Biomedical subjects
Publications and source records attributed to A Nandy.
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Chloroquine-resistant Plasmodium vivax malaria was first reported in India in 1995. This report led to the round-the-year monitoring, in Calcutta (West Bengal) and Mayurbhanj district (northern Orissa), of the in-vivo sensitivity of local P. vivax to chloroquine (CQ). Between January 1998 and December 2001, 800 cases with microscopically confirmed P. vivax malaria were enrolled in the study. Each was given CQ in the regimen recommended both by the Government of India and the World Health Organization (i.e. a total of 25 mg/kg, over 3 days). Only six cases, of the 480 who completed the scheduled 28 days of follow-up, failed to clear their parasitaemias by day 5. Even these six cases had only low-level parasitaemias on day 5, and all were aparasitaemic by day 7. In the study area, despite the wide-spread abuse of CQ and the increasingly frequent reports of CQ-resistant P. falciparum, CQ appears to remain an effective drug in the treatment of P. vivax.
The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.
BACKGROUND: The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed. METHODS: A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison. RESULTS: Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE. CONCLUSIONS: The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.
We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.
Different concentrations (50, 100, 150 microg/mL) of the methanol extract of the roots of Asparagus racemosus Willd. showed considerable in vitro antibacterial efficacy against Escherichia coli, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Pseudomonas putida, Bacillus subtilis and Staphylococcus aureus. The effects produced by the methanol extract were compared with chloramphenicol.
Advances in radiological and endoscopic techniques have allowed many biliary complications after orthotopic liver transplantation (OLT) to be managed without surgery. The influence of nonsurgical management on the outcome of patients requiring surgical revision has not been addressed. We reviewed our 10-year experience (October 1988 to January 1998) of Roux-en-Y choledochojejunostomy (CDJ) to treat biliary complications after OLT. Forty-six patients underwent CDJ for biliary complications (32 men, 14 women; age, 22 to 65 years; median, 60 years). Biliary reconstruction at the time of OLT was duct to duct in 41 patients, primary CDJ in 3 patients, and gall bladder conduit in 2 patients. T-tubes were used only in patients with gallbladder conduit. The indication for CDJ was biliary leak (23 patients), stricture (20 patients), biliary stones (2 patients), and biliary sludge (1 patient). Two patients (4.3%) had associated hepatic artery thrombosis. The bile leaks were diagnosed at a median of 29 days post-OLT (range, 2 to 65 days) and strictures at a median of 2 years (range, 33 days to 6.5 years) post-OLT. Before surgery, 25 patients (54%) underwent an attempt at radiological or endoscopic therapeutic intervention that failed. Median follow-up was 5 years (range, 9 months to 10 years). Early complications occurred in 12 patients (26%); the most common was chest infection (4 patients). There were 3 perioperative deaths (6%); 1 death was directly related to surgery. Late complications, mainly anastomotic strictures, occurred in 10 patients (22%), half of which were successfully treated by biliary balloon dilatation. The complication rate post-CDJ was less in those who underwent a failed nonsurgical approach than those proceeding straight to surgery (9 of 25 patients; 36% v 13 of 21 patients; 62%; P =.21, not significant). The procedure-related mortality for surgical revision of biliary complications after OLT is low, but early and late complications are common. A failed attempt at nonsurgical management does not increase the complications of reconstructive surgery. Strictures after CDJ should be considered for biliary balloon dilatation.
Medium-chain acyl-CoA dehydrogenase (MCADH) deficiency, an autosomal recessive inherited disorder, is the most common genetic disorder in mitochondrial beta-oxidation in humans. In addition to one prevalent disease-causing mutation (K304E), a series of rarer mutations has been reported, but none of these has yet been characterized in detail. We report here on the biochemical characterization of the purified recombinant mutant protein in which threonine is replaced by alanine at position 168 of the mature protein (T168A-MCADH). It is the first mutation to be found in patients that is located in the active site of the enzyme. Thr-168 is hydrogen-bonded to the flavin N(5) of the cofactor FAD. The thermostability of T168A-MCADH is markedly decreased compared with human wild-type MCADH (hwt-MCADH). Catalytic activity with ferricenium as acceptor is lowered by 80% and with the natural acceptor electron-transferring flavoprotein by over 90% compared with hwt-MCADH. In the mutant the extent of flavin semiquinone formed on reduction is approx. 50% that of hwt-MCADH. The pK reflected by the pH-dependence of Vmax is shifted from approx. 8.2 (hwt-MCADH) to approx. 7 (T168A-MCADH) and the rates of enzyme flavin reduction (stopped-flow measurements) are only approx. 1/10 those of the parent enzyme. These properties are discussed in the light of the possible mechanisms leading to disease in humans.
Before field application of the direct agglutination test (DAT) for leishmaniasis, it was assessed as a diagnostic tool. Fifteen confirmed visceral leishmaniasis cases (bone marrow aspiration positive), 120 tuberculosis, 58 leprosy, 15 malaria, 26 intestinal parasitic infection cases, 24 endemic healthy controls from adjacent to the study area, and 18 controls from Kathmandu (who had never visited the VL endemic areas) were tested for anti-leishmanial antibody agglutination titers. Two of the tuberculosis cases were positive for anti-leishmanial agglutinating antibodies at 1:800. All the visceral leishmaniasis confirmed cases were reactive to anti-leishmanial antibody at > or = 1:3,200. Other specimens were negative for serology. The sensitivity of the direct agglutination test was 100% and the specificity was 99.2%. The direct agglutination test had positive and negative predictive values of 100% and 99.2% respectively. The direct agglutination test has been found to be simple, rapid, reliable, economic, safe and adaptable to micro-techniques using microtiter plates. It is specific and sensitive. The direct agglutination test is simple enough for it to be performed in a field laboratory.
Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.
Recurrence of kala-azar after post kala-azar dermal leishmaniasis (PKDL) has remained uncommon. We report here two patients with recurrence of kala-azar (KA) after development of PKDL. In one case the second attack of KA was preceded by repeated attacks of malaria and tuberculosis, and in the other the recurrence of KA followed an attack of measles. While measles has earlier been suggested as co-factor in inducing transformation from sub-clinical to clinical kala-azar, malaria was demonstrated to enhance the virulence and invasiveness of Leishmania in an experimental model as well as under natural condition. We propose that in our cases, measles and repeated attacks of malaria or tuberculosis led to immunosuppression and recurrence of visceral leishmaniasis (VL).
Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ([Glu304]MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity. The crucial factor leading to the production of active [Glu304]MCADH protein is the expression in E. coli cells at reduced temperature (28 degrees C). Expression in the same system at 37 degrees C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH. Although [Glu304]MCADH has approximately the same rate of substrate reduction with dodecanoyl-CoA and the same V(max) as human MCADH with the best substrate for the latter, octanoyl-CoA, the K(m) in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, V(max) obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The V(max)/K(m) versus chain-length profile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl-CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrogenase. The purified mutant enzyme exhibits a reduced thermal stability compared to human wild-type MCADH. The major difference between the two proteins expressed in E. coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases. Differences between tetrameric [Glu304]MCADH which survives the first step(s) of purification and corresponding MCADH are minor. The overall differences in properties of [Glu304]MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation.
Post kala-azar dermal leishmaniasis (PKDL) involving the mucus membranes is relatively rare on the Indian sub-continent. We describe 3 cases of PKDL presenting with hoarseness of voice. In one case the skin, nasal, oral, oropharyngeal and laryngeal mucosa had nodular and nodulo-ulcerative lesions; in the 2 other cases, genitalia and anorectal mucosa were also affected. Laryngoscopic examination revealed nodular lesions on the vocal cords. Biopsy smear and culture confirmed their leishmanial origin.
The present study was undertaken to determine the efficacy of ketoconazole in comparison to sodium antimony gluconate (SAG) in the treatment of kala-azar. The study was conducted at two centres: All India Institute of Medical Science, New Delhi and Calcutta School of Tropical Medicine, Calcutta. A total of 180 patients with proven kala-azar were recruited. After preliminary investigations, the patients were randomly divided into 2 groups: One group received ketoconazole in a dose of 600 mg/ day in 3 divided doses for 4 weeks while the other group was treated with SAG at a dose of 20 mg/kg/day up to a maximum of 850 mg/day for 4 weeks. The patients were followed up by clinical examination, liver functions, haemogram and the bone marrow/splenic aspiration. Responders were followed up at 3 and 9 months of intervals. Of 90 cases in SAG, 78 (81.7%) got cured initially while under ketoconazole group, only 26 (33.3%) of 78 patients responded initially (p < 0.001). After 3 months of follow up, 75 of 78 SAG-responders (96.2%) and 24 of 26 ketoconazole-responders (92.3%) continued to be in remission. Despite the fact that 2 patients in each group were lost to follow up at 9 months, similar observations were noted with only one relapse in SAG group. The response to SAG was comparable at the two centres. However, the response to ketoconazole was better at Delhi centre as compared to that at Calcutta. There were no significant side effects or hormonal changes in any of the patients in ketoconazole group at Delhi centre. Significantly higher side effects were reported at Calcutta centre in ketoconazole group (P < 0.05). No satisfactory explanation can be given for this difference in response to ketoconazole at two centres. However, it is known that leishmanial parasites of different geographical origin differ in their response to different drugs and this could be one of the reasons for difference observed in response rate to ketoconazole as the study involved different populations of people.
The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].
Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 A resolution, respectively. The catalytic base responsible for the alpha-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its omega-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C alpha atom of the substrate and the distance between the Glu255 carboxylate and the C alpha atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C alpha-C beta bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O2 accessibility in the mutant which might account for the higher oxygen reactivity.
Mice homozygous for the scid mutation bear a severe defect in their ability to rearrange V(D)J gene segments to yield active genes for immunoglobulin and T cell receptor molecules. In older animals few clones of B and T cells can arise at random, a phenomenon called leakyness of the scid mutation. We established scid mice carrying as a transgene the rearranged heavy chain of the IgM/lambda1 antibody MOPC 104E with specificity for the alpha(1,3) glucosidic linkages in Dextran. Despite the scid defect one-third of these mice immunized with the thymus independent antigen Dextran at 2 weeks of age, and all of those immunized at 6 weeks responded with anti-Dextran antibodies bearing the lambda light chain. This indicates that despite the scid mutation these animals had at least once successfully rearranged their endogenous lambda1 light chain gene segments and harbor Dextran specific B cells. These mice thus provided for the first time the opportunity to study the immune response of B cells of a single specificity in an environment that should, as we shall argue, be devoid of regulatory B and T cells able to recognize the idiotype of the responding cells. One week after immunization the anti-Dextran response of 5- to 6-week-old mu-transgenic scid mice amounted to 30% of the response of mu-transgenic non-scid mice but in essence both responses followed the same kinetics, reaching antibody concentrations indistinguishable from each other 8 weeks after a single dose of Dextran. Furthermore, the ready response of young mu-transgenic scid mice to this antigen by employment of endogenously rearranged lambda1 light chains allowed experiments to be done to compare the frequency of lambda1 light chain rearrangements in mu-transgenic scid mice to that in mu-transgenic non-scid mice. This was done in limiting dilution assays counting B cell precursors responsive to mitogen and differentiating in vitro to produce antibodies toward Dextran. Specific precursors were reduced to about 1% in the spleen of mu-transgenic scid mice when compared to the spleen of mu-transgenic non-scid mice; those in the peritoneal cavity lymphocyte population were reduced to about 12%.
Distribution of T cells in the peripheral blood and lymph nodes of Indian kala-azar (KA) patients was studied by using appropriate phenotypic markers for CD2+, CD4+ and CD8+ cells. Significant reduction in the CD2+, CD4+ cell numbers as well as CD4+/CD8+ cell ratio was noted in the peripheral blood of active KA cases. Such alteration in the T cell population appeared to be a manifestation of the disease process as it showed a tendency to return close to normalcy several months after successful chemotherapy. Histopathological studies of KA patients with lymphadenopathy demonstrated gradual destruction of lymph node follicular architecture which correlated well with the severity and duration of illness. Massive infiltration of CD2+ cells in the cortical region of lymph node was evident. The observed preponderance of CD4+ cells over CD8+ ones in these infiltrates was in sharp contrast to the distribution pattern of these cells in the periphery. Significance of these findings is discussed in relation to the current concepts on the immunology of leishmaniasis and related diseases.