Role of hydrophilic residues in proton transfer during catalysis by human carbonic anhydrase II

Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.     

Effect of immobilization on kinetic and thermodynamic characteristics of sulfide oxidase from Arthrobacter species

In order to determine the impact of immobilization on biocatalytic efficacy of sulfide oxidase, the kinetic and thermodynamic properties of native and DEAE-cellulose immobilized sulfide oxidase from Arthrobacter species FR-3 were evaluated. Immobilization increased the catalytic efficiency of sulfide oxidase by producing a lower Michaelis-Menten constant (Km) and a higher rate of catalysis (Vmax) at different temperatures. The first-order kinetic analysis of thermal denaturation demonstrated that the values of enthalpy (delta H*d) and entropy (delta S*d) of immobilized sulfide oxidase were lower than the native enzyme, confirming the thermal stabilization of sulfide oxidase by immobilization. The delta H*d and delta S*d of the immobilized enzyme at 35 degrees C were 138.07 kJ/mol and 122.04 J/K/mol, respectively. These results suggest that immobilization made the sulfide oxidase from Arthrobacter sp. FR-3 thermodynamically more efficient for catalysis of sulfide oxidation.     

Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II

The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹⁸O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO₂, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.     

Effect of irrigation,plant density and fertilisers on yield and NPK uptake by groundnut

Summary Three levels of irrigation, three plant densities and three fertiliser schedules were tested in 3³ factorial confounded design with two replications in winter on sandy loam soils of Tirupati campus of Andhra Pradesh Agricultural University. Pod yield did not vary due to the plant densities and the fertiliser schedules when the crop was sown in optimum time during 1980. When the sowings were delayed, as in 1979, 444000 plants per ha and 60 kg N ha⁻¹, 40 kg P ha⁻¹ and 100 kg K ha⁻¹ was optimum. Scheduling irrigation at 25 or 50% DASM (depletion of available soil moisture) was optimum for the groundnut crop. There was no difference in the uptake of NPK due to irrigations at 25 and 50% DASM in both the years. Low plant density was as effective as high plant density for efficient use of fertilisers. Uptake of NPK by the crop was relatively high at higher fertiliser levels. However, this higher uptake did not contribute to high pod yield, probably due to utilisation of absorbed nutrients for rank vegetative growth.     

The State-Led Large Scale Public Private Partnership ‘Chiranjeevi Program’ to Increase Access to Institutional Delivery among Poor Women in Gujarat,India: How Has It Done? What Can We Learn?

Background

Many low-middle income countries have focused on improving access to and quality of obstetric care, as part of promoting a facility based intra-partum care strategy to reduce maternal mortality. The state of Gujarat in India, implements a facility based intra-partum care program through its large for-profit private obstetric sector, under a state-led public-private-partnership, the Chiranjeevi Yojana (CY), under which the state pays accredited private obstetricians to perform deliveries for poor/tribal women. We examine CY performance, its contribution to overall trends in institutional deliveries in Gujarat over the last decade and its effect on private and public sector deliveries there.

Methods

District level institutional delivery data (public, private, CY), national surveys, poverty estimates, census data were used. Institutional delivery trends in Gujarat 2000–2010 are presented; including contributions of different sectors and CY. Piece-wise regression was used to study the influence of the CY program on public and private sector institutional delivery.

Results

Institutional delivery rose from 40.7% (2001) to 89.3% (2010), driven by sharp increases in private sector deliveries. Public sector and CY contributed 25–29% and 13–16% respectively of all deliveries each year. In 2007, 860 of 2000 private obstetricians participated in CY. Since 2007, >600,000 CY deliveries occurred i.e. one-third of births in the target population. Caesareans under CY were 6%, higher than the 2% reported among poor women by the DLHS survey just before CY. CY did not influence the already rising proportion of private sector deliveries in Gujarat.

Conclusion

This paper reports a state-led, fully state-funded, large-scale public-private partnership to improve poor women’s access to institutional delivery - there have been >600,000 beneficiaries. While caesarean proportions are higher under CY than before, it is uncertain if all beneficiaries who require sections receive these. Other issues to explore include quality of care, provider attrition and the relatively low coverage.     

The prevalence of blinding trachoma in northern states of Sudan

Background

Despite historical evidence of blinding trachoma, there have been no widespread contemporary surveys of trachoma prevalence in the northern states of Sudan. We aimed to conduct district-level surveys in this vast region in order to map the extent of the problem and estimate the need for trachoma control interventions to eliminate blinding trachoma.

Methods and Findings

Separate, population based cross-sectional surveys were conducted in 88 localities (districts) in 12 northern states of Sudan between 2006 and 2010. Two-stage cluster random sampling with probability proportional to size was used to select the sample. Trachoma grading was done using the WHO simplified grading system. Key prevalence indicators were trachomatous inflammation-follicular (TF) in children aged 1–9 years and trachomatous trichiasis (TT) in adults aged 15 years and above. The sample comprised 1,260 clusters from which 25,624 households were surveyed. A total of 106,697 participants (81.6% response rate) were examined for trachoma signs. TF prevalence was above 10% in three districts and between 5% and 9% in 11 districts. TT prevalence among adults was above 1% in 20 districts (which included the three districts with TF prevalence >10%). The overall number of people with TT in the population was estimated to be 31,072 (lower and upper bounds = 26,125–36,955).

Conclusion

Trachoma mapping is complete in the northern states of Sudan except for the Darfur States. The survey findings will facilitate programme planning and inform deployment of resources for elimination of trachoma from the northern states of Sudan by 2015, in accordance with the Sudan Federal Ministry of Health (FMOH) objectives.     

RNA helicase, CrhR is indispensable for the energy redistribution and the regulation of photosystem stoichiometry at low temperature in Synechocystis sp. PCC6803

We investigated the role of a cold-inducible and redox-regulated RNA helicase, CrhR, in the energy redistribution and adjustment of stoichiometry between photosystem I (PSI) and photosystem II (PSII), at low temperature in Synechocystis sp. PCC 6803. The results suggest that during low temperature incubation, i.e., when cells are shifted from 34°C to 24°C, wild type cells exhibited light-induced state transitions, whereas the mutant deficient in CrhR failed to perform the same. At low temperature, wild type cells maintained the plastoquinone (PQ) pool in the reduced state due to enhanced respiratory electron flow to the PQ pool, whereas in ?crhR mutant cells the PQ pool was in the oxidized state. Wild type cells were in state 2 and ?crhR cells were locked in state 1 at low temperature. In both wild type and ?crhR cells, a fraction of PSI trimers were changed to PSI monomers. However, in ?crhR cells, the PSI trimer content was significantly decreased. Expression of photosystem I genes, especially the psaA and psaB, was strongly down-regulated due to oxidation of downstream components of PQ in ?crhR cells at low temperature. We demonstrated that changes in the low temperature-induced energy redistribution and regulation of photosystem stoichiometry are acclimatization responses exerted by Synechocystis cells, essentially regulated by the RNA helicase, CrhR, at low temperature.     

1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D

Several studies have demonstrated that excess of vitamin D3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D3 (1,25(OH)2 D3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH)2 D3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH)2D3 receptor and increased calcium uptake in smooth muscle cells of the arteries.     

Temporal association of Ca(2+)-dependent protein kinase with oil bodies during seed development in Santalum album L.: its biochemical characterization and significance

Calcium-dependent protein kinase (CDPK) is expressed in sandalwood (Santalum album L.) seeds under developmental regulation, and it is localized with spherical storage organelles in the endosperm [Anil et al. (2000) Plant Physiol. 122: 1035]. This study identifies these storage organelles as oil bodies. A 55 kDa protein associated with isolated oil bodies, showed Ca(2+)-dependent autophosphorylation and also cross-reacted with anti-soybean CDPK. The CDPK activity detected in the oil body-protein fraction was calmodulin-independent and sensitive to W7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide) inhibition. Differences in Michaelis Menton kinetics, rate of histone phosphorylation and sensitivity to W7 inhibition between a soluble CDPK from embryos and the oil body-associated CDPK of endosperm suggest that these are tissue-specific isozymes. The association of CDPK with oil bodies of endosperm was found to show a temporal pattern during seed development. CDPK protein and activity, and the in vivo phosphorylation of Ser and Thr residues were detected strongly in the oil bodies of endosperm from maturing seed. Since oil body formation occurs during seed maturation, the observations indicate that CDPK and Ca(2+) may have a regulatory role during oil accumulation/oil body biogenesis. The detection of CDPK-protein and activity in oil bodies of groundnut, sesame, cotton, sunflower, soybean and safflower suggests the ubiquity of the association of CDPKs with oil bodies.     

Characterization of somatic embryogenesis in sandalwood (Santalum album L.)

In the synchronous embryogenesis system of sandalwood developed in our laboratory, we observed that the early events of differentiation from freshly induced callus (stage 0) are accomplished in three distinct stages viz., preglobular masses (stage 1), globular embryos (stage 2), and bipolar embryos (stage 3). Transition from stage 0 to 1 was accomplished using 2,4-D and involves a stage specific appearance of two polypeptides of 15 and 30 kDa molecular weight. A 24 kDa polypeptide that was detected as a marked band in extracts of primary callus was not detected in stages 1, 2, and 3. Further, the tissue level of a 50 kDa glycoprotein decreased during transition from stage 2 to stage 3. However, the levels of glycoproteins in the medium were markedly higher in stage 0 cultures compared to those in stage 1. The activities of a protein kinase, glycosidase, and xylanase increased markedly with progressing embryogenesis. Our observations suggest that in addition to being controlled at the level of stage-specific gene expression, somatic embryogenesis in sandalwood is also regulated at the level of controls on cell wall flexibility and posttranslational changes in the pool of preexisting proteins.