original sources
PubMed2006

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads.

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.

Alginates↗
PubMed2005

Immune cascade of Spodoptera litura: cloning, expression, and characterization of inducible prophenol oxidase.

Haemolymph associated phenol oxidase is a critical component of invertebrate immune reaction and cuticle sclerotization. Phenol oxidase catalyses the conversion of mono-phenols to diphenols and quinones which finally leads to melanin formation. We have cloned the c-DNA encoding phenol oxidase from the haemocytes of Spodoptera litura and expressed it in Escherichia coli. The encoding gene is 2452bp with an open reading frame of 2091 bp translating into a 697 amino acid protein. Multiple alignment analysis of the predicted protein sequence shows close homology to other lepidopeteran PPOII type genes. The transcription of the gene is induced upon microbial challenge of 6th instar larvae with E. coli and is unresponsive to injury. Cloning of the ORF of SLPPO in-frame in the E. coli expression vector pQE30 resulted in its expression. Enzymatic analysis of the recombinant protein reveals that the recombinant protein is catalytically active on 4-methyl pyrocatechol upon activation by cetyl pyridinium chloride.

Amino Acid Sequence↗
PubMed2005

Glutathione S-transferase T1 polymorphisms are associated with outcome in colorectal cancer.

Colorectal cancer (CRC) remains a significant cause of mortality accounting for approximately 10% of all deaths from malignancy in the western world. Polymorphism in the glutathione S-transferase GSTT1 gene has been associated with CRC risk in some but not all studies. In this study, we examined associations between GSTT1 genotypes and CRC risk, and prognosis in 361 cases and 881 unrelated controls. GSTT1 null was associated with a small but significant increase in risk (P = 0.0006, odds ratio (OR) = 1.65, 95% confidence interval (CI) = 1.22-2.24). GSTT1 null was also associated with a significantly younger age at diagnosis (mean 65.2 years) compared with GSTT1 A (mean 67.6 years, P = 0.031). There were no significant associations between GSTT1 genotypes and clinical factors (e.g. Dukes stage, differentiation and tumour node metastasis classification) in the total case group. However, following stratification by age (<70 versus > or =70 years at diagnosis), in the patients diagnosed <70 years of age, GSTT1 null was more common in Dukes grade A/B tumours (P = 0.046), stage T1/T2 tumours (P = 0.053) and those with a pushing margin (P = 0.066). We also identified associations between GSTT1 null and increased prevalence of host lymphocyte response, particularly in the younger patients (P = 0.036). Furthermore, GSTT1 null was associated with improved survival in younger patients (P = 0.017, hazards ratio (HR) = 0.52, 95% CI = 0.31-0.89) but poorer survival in older patients (P = 0.017, HR = 1.89, 95% CI = 1.12-3.20). We proposed a model based on the dual functionality of GSTT1 to explain these contrasting results. We suggest that the null genotype is associated with improved immune response in younger patients, but poorer detoxification in older patients. These findings may also provide an explanation for the contrasting finding of other studies on the role of this gene in CRC.

Aged↗
PubMed2005

Thrombectomy and distal protection devices.

Visible distal embolization is a relatively rare complication during percutaneous coronary interventions but it may occur in 15-20% of angioplasties in saphenous vein grafts (SVG) and in thrombus containing lesions of native coronary arteries, especially in the setting of acute myocardial infarction (AMI). Mechanical thrombectomy devices and distal protection devices have been introduced to reduce the incidence of distal embolisation. Angiojet thrombectomy has shown positive outcomes in thrombus containing lesions when compared with intracoronary thrombolysis but a randomised trial failed to show benefit in the context of AMI. The X-Sizer, Excimer LASER and various simple aspiration thrombectomy catheters show promise in treatment of AMI with improvement shown in surrogate end-points such as resolution of ST-elevation but there have been no randomised trials large enough to demonstrate changes in hard clinical end-points. Distal protection devices have come to be routinely used during SVG treatment and, after the results of the SAFER and FIRE trials; they became a treatment recommendation in the recently issued European Society of Cardiology (ESC) guidelines. For AMI, these devices showed great promise in early single-centre studies which have been offset by the lack of clinical benefit in the multi-centre EMERALD trial. Proximal occlusion devices may further decrease the incidence of distal embolisation, especially in the treatment of totally occluded SVGs or native arteries, by reversing the blood flow and offering protection at a very early stage of the procedure before wire passage. Despite continuous technical improvement and a growing number of miniaturised thrombectomy devices and filters, because of the paucity of controlled large studies, it remains unclear whether these devices will remain limited to niche' applications or will become a regular companion to balloons and stents in mainstream angioplasty procedures.

Angioplasty, Balloon, Coronary↗
PubMed2003

Molecular characterization of chitinase from polyphagous pest Helicoverpa armigera.

Chitinase from a polyphagous pest, Helicoverpa armigera, has been cloned and expressed. The Helicoverpa chitinase cDNA is 2870 bp in length and contains an open reading frame of 1767 bp. The cDNA encodes a polypeptide of 588 residues with a predicted molecular weight of 66 kDa and a pI of 5.99. The polypeptide has distinct catalytic and substrate binding domains at the N- and the C termini, respectively. The two domains are held together by a proline, threonine rich linker region. The catalytic and the substrate binding domains shared a high level of homology with other lepidopteran chitinases, but the proline and threonine rich region is longer in H. armigera chitinase than in other lepidopteran chitinases. The transcription of chitinase at different developmental stages and in different tissues was analysed by RT-PCR. Chitinase transcript was found in the integument, gut, and fat bodies but was absent in the haemocytes. The levels of chitinase mRNA were abundant at the moulting stages and a basal level of transcript was maintained throughout the development of the insect. Interestingly, Western blot analysis of total proteins from the integument and the gut showed the presence of chitinase in the moulting stages but was absent in the intermoult periods, suggesting post-transcriptional control. The chitinase cDNA was expressed in bacteria and in insect cells. The insect cell expressed chitinase was glycosylated and catalytically active against the simple and complex substrates. The chitinase gene spans about 6.8 kb of genomic DNA and is organized into 10 exons and 9 introns. The 6.8 kb genomic clone of chitinase revealed a high degree of conservation in the position and size of the exons with other lepidopteran insects.

Amino Acid Sequence↗
PubMed2003

A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1.

A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.

Amino Acid Sequence↗
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↗
PubMed2003

Ultrasound promoted para-selective nitration of phenols in ionic liquid.

Ultrasound promoted nitration of phenol and substituted phenols using ferric nitrate and Clayfen respectively in the ionic liquid ethyl ammonium nitrate has exhibited significant enhancement in rates of reaction as well as high para selectivity as compared to corresponding reactions performed without ultrasound.

Journal Article↗
PubMed2002

Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor.

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn double-stranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins.

Animals↗
PubMed2002

Ultrasound promoted Suzuki cross-coupling reactions in ionic liquid at ambient conditions.

Palladium catalyzed Suzuki cross-coupling reactions of halobenzenes including chlorobenzenes with phenylboronic acid have been achieved at ambient temperature (30 degrees C) in the absence of a phosphine ligand using the ionic liquid 1,3-di-n-butylimidazolium tetrafluoroborate [bbim][BF4] with methanol as co-solvent under ultrasonic irradiation.

Journal Article↗
PubMed2001

Phenol sulphotransferase SULT1A1*1 genotype is associated with reduced risk of colorectal cancer.

Sulphation is an important detoxification pathway for numerous xenobiotics; however, it also plays an important role in the metabolism and bioactivation of many dietary and environmental mutagens, including heterocyclic amines implicated in the pathogenesis of colorectal and other cancers. A major sulphotransferase (SULT) enzyme in humans, SULT1A1, is polymorphic with the most common variant allele, SULT1A1*2, occurring at a frequency of about 32% in the Caucasian population. This allele codes for an allozyme with low enzyme activity and stability compared to the wild-type (SULT1A1*1) enzyme, and therefore SULT1A1 genotype may influence susceptibility to mutagenicity following exposure to heterocyclic amines and other environmental toxins. Previously, a significant association of SULT1A1*1 genotype with old age has been observed, suggesting a 'chemoprotective' role for the high-activity phenotype. Here we have compared the frequencies of the most common SULT1A1 alleles in 226 colorectal cancer patients and 293 previously described control patients. We also assessed whether SULT1A1 genotype was related to various clinical parameters in the patient group, including Duke's classification, differentiation, site, nodal involvement and survival. There was no significant difference in allele frequency between the control and cancer patient populations, nor was there a significant association with any of the clinical parameters studied. However, when the age-related difference in allele frequency was considered, a significantly reduced risk of colorectal cancer (odds ratio = 0.47; 95% confidence interval = 0.27-0.83; P = 0.009), was associated with homozygosity for SULT1A1*1 in subjects under the age of 80 years. These results suggest that the high activity SULT1A1*1 allozyme protects against dietary and/or environmental chemicals involved in the pathogenesis of colorectal cancer.

Adolescent↗
PubMed2001

Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis.

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.

Animals↗