Antigenic domains of the streptococcal Pep M5 protein. Localization of epitopes crossreactive with type 6 M protein and identification of a hypervariable region of the M molecule.

Pep M5, the pepsin-derived N-terminal half of the group A streptococcal type 5 M protein exhibits immunologic crossreaction with type 6 M protein, localizing some of the M6-crossreactive epitope(s) within this segment of the M5 protein. Based on the amino acid sequence of the Pep M5 protein, two structurally distinct domains have been recognized within its coiled-coil structure. We have now found that peptides derived from both the structurally distinct domains of the Pep M5 protein contain antigenic epitopes. Furthermore, only the peptides from the C-terminal domain of the Pep M5 protein crossreacted with rabbit anti-M6 sera, whereas those from the N-terminal domain did not. Consistent with this, sequence analyses of the arginyl peptides of the Pep M6 protein, the pepsin-derived N-terminal half of the M6 protein, revealed extensive homology of some of these peptides with regions within the C-terminal domain of the Pep M5 molecule. While an arginyl peptide of the Pep M6 protein exhibits 84% homology with region 150-186 of the Pep M5 protein, the C-terminal hexadecapeptide of the Pep M6 protein is virtually identical with the corresponding region of the Pep M5 protein. These results are suggestive of conformational similarities in the region around the pepsin-susceptible site within the M5 and M6 proteins. In addition, one or more epitopes of the M5 protein that are crossreactive with the M6 protein may be placed close to the pepsin-susceptible site of the M5 protein. Previous studies have suggested the N-terminal half of the M proteins to be the variable part of the molecule among the different M protein serotypes. The present results suggest that the N-terminal quarter of the M protein may represent the hypervariable domain of the M molecule.

Epidemiological features and genetic characterization of virus strains in rotavirus associated gastroenteritis in children of Odisha in Eastern India.

We have studied the clinical characteristics, severity and seasonality of rotavirus infection and prevalent genotypes in 652 non-rota vaccinated children in Odisha in eastern India. P genotypes were analysed for their association with host blood group antigens. P type of the virus is determined by the VP8* gene, and specific recognition of A - type of Histo - blood group antigen by P[14]VP8* has been reported. VP4, VP7 and VP6 genes of commonly identified G1P[8] strain were compared with genes of the same strain isolated from other parts of India, elsewhere and strains used for Rotarix and Rotateq vaccines. In 54.75% of children with gastroenteritis, rota virus was found. 9.65% of children had moderate, 78.07% severe, and 12.28% very severe disease as assessed using the Vesikari scoring system. The incidence of infection was highest during winter months. There was no association between any blood group and specific P genotypes. G1P[8] was the commonest cause of gastroenteritis, followed by G1P[11], G3P[8], G9P[8], G2P[4], G2P[6], G9P[4], G9P[11] and G1P[6]. Predominant G genotypes identified were G1 (72.9%), G9 (10.81%), G2 (8.10%) and G3 (8.10%). Sequence analysis of the VP7 gene, placed the G1P[8] strain in lineage 1 and of VP6 gene placed nine G1P[8] strains in subgroup II and one in subgroup I. The VP7 gene segment of two Odisha G1P[8] strains were found to cluster relatively close to the VP7 sequences of Rotarix vaccine. Antigenic differences were found with vaccine strains. Ten G1P[8] strains sequenced for the VP4 gene had 91-93% nucleotide and 92-96% amino acid identity with Rotateq vaccine P[8]). Rotarix vaccine VP4 had 89-91% nucleotide and 90-92% amino acid identity. Our findings indicate genetic variability of rotavirus strains circulating in the region and are significant, given the introduction of rota vaccination in the State.

Characterization of the particles produced by exposure of ribosomal subunits to urea.

When Escherichia coli 50-S ribosomal subunits are treated with increasing concentrations of urea partial deproteination occurs. Furthermore, we observed that the number of sulfhydryl groups which react with Ellman's reagent is a sigmoidal function of the urea concentration. These results are similar to those previously reported for the 30-S subunit (Acharya, A.S. and Moore, P.B. (1973) J. Mol. Biol. 76, 207-221). For both subunits we identify the proteins which dissociate (split proteins) or are recoverable in a ribonucleoprotein particle (core proteins) under the action of 6 M urea in a buffer of moderate ionic strength.

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.

Precontrol observations on lymphatic filariasis & geo-helminthiases in two coastal districts of rural Orrisa.

BACKGROUND & OBJECTIVE: Lymphatic filariasis (LF) is a major public health problem in India, accounting for 40 per cent of the global burden. The World Health Organization has launched a global programme to eliminate LF by 2020 and India is a signatory to it. Orissa, an eastern Indian State has long been known to be endemic for LF. Prior to implementation of mass drug administration programme it is important to collect baseline data on filariasis and geo-helminthiases in the State. The present cross-sectional survey was therefore carried out between February and December 2001 to obtain baseline information on both LF and geo-helminthiases before application of the control measures. METHODS: The study was carried out in rural areas of Puri and Ganjam districts in two phases. In phase I, the distribution of microfilaraemia in two district was mapped out in randomly selected primary health centres (PHCs), and 12 microfilaraemic villages were identified in each district by cluster analysis for the phase II study. In phase II, detailed clinical and parasitological survey for LF and geo-helminthiases was carried out following the standard procedures. RESULTS: Wuchereria bancrofti was found to be widely prevalent in Puri district with certain pockets of Brugia malayi while W. bancrofti was the only species in Ganjam district. The microfilaraemia (Mf) rate was found to be 9.5 and 11.1 per cent; and circulating filarial antigenaemia (CFA) was 16.8 and 17.8 per cent in Puri and Ganjam respectively. The geometric mean intensity (GMI) of Mf per ml of blood among positive individuals was 387 in Puri and 454 in Ganjam. The overall disease rate in Puri was 7.9 and 8.9 per cent in Ganjam. The prevalence of chronic manifestations was found to be significantly higher (P<0.001) than the acute manifestations in both the districts. The prevalence of geo-helminthiases was 31.8 per cent in Puri and 42.1 per cent in Ganjam; and the heavy infection was found to be significantly higher (P<0.001) in Ganjam compared to Puri district. INTERPRETATION & CONCLUSION: The present study identified LF and geo-helminthiases as widely distributed health problem in rural areas of coastal Orissa which warrants intervention measures along the lines recommended by the global programme for elimination of LF and geo-helminthiases to reduce the disease burden.

Chimeric hemoglobins--hybrids of human and swine hemoglobin: assembly and stability of interspecies hybrids.

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely H alpha 2 P beta 2 (H = human, P = swine). In an attempt to establish whether the absence of the swine alpha and human beta (P alpha 2 H beta 2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine alpha human beta chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine alpha human beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P alpha chain of P alpha 2 H beta 2 hybrid occurs in the presence of H alpha chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P alpha 2 H beta 2 hybrid is lower compared to that of HbA. In contrast, P beta chain of H alpha 2 P beta 2 hybrid is refractory to exchange with H beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H alpha 2 P beta 2 is higher compared to that of HbA (H alpha 2 H beta 2). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha-chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P alpha H beta heterodimer (compared to the homodimers H alpha H beta and P alpha P beta) facilitates its segregation into the homodimers by subunit exchange reaction involving either H alpha or P beta. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P alpha 2 H beta 2 hybrid in transgenic swine expressing HbA.

The economic loss due to treatment costs and work loss to individuals with chronic lymphatic filariasis in rural communities of Orissa, India.

This paper is based on 1 year round case control study to investigate the economic burden, in terms of treatment costs and loss of work to people affected with chronic lymphatic filariasis in rural communities of Orissa, Eastern India. Around three-fourths of the chronic patients have sought treatment for their condition and spent, on average an amount of INR 396 (approximately US$ 8.7) per year. The major component of the expenditure is the cost of medicines. The data on loss of work time due to chronic condition reveal that the total absenteeism to the work is significantly higher among chronic filariasis patients than controls. The total number of working hours spent per day by patients and controls are 4.94 and 6.06, respectively with a significant difference. The total absenteeism and the total number of working hours per day are influenced significantly by disease condition and other personal characteristics, namely age, sex and family type. The chronic patients lose a total of 68 days of work per year, which is equivalent to 19% of the total working time of the year. The present results show that the chronic conditions of lymphatic filariasis pose considerable burden on the patient, family and community.

Seroprevalence of HIV infection among jail inmates in Orissa.

Seroprevalence of human immunodeficiency virus (HIV) infection was carried out in three prisons in Orissa from March 1994 to December 1995. All the prisoners of Indian origin (300), housed in these jails tested negative for HIV infection. On the other hand, 33.8 per cent (26/77) of jail inmates from foreign countries (Thailand and Myanmar), serving short terms in Orissa jails were found positive for HIV infection. While all the 26 HIV positive prisoners had HIV-1 infection, five of them had antibodies for HIV-1 and HIV-2 viruses. Such a high prevalence of HIV infection and detection of HIV-2 virus is being reported for the first time from Orissa.

A clinico-epidemiological perspective of lymphatic filariasis in Satyabadi block of Puri district, Orissa.

BACKGROUND & OBJECTIVES: Lymphatic filariasis is a major public health problem in the coastal district of Orissa. However, no systematic studies have been done to document the prevalence of microfilaraemia/disease in different regions of the State. Therefore, the present cross sectional study was undertaken during 1996-97 to obtain information on the clinical and epidemiological status of the disease in Satyabadi block area of Puri district, known to be endemic for filariasis. METHODS: Night blood smear survey and clinical examinations were performed on 4646 individuals aged 0-> or = 60 yr from systematically selected households of 17 randomly selected villages of the Block. Microfilaraemia was detected by thick drop technique using 20 microliters of peripheral blood and microfilariae (mf) density by nucleopore filtration technique collected during 1900-2300 h. RESULTS: The prevalence of microfilaraemia was observed to be 14.8 per cent; 13.3 per cent Wuchereria bancrofti, 1.4 per cent Brugia malayi and 0.09 per cent had mixed infections. Geometric mean microfilaraemia density (infected persons only) was found to be 1288 per ml in case of W. bancrofti and 204 per ml in case of B. malayi. The disease rate was observed to be 19.8 per cent; 12.85 per cent had acute manifestations and 6.97 per cent had chronic manifestations. INTERPRETATION & CONCLUSION: The rate of acute disease manifestations was observed to be significantly higher (P < 0.001) than chronic manifestations. There was a male preponderence among the affected individuals (P < 0.001). The interesting observations of the study were the occurrence of occult filarial manifestations viz., tropical pulmonary eosinophilia (TPE) in 0.47 per cent cases and other associated manifestations like asymptomatic microscopic haematuria, monoarticular arthritis and filarial associated respiratory diseases in 0.50, 0.24 and 0.19 per cent of cases respectively. The present study indicates that the area is highly endemic for lymphatic filariasis with active transmission.

Nutritional status of preschool children in the drought affected Kalahandi district of Orissa.

A community-based cross-sectional study was undertaken to determine the magnitude of undernutrition and protein-energy malnutrition among young children during 1996-97 in drought affected Kalahandi district of Orissa. A total of 751 children aged 0-5 yr were studied for anthropometry and clinical signs of nutritional deficiencies from 15 Gram Panchayats selected using probability proportionate to size sampling. There was no significant difference between boys and girls for nutritional status. According to weight-for-age, 57.1 per cent of the children were suffering from underweight (< median -2SD) and 21.3 per cent of children had very low body weights which were < -3SD of standard. Height-for-age and weight-for-height data showed that 41.8 per cent of children suffered from stunting and 27.9 per cent recorded wasting. The children below one year of age had relatively lower prevalence of malnutrition than the other age groups. The prevalence of clinical PEM in the form of marasmus was found in 0.7 per cent of children, while kwashiorkor was absent. This study showed that malnutrition is still a leading problem among preschool children of Kalahandi district and this has not improved in spite of nutrition intervention programmes which are currently in operation.

Software for estimating LD50 and LD90 by logit analysis.

We describe a user-friendly software package using Turbo Pascal language under MS-DOS environment for estimating LD50 and LD90 by Logit Analysis with a X2 test of goodness of fit for the model.

Transmission dynamics of filariasis in Khurdha district of Orissa, India.

A three year longitudinal study was carried out to quantify the different parameters of filarial transmission in an endemic area of Orissa State, India. Parasitological surveys revealed mean microfilaria rate, microfilaria density and median microfilaria density (MFD-50) to be 9.41, 19.23 and 7.33, respectively. The per man hour density of the vector, Culex quinquefasciatus varied from 24.2 to 66.0 with a peak in January. Infection rate varied from 0.9 to 27.5%, while infectivity rate ranged between 0.0 and 15.2%. Infectivity rate showed high correlation with microfilaria rate and per man hour density of adult mosquito. The highest numbers of first stage larvae (L1), second stage larvae (L2) and third stage larvae (L3) per mosquito were found to be 25, 22 and 11, respectively. Average L3 load per infective mosquito ranged from 1.0 to 7.2. L3 load showed high correlation with microfilaria rate (r = 0.845, p < 0.01) while no correlation was seen with microfilaria density.

Lymphatic filariasis in Khurda district of Orissa, India: an epidemiological study.

A cross-sectional survey was undertaken to determine the prevalence of disease due to lymphatic filariasis in Khurda district of Orissa, India. The total disease attributable to filariasis was significantly higher in males (14.79%) than females (10.04%). However, elephantiasis is more prevalent in females, and adenolymphangitis is more prevalent in males than their counterparts. The prevalences of various forms of the disease are age dependent in both sexes. About one-seventh of men and women of higher age groups suffered from chronic debilitation forms of the disease. The study suggests that overt clinical forms of lymphatic filariasis constitute a major public health problem in the study area.

An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system.

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.

Basic carboxyl groups of hemoglobin S: influence of oxy-deoxy conformation on the chemical reactivity of Glu-43(beta).

The gamma-carboxyl groups of Glu-43(beta) and Glu-22(beta) of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide at pH 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence of N-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22(beta) and Glu-43(beta) with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43(beta) with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43(beta) (i.e., chemical reactivity) as a function of the pH in the region of 5.5-7.5, the apparent pKa of its gamma-carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43(beta) of HbS at pH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(beta), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the gamma-carboxyl group and could be one of the crucial preludes to the polymerization process.