Activation and enzymatic characterization of recombinant human kallikrein 8.

Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM

Reproductive health issues in rural Western Kenya

<p>Abstract</p> <p>Background</p> <p>We describe reproductive health issues among pregnant women in a rural area of Kenya with a high coverage of insecticide treated nets (ITNs) and high prevalence of HIV (15%).</p> <p>Methods</p> <p>We conducted a community-based cross-sectional survey among rural pregnant women in western Kenya. A medical, obstetric and reproductive history was obtained. Blood was obtained for a malaria smear and haemoglobin level, and stool was examined for geohelminths. Height and weight were measured.</p> <p>Results</p> <p>Of 673 participants, 87% were multigravidae and 50% were in their third trimester; 41% had started antenatal clinic visits at the time of interview and 69% reported ITN-use. Malaria parasitemia and anaemia (haemoglobin < 11 g/dl) were detected among 36% and 53% of the women, respectively. Geohelminth infections were detected among 76% of the 390 women who gave a stool sample. Twenty percent of women were underweight, and sixteen percent reported symptoms of herpes zoster or oral thrush in the last two months. Nineteen percent of all women reported using a contraceptive method to delay or prevent pregnancy before the current pregnancy (injection 10%, pill 8%, condom 0.4%). Twenty-three percent of multigravidae conceived their current pregnancy within a year of the previous pregnancy. More than half of the multigravidae (55%) had ever lost a live born child and 21% had lost their last singleton live born child at the time of interview.</p> <p>Conclusion</p> <p>In this rural area with a high HIV prevalence, the reported use of condoms before pregnancy was extremely low. Pregnancy health was not optimal with a high prevalence of malaria, geohelminth infections, anaemia and underweight. Chances of losing a child after birth were high. Multiple interventions are needed to improve reproductive health in this area.</p

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates

Global report on preterm birth and stillbirth (4 of 7): delivery of interventions

<p>Abstract</p> <p>Background</p> <p>The efficacious interventions identified in the previous article of this report will fail unless they are delivered at high and equitable coverage. This article discusses critical delivery constraints and strategies.</p> <p>Barriers to scaling up interventions</p> <p>Achieving universal coverage entails addressing major barriers at many levels. An overarching constraint is the lack of political will, resulting from the dearth of preterm birth and stillbirth data and the lack of visibility. Other barriers exist at the household and community levels, such as insufficient demand for interventions or sociocultural barriers; at the health services level, such as a lack of resources and trained healthcare providers; and at the health sector policy and management level, such as poorly functioning, centralized systems. Additional constraints involve weak governance and accountability, political instability, and challenges in the physical environment.</p> <p>Strategies and examples</p> <p>Scaling up maternal, newborn and child health interventions requires strengthening health systems, but there is also a role for focused, targeted interventions. Choosing a strategy involves identifying appropriate channels for reaching high coverage, which depends on many factors such as access to and attendance at healthcare facilities. Delivery channels vary, and may include facility- and community-based healthcare providers, mass media campaigns, and community-based approaches and marketing strategies. Issues related to scaling up are discussed in the context of four interventions that may be given to mothers at different stages throughout pregnancy or to newborns: (1) detection and treatment of syphilis; (2) emergency Cesarean section; (3) newborn resuscitation; and (4) kangaroo mother care. Systematic reviews of the literature and large-scale implementation studies are analyzed for each intervention.</p> <p>Conclusion</p> <p>Equitable and successful scale-up of preterm birth and stillbirth interventions will require addressing multiple barriers, and utilizing multiple delivery approaches and channels. Another important need is developing strategies to discontinue ineffective or harmful interventions. Preterm birth and stillbirth interventions must also be placed in the broader maternal, newborn and child health context to identify and prioritize those that will help improve several outcomes at the same time. The next article discusses advocacy challenges and opportunities.</p

Angiogenèse et cancer de vessie: marqueur tumoral à la mode ou nouvelle cible thérapeutique [Angiogenesis and bladder cancer: trendy prognostic factor or new therapeutic target?].

Tumor-associated angiogenesis is emerging as an important prognostic factor and represents a hopeful potential therapeutic target for the cancer treatment. Bladder tumors, as all solid tumors, require an active angiogenesis to support their growth and progression. The angiogenic phenotype observed within a tumor is determined in large part by the balance between stimulatory and inhibitory inputs to the endothelial cells. Clinically, the importance of the angiogenic response observed within a tumor should be considered as an independent prognostic factor for superficial as well as invasive bladder tumors. The analysis of the angiogenic response could in the future influence the therapeutic strategy. Angiogenesis also represents a promising new therapeutic target, and is at the moment intensively tested in experimental and clinical trials. In this article, we will first review the mechanism of angiogenesis, and then its implication in bladder cancer, and as a potential therapeutic target

Maternal and congenital syphilis programmes: case studies in Bolivia, Kenya and South Africa

Preventing congenital syphilis is not technically difficult, however operational difficulties limit the effectiveness of programmes in many settings. This paper reports on programmes in Bolivia, Kenya, and South Africa. All three countries have established antenatal syphilis control programmes. Early antenatal syphilis screening and management of positive cases were difficult to implement since most women presented for their first antenatal clinic visit after 6 months of pregnancy. Most women had rapid plasma reagin (RPR) testing; results were available on the same day in some clinics but took up to 4 weeks in others. No clinic had a system for tracking RPR-reactive women who did not return for their results. There were no guidelines for providers in Kenya and Bolivia. In all countries, supplies, drugs, notification cards, and other consumables were often unavailable. Health-care providers were unmotivated in Kenya and reported an excessive client load. In South Africa and Kenya some clients reported at their exit interview that they had never heard of syphilis nor had they been informed why blood was collected. Several prevention strategies could be implemented at the clinic level. These include encouraging women to attend for antenatal care before the fourth month of pregnancy, providing point-of-care testing so that results are available immediately and women who test positive can be treated, implementing presumptive treatment of sexual partners of women who test positive, adding a second test later in pregnancy so that incident cases can be managed, and improving the quality of syphilis care during pregnancy, delivery, and the neonatal period

Maternal and congenital syphilis programmes: case studies in Bolivia, Kenya and South Africa.

Preventing congenital syphilis is not technically difficult, however operational difficulties limit the effectiveness of programmes in many settings. This paper reports on programmes in Bolivia, Kenya, and South Africa. All three countries have established antenatal syphilis control programmes. Early antenatal syphilis screening and management of positive cases were difficult to implement since most women presented for their first antenatal clinic visit after 6 months of pregnancy. Most women had rapid plasma reagin (RPR) testing; results were available on the same day in some clinics but took up to 4 weeks in others. No clinic had a system for tracking RPR-reactive women who did not return for their results. There were no guidelines for providers in Kenya and Bolivia. In all countries, supplies, drugs, notification cards, and other consumables were often unavailable. Health-care providers were unmotivated in Kenya and reported an excessive client load. In South Africa and Kenya some clients reported at their exit interview that they had never heard of syphilis nor had they been informed why blood was collected. Several prevention strategies could be implemented at the clinic level. These include encouraging women to attend for antenatal care before the fourth month of pregnancy, providing point-of-care testing so that results are available immediately and women who test positive can be treated, implementing presumptive treatment of sexual partners of women who test positive, adding a second test later in pregnancy so that incident cases can be managed, and improving the quality of syphilis care during pregnancy, delivery, and the neonatal period