Netting the malaria menace: Distribution and utilization of long-lasting insecticidal net in a malaria endemic area in Bankura, West Bengal.

BACKGROUND & OBJECTIVES: Long-lasting insecticidal net (LLIN) is considered to be a highly effective intervention against malaria under National Vector Borne Disease Control Programme in India. A cross-sectional study was undertaken to assess the coverage and utilization of LLIN and the factors related thereto. METHODS: A survey of 1300 households was carried out in Ranibandh block of Bankura district in West Bengal, India, using lot quality assurance sampling (LQAS) method. Coverage/utilization of 80% was considered as minimum acceptable norm. The weighted sample size was calculated from each village of the block. The sociodemographic, economic information of the household along with the availability and use of LLIN was collected through interview and observation. RESULTS: In total, 7320 individuals including 840 children ≤ 5 yr were visited. Overall coverage of adequate LLIN was 65.4% (± 1.5%) and for children ≤ 5 yr, it was 60.5% (± 1.3%). Overall, 66.1% (± 1.4%) people of all ages and 63.7% (± 1.4%) children ≤ 5 yr slept under LLINs in the night before the survey. Out of 26 sub-centres, distribution of LLINs in 10 sub-centres was below the accepted norm, whereas utilization was sub-optimal in 19 sub-centres. In only 18.2% (± 0.5%) households, LLINs remained hanging during daytime. Poverty, caste, education, perception regarding malarial morbidity and preventive action of LLIN were associated significantly with the distribution of LLIN. Similarly, poverty (AOR = 2.14), threat perception regarding malarial morbidity (AOR = 1.51) and mortality (AOR = 2.52) were positively associated with the use of LLIN. Full utilization of bednets by under-fives of the households was higher in villages with sub-centres. INTERPRETATION & CONCLUSION: Around two-third population of the study area was effectively covered with LLIN. Higher proportion of socially marginalized people received LLIN. Threat perception regarding malaria was directly associated with both receipt and use of LLIN. Behaviour change communication on utilization along with adequate access to LLIN needs to be strengthened.

Hand washing practices in urban and rural communities in and around Kolkata, West Bengal.

BACKGROUND: Public health importance of hand washing was known since 19th century. Many researchers also highlighted how hand washing could bring down the incidence of diarrhea, ARI & other diseases. OBJECTIVE: To find out the extent of hand washing as practiced by the community, to what extent suggested steps of hand washing was followed and to assess the changes in bacteriological count of hand before and after hand washing. METHOD: A community based cross sectional study was carried out during January-February 2007 among 161 respondents in and around Kolkata through interview, observation of hand washing in some situations as well as carrying out microbiological test. RESULT: 100% respondents interviewed practiced hand washing after defecation either with soap (59%) or with plain water, ash & mud (41%). But 64%, 51.6% and only 21.7% practiced hand washing before preparation of food; after using urinals; after changing the babies' nappies and disposing their feces respectively. Only 16.1% respondents washed their hands as per the recommended time of 15-30 seconds. Majority (75%) dried hands with apparently unclean materials. 90.7% followed step1 but none followed all the steps. Swab collection before and after hand washing revealed a decrease in colony count in 60% of the samples. CONCLUSION: It can be concluded that extent of desirable practices regarding hand washing is still lacking and needs to be emphasized.

A pilot survey on hand washing among some communities of West Bengal.

A cross-sectional observational study was carried out between April to May 2006 by interview method and observation technique with the objective to know the knowledge regarding hand washing in the community and it was done in the slum and nonslum urban areas and also one rural area. The result shows that in urban slum area 98% washed their hands with soap after defecation; Only 36%, 16% and 2% washed their hands with soap before meal, before serving food and before cooking respectively. However, it was observed that 69% used soap and water for hand washing after cleaning the child's faeces. In rural area 71% used soap and water after defecation while 26% used mud or ash. Only 13%, 1%, 1% and 5% used soap and water before meal, before serving food, before cooking and after cleaning the child's faeces. 82.35% of respondents in non slum area and 89% of respondents in rural area considered that diarrhoea and dysentery could be prevented by hand washing while they did not give importance to hand washing in prevention of diarrhoea over other methods like cleanliness, boiling and purification of water. ARI was much higher (25.72%) in rural area followed by slum area (13.77%) and non-slum area (3.87%). Out of 30 observations among 302 interview made on hand-washing only first step i.e. palm washing (transient rubbing the palm with soap) was followed by all the participants observed. Time taken for such hand-washing was only around five seconds (ideal 15-30 seconds) in urban slum and rural areas while in non slum area it varied between 7-10 seconds on an average. No one followed any other steps of hand-washing, recommended by IFH.

Purification and characterization of avian glycolipid: beta-galactosyltransferases (GalT-4 and GalT-3): cloning and expression of truncated betaGalT-4.

Acidic glycolipid of ganglio-(containing sialic acid) and sialyl-lactofucosyl-type, SA-Lex (containing sialic acid and fucose) are developmentally regulated and appear to be ubiquitous on neuronal and cancer cell surfaces of animals. Two glycolipid: beta-galactosyltransferases, GalT-3 and GalT-4, were characterized in embryonic chicken brain (ECB). Based on substrate competition experiments, these two activities were believed to be due to expression of two gene products. A cDNA fragments (about 600 bp) encoding the catalytic domain of the GalT-4 (UDP-Gal:LcOse3Cer beta1,4galactosyltransferase) from ECB and human Colo-204 were isolated. These cDNAs were expressed as a soluble glutathione-S-transferase fusion protein (48 kDa) in Escherichia coli. Interactions between GlcNAc-, UDP-hexanolamine-, and alpha-lactalbumin were studied with the purified fusion protein (recombinant and truncated). Functionally it was similar to that of native GalT-4 purified (40000-fold) from 11-day-old ECB. GalT-3 (UDP-Gal:GM2beta1,3galactosyltransferase) was purified from 19-day-old ECB, and a polyclonal antibody was produced against the peptide backbone for immunoscreening of a lambdaZAP ECB cDNA expression library. Each of the GalT-3 peptides (62 and 65 kDa was analyzed by protein fingerprinting analysis indicating a similar peptide mapping pattern.

Biosynthesis and regulation of Le(x) and SA-Le(x) glycolipids in metastatic human colon carcinoma cells.

This report concerns the stepwise biosynthesis in vitro of Sialyl Lewis X, (SA-Le(x)), a carcinoembryonic antigen, in human colon carcinoma KM12 cells exhibiting different metastatic behaviors. The significance of SA-Le(x) has become even more apparent since the detection of its terminal epitope NeuAc(alpha 2-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, as the binding ligand of the selectin family member ELAM-1. The activity level of galactosyltransferase GalT-4 which catalyzes the formation of core nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) is very high in all the metastatic lines tested with highly metastatic lines (KM12-SM) exhibiting the highest activity. The same activity pattern for galactosyltransferase is also observed when tested with iLcOse5Cer (GlcNAc beta 1-3nLcOse4Cer), the precursor for polylactosamine glycolipid. Sialyltransferase SAT-3 which catalyzes the formation of LM1 (NeuAc alpha 2-3nLcOse4Cer), the precursor for SA-Le(x), is also present in all the metastatic cell lines although the activity levels are much lower compared to galactosyltransferase. The fucosyltransferase FucT-3, which catalyzes the formation of R'-Gal-Fuc(alpha 1-3)GlcNAc-R linkage, is active with both nonsialylated substrate, nLcOse4Cer, and sialylated substrate, LM1 (NeuAc alpha 2-3nLcOse4Cer) with the formation of either Le(x) (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer) or SA-Le(x) (NeuAc alpha 2-3nLcOse4Cer). However, the sialylated substrate LM1 is preferred to enzymatic activity since it exhibited lower Km (46 microM) than that of nLcOse4Cer (67 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of SAT-3 involved in SA-Le(x) biosynthesis: inhibition studies with polyclonal antibody against GST-SAT-3 fusion protein.

The SAT-3 activity (CMP-NeuAc:Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4Glc-ceramide alpha 2-3 sialytransferase) involved in the biosynthesis of sialy Le(x) has been characterized in human colon carcinoma cells and embryonic chicken brains. Using RT-PCR-based strategy, we have isolated partial cDNA clones of SAT-3 from ECB and Colo-205 mRNAs. Suitable primers from sialylmotif and N-terminal sequence of human placenta SAT-3 (HP-SAT-3) were used. The 800 bp cDNA fragment encoding a region (90%) of alpha 2-3 sialyltransferase (SAT-3) catalytic domain from ECB has been expressed as a glutathione S-transferase (GST) soluble fusion protein (62 kDa) in E. coli and purified over glutathione-agarose affinity matrix. Polyclonal antibody has been produced against affinity-purified catalytic domain of SAT-3 (GST-SAT-3 fusion protein). A concentration-dependent polydonal antibody binding to native SAT-3 has also been demonstrated by measuring the residual SAT-3 activity in the enzyme fractions from Colo-205. The marked inhibition (> 80%) of SAT-3 activity and relatively less inhibition (< 20%) of SAT-4 activity (CMP-NeuAc:GgOse4Cer alpha 2-3sialyl transferase) suggests strongly the existence of two different gene products (SAT-3 and SAT-4) in human colon carcinoma Colo-205 cells and in embryonic chicken brains (ECB).

Regulation of expression of cell surface neolacto-glycolipids and cloning of embryonic chicken brain GalT-4 (UDP-Gal: GlcNAc-R beta 1-4 galactosyltransferase).

The biosynthesis of GM1 ganglioside (Gal beta 1-3GalNAc beta 1-4 (NeuAc alpha 2-3) Gal beta 1-4Glc-Cer) and nLcOse4Cer is catalyzed by two different beta-galactosyltransferases GalT-3 (UDP-Gal: GM2 beta 1-3GalT) and GalT-4 (UDP-Gal: Lc3 beta 1-4GalT) respectively. Solubilized GalT-3 and GalT-4 have been purified 3,000-fold and 22,000-fold, respectively, from 11-19-day-old embryonic chicken brain. The purified GalT-3 transfers galactose to GM2 very actively (Km 33 microM), whereas acetyl GM2 is not an active substrate (Km 350 microM), GalT-3 and GalT-4 are classified as HYCARS (hydrophobic recognition site) and CARS (carbohydrate recognition sites), respectively. An anion-transport inhibitor DIDS (diisothiocyanato stilbene 4,4'-disulphonate), irreversibly inhibits both GalT-3 and GalT-4 activities by binding to a UDP binding site. Polyclonal antibodies against purified GalT-3 and GalT-4 inhibited these two purified activities and showed no cross reactivity on the western blots. RNA-PCR of 11-day-old embryonic chicken brain mRNA with PCR primers designed from the homologous coding regions of cloned sequences of beta 1-4 GalT of human, bovine, and murine-tissues produced a -600 bp cDNA fragment. Dideoxy-sequences of this fragment reveals it to be 74% similar to the nucleotide sequences of the cloned beta 1-4GalT from human liver. The cloned-600 bp cDNA was used to identify two mRNA transcripts (1.4 and 2.3 kb) from ECB by Northern blot analysis and four genomic DNA EcoRI fragments (18.6, 12.9, 10.5 and 3.7 kb) on a Southern blot analysis.

Isolation of a cDNA clone for beta 1-4 galactosyltransferase from embryonic chicken brain and comparison to its mammalian homologs.

Based on the observed biochemical and immunological similarities between bovine beta 1-4 galactosyltransferase (GalT) and a developmentally regulated galactosyltransferase (GalT-4; UDP-Gal:Lc3 beta 1-4 GalT) from embryonic chicken brain, a genetic similarity between the two enzymes has been postulated. To test our hypothesis, we have employed a reverse transcription-polymerase chain reaction (RT-PCR)based approach and isolated a approximately 600 bp cDNA clone from embryonic chicken brain mRNA. Our results indicate that, within the approximately 600 bp fragment, the avian cDNA nucleotide sequence is 74% homologous to the human beta 1-4 galactosyltransferase cDNA. Similarity and identity between the predicted amino acid sequences of the two galactosyltransferases are 75% and 61%, respectively. Similar to its mammalian counterparts, the embryonic chicken brain galactosyltransferase gene appears to encode multiple mRNA transcripts (2.3 and 1.4 kb) and shows multiple bands on a Southern blot (18.6, 12.9, 10.5 and 3.7 kb) indicating that the avian gene is either polyexonic and/or it belongs to a multiple gene family.

A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid.

Galacturonic acid (GalA) is a major component of plant cell-wall-derived pectins. It can be also found in the cell-surface polysaccharides of different microorganisms, including several symbiotic and pathogenic bacteria. Uridine diphosphogalacturonic acid (UDP-GalA) is a likely donor for GalA during the biosynthesis of these polysaccharides. A highly efficient, yet simple, method is presented for generating and purifying UDP-[14C]GalA. Commercially available UDP-[14C]-galactose was quantitatively oxidized (>95% conversion) to UDP-[14C]GalA in the presence of high levels of galactose oxidase and catalase, at prolonged incubation times. Following this one-step enzymatic oxidation, UDP-[14C]GalA was purified using a polyethyleneimine cellulose column with a single-step 1 M NaCl elution. The authenticity of the purified UDP-[14C]GalA was verified by its relative mobility on thin-layer chromatograms, analysis of its chemical hydrolysis products, and 1H NMR spectroscopy. Our yield of >90% is much higher than by previously described methods. The method may serve as a prototype for the preparation of other radiolabeled uronic acids and their nucleotide derivatives.

A phosphotransferase that generates phosphatidylinositol 4-phosphate (PtdIns-4-P) from phosphatidylinositol and lipid A in Rhizobium leguminosarum. A membrane-bound enzyme linking lipid a and ptdins-4-p biosynthesis.

Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unusual phosphate-deficient lipid A found in this organism. The enzyme has been solubilized with Triton X-100 and purified 80-fold. As shown by co-purification and thermal inactivation studies, the 4'-phosphatase catalyzes not only the hydrolysis of (Kdo)2-[4'-32P]lipid IVA but also the transfer the 4'-phosphate of Kdo2-[4'-32P]lipid IVA to the inositol headgroup of phosphatidylinositol (PtdIns) to generate PtdIns-4-P. Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizobium meliloti, or the nodulation-defective mutant 24AR of R. leguminosarum. The specific activity for the phosphotransferase reaction is about 2 times higher than that of the 4'-phosphatase. The phosphotransferase assay conditions are similar to those used for PtdIns kinases, except that ATP and Mg2+ are omitted. The apparent Km for PtdIns is approximately 500 microM versus 20-100 microM for most PtdIns kinases, but the phosphotransferase specific activity in crude cell extracts is higher than that of most PtdIns kinases. The phosphotransferase is absolutely specific for the 4-position of PtdIns and is highly selective for PtdIns as the acceptor. The 4'-phosphatase/phosphotransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns. A phosphoenzyme intermediate may account for the dual function of this enzyme, since a single 32P-labeled protein species (Mr approximately 68,000) can be trapped and visualized by SDS gel electrophoresis of enzyme preparations incubated with Kdo2-[4'-32P]lipid IVA. Although PtdIns is not detected in cultures of R. leguminosarum/etli (CE3), PtdIns may be synthesized during nodulation or supplied by plant membranes, given that soybean PtdIns is an excellent phosphate acceptor. A bacterial enzyme for generating PtdIns-4-P and a direct link between lipid A and PtdIns-4-P biosynthesis have not been reported previously.

Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line.

Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.

Analysis of glycosphingolipids by fluorophore-assisted carbohydrate electrophoresis using ceramide glycanase from Mercenaria mercenaria.

Polyacrylamide gel electrophoresis of fluorophore-labeled saccharides is a simple and sensitive separation method for glycan analysis. This method requires an aldehydic reducing carbon on the saccharide in order to react with the amine group of the fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid or 2-aminoacridone). We have used exoglycosidase-free ceramide glycanase from hard-shelled clam to expose the reducing terminal of the glycan in glycosphingolipid by cleaving the linkage between the glycan and the ceramide. The released glycan was used for fluorophore-assisted carbohydrate electrophoresis, in order to qualitatively determine its monosaccharide composition, chain length, and glycosidic linkages using linkage-specific glycosidases, including a beta 1-3,4 galactosidase from clam.

A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors.

Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4'. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.