Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Lysine overproducing Corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states

A homoserine auxotroph strain of Corynebacterium glutamicum accumulates storage compound trehalose with lysine when limited by growth. Industrially lysine is produced from C. glutamicum through aspartate biosynthetic pathway, where enzymatic activity of aspartate kinase is allosterically controlled by the concerted feedback inhibition of threonine plus lysine. Ample threonine in the medium supports growth and inhibits lysine production (phenotype-I) and its complete absence leads to inhibition of growth in addition to accumulating lysine and trehalose (phenotype-II). In this work, we demonstrate that as threonine concentration becomes limiting, metabolic state of the cell shifts from maximizing growth (phenotype-I) to maximizing trehalose phenotype (phenotype-II) in a highly sensitive manner (with a Hill coefficient of 4). Trehalose formation was linked to lysine production through stoichiometry of the network. The study demonstrated that the net flux of the population was a linear combination of the two optimal phenotypic states, requiring only two experimental measurements to evaluate the flux distribution. The property of linear combination of two extreme phenotypes was robust for various medium conditions including varying batch time, initial glucose concentrations and medium osmolality.     

Two new fossil woods from the early Miocene of Kutch,Gujarat, India and their significance

The Kutch region of western India (Gujarat State) is today arid to semiarid and characterised by mostly ephemeral streams which carry water during the monsoon. The uneven distribution of rainfall and disturbed topography are the result of climate change during the Cenozoic period. Two fossil woods, namely Bauhinium palaeomalabaricum Prakash and Prasad (Fabaceae) and Ebenoxylon indicum Ghosh and Kazmi (Ebenaceae), are described from Kutch in order to provide insights into the palaeovegetation and palaeoclimate. Because the modern representatives of the present and previously described taxa from the same horizon are thermophilic in nature and grow in evergreen to deciduous forests, a warm and humid climate is interpreted. Furthermore, the finding of some mangrove taxa in the assemblage indicates the lagoonal to intertidal environment at the time of deposition.     

An Analysis of Pregnancy-Related Mortality in the KEMRI/CDC Health and Demographic Surveillance System in Western Kenya

Background

Pregnancy-related (PR) deaths are often a result of direct obstetric complications occurring at childbirth.

Methods and Findings

To estimate the burden of and characterize risk factors for PR mortality, we evaluated deaths that occurred between 2003 and 2008 among women of childbearing age (15 to 49 years) using Health and Demographic Surveillance System data in rural western Kenya. WHO ICD definition of PR mortality was used: “the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the cause of death”. In addition, symptoms and events at the time of death were examined using the WHO verbal autopsy methodology. Deaths were categorized as either (i) directly PR: main cause of death was ascribed as obstetric, or (ii) indirectly PR: main cause of death was non-obstetric. Of 3,223 deaths in women 15 to 49 years, 249 (7.7%) were PR. One-third (34%) of these were due to direct obstetric causes, predominantly postpartum hemorrhage, abortion complications and puerperal sepsis. Two-thirds were indirect; three-quarters were attributable to human immunodeficiency virus (HIV/AIDS), malaria and tuberculosis. Significantly more women who died in lower socio-economic groups sought care from traditional birth attendants (p = 0.034), while less impoverished women were more likely to seek hospital care (p = 0.001). The PR mortality ratio over the six years was 740 (95% CI 651–838) per 100,000 live births, with no evidence of reduction over time (χ² linear trend = 1.07; p = 0.3).

Conclusions

These data supplement current scanty information on the relationship between infectious diseases and poor maternal outcomes in Africa. They indicate low uptake of maternal health interventions in women dying during pregnancy and postpartum, suggesting improved access to and increased uptake of skilled obstetric care, as well as preventive measures against HIV/AIDS, malaria and tuberculosis among all women of childbearing age may help to reduce pregnancy-related mortality.     

Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.     

Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied.     

User and Provider Acceptability of Intermittent Screening and Treatment and Intermittent Preventive Treatment with Dihydroartemisinin-Piperaquine to Prevent Malaria in Pregnancy in Western Kenya

Background

The World Health Organization recommends intermittent preventive treatment in pregnancy (IPTp) with sulphadoxine-pyrimethamine (SP) alongside long-lasting insecticide-treated nets (LLIN) and case management for reducing the risks associated with malaria in pregnancy in areas of moderate-to-high transmission in sub-Saharan Africa. Due to increasing Plasmodium falciparum resistance to SP, the search for alternative drugs or strategies to control malaria in pregnancy is a priority. We assessed the acceptability among pregnant women and health providers of intermittent screening and treatment (ISTp) and IPTp with dihydroartemisinin-piperaquine (DP) as alternative strategies in the context of an un-blinded clinical trial.

Methods

Qualitative data were collected through ten focus group discussions with women participating in a randomized controlled trial to evaluate ISTp or IPTp with DP (multi-day regimen) versus IPTp with SP (single dose) in western Kenya. Individual in-depth interviews were conducted with 26 health providers working in the trial facilities and trial staff.

Results

Women appreciated the advantages of being tested with a rapid diagnostic test (RDT) at every ANC visit (although a few women disliked finger pricks) and accepted that they would not receive any antimalarial when tested RDT-negative. There were differences in women’s experiences of the efficacy of antimalarials between the trial arms, with more women in the IPTp-SP arm reporting they had experienced malaria episodes. Side effects were experienced among women taking DP and SP. Although women and trial staff reported adherence to the full DP regimen within the trial, health providers were not confident that women would adhere to multi-day regimens in non-trial settings. Health providers recognized the advantages of ISTp in reducing unnecessary exposure to drugs, but lacked confidence in the reliability of RDTs compared to microscopy.

Conclusions

Our findings indicate that, within a trial context, ISTp-DP and IPTp-DP were generally acceptable among both users and providers and were regarded as potentially valuable alternatives to IPTp-SP. Several challenges were identified the most important of which was concerns with achieving adherence to DP in non-trial settings, requiring operational feasibility studies in routine health systems. Policy adoption of ISTp with RDTs would require a major shift in thinking among health providers due to lack of confidence in RDTs.