Purification of heparin binding oviduct specific proteins and its effect on in vitro embryo development in cattle.

The objective of this study was to see the effect of purified heparin binding oviduct specific proteins (OSP) as media supplement on in vitro embryo developmental competence in cattle. The oviduct specific proteins were isolated from abattoir cattle oviducts and precipitated, dialyzed and at the end purified by high performance liquid chromatography system. The SDS-PAGE profile of eluted heparin binding protein (HBP) fraction showed bands between ~66 - ~97 kDa, while heparin unbinding protein (HUBP) fraction showed two bands at ~66 kDa and in total protein (TP) bands were ~60 - ~95 kDa. Collected all three OSP fractions were used as a media supplement in three different concentrations (0, 5 and 20 µg/mL) for in vitro maturation of immature oocytes, in vitro fertilization and culture of presumptive embryos at 38.5 ºC in 5% CO2 incubator with maximum humidity. The highest cleavage rate (73.40±2.36%) was observed at 5 µg/mL concentration level and lowest cleavage rate (27.63±1.89%) was obtained in 20 μg/mL total protein (TP) fraction. The highest blastocyst formation (26.47±1.47%) also occurred in 5 µg/mL concentration of total protein (TP) fraction and the lowest blastocyst rate (3.60±1.80%) was achieved at 20 µg/mL HBP fraction. The highest cleavage rate in the control group was 60.45±2.66% in TP fraction and blastocyst formation was 11.66±2.54% in HUBP fraction which was not significantly differ from HBP fraction. These results indicate that at 5 µg/mL of total OSP fraction (TP) and HBP used as media supplement increased the cleavage rate significantly as compared to HUBP fraction, and total OSP fraction (TP) increased blastocyst formation significantly (P<0.05) as compared to HBP & HUBP fraction.

Retrospective study of chikungunya outbreak in urban areas of India.

Background & objectives: A retrospective study on chikungunya outbreak in India in five States viz. Delhi, Madhya Pradesh, Orissa, Maharashtra and Kerala was conducted in 2007-2008 to know the distribution and determinants of chikungunya fever outbreak in India. Methods: On the basis of high and low incidence of chikungunya fever, two districts from each State and two wards from the selected district were taken for random selection of 1000 households from 10 districts and 5 States. Semi-structured questionnaires were administered to individuals, patients, qualified health professionals and to stakeholders for collecting information. Results: The educational background and occupation of the respondents showed variations across the study States. Only in high incidence ward of Maharashtra, water storage period for 3-6 days and emptying, drying of water containers on weekly basis was noted. The study through knowledge, attitude, belief, practice (KABP) obtained individual's perception of chikungunya fever, its prevention and control. Patients’ expenditure on treatment was mainly recorded less than Rs 500 across study States. Health facility survey obtained an overview of the capacity of local health facilities. Stakeholders’ perception regarding chikungunya fever was also noted. Interpretation & Conclusions: The study revealed differences in awareness of chikungunya, cause of the disease, vector responsible, mode of transmission, biting time and elimination of breeding of mosquitoes statistically significant among high and low incidence wards of all the States. Expenditure on treatment was independent of economically active status and loss of man-days across all the States. Education and occupation did not have any relation with emptying/drying of water containers in high incidence wards. Strengthening of surveillance, information, education and communication (IEC) activities along with case management facilities may be provided by the State health department for prevention of chikungunya outbreaks in future. Stakeholders should be more involved in outbreak management and future planning.

Human carbonic anhydrase I: Effect of specific site mutations on its function.

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, HislO7, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, GlulO6Gln, Tyr7Trp, Glu.117Gln, and His I 07Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu 117Gln and His I 07Val mutant proteins in Escherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and GlulO6 but not for Tyr7 in the catalytic function of HCAI.