Detection of serine proteases in extracts of the domestic mite Blomia tropicalis

Previous studies have shown that the domestic mites Dermatophagoides pteronyssinus and D. farinae contain allergens with serine protease activity. These proteolytic allergens include trypsin, chymotrypsin, elastase, kallikrein, and C3/C5 convertase. However, it is not known whether the domestic mite Blomia tropicalis shares with other mite species the serine protease activities. The enzymatic activity present in extracts obtained from food-free B. tropicalis was investigated using specific substrates and inhibitors. Based upon the concentration response and inhibition profiles, and the digestion of specific substrates our data demonstrate that extracts from B. tropicalis exhibit several serine-protease-like activities. The enzyme activities detected in the B. tropicalis extracts are trypsin, elastase, chymotrypsin, kallikrein, C3/C5 convertase, and mast cell protease. Our results also demonstrate that kallikrein and C3/C5 convertase-like activities were not significantly affected by the α₁-antiprotease, a naturally occurring serine protease inhibitor which protects lung mucosa from the enzymatic action. These data strongly suggest that the Echymyopodidae mite B. tropicalis shares at least five serine proteases with members of other mite families, the Glycyphagidae and Pyroglyphidae. In addition, our data demonstrate the potential use of biochemical methods to detect serine proteases for evaluation of mite growth in vitro, or to detect environmental exposures to these enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

A pilot randomised trial of induced blood-stage Plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs

Background

Critical to the development of new drugs for treatment of malaria is the capacity to safely evaluate their activity in human subjects. The approach that has been most commonly used is testing in subjects with natural malaria infection, a methodology that may expose symptomatic subjects to the risk of ineffective treatment. Here we describe the development and pilot testing of a system to undertake experimental infection using blood stage Plasmodium falciparum parasites (BSP). The objectives of the study were to assess the feasibility and safety of induced BSP infection as a method for assessment of efficacy of new drug candidates for the treatment of P. falciparum infection.

Methods and Findings

A prospective, unblinded, Phase IIa trial was undertaken in 19 healthy, malaria-naïve, male adult volunteers who were infected with BSP and followed with careful clinical and laboratory observation, including a sensitive, quantitative malaria PCR assay. Volunteers were randomly allocated to treatment with either of two licensed antimalarial drug combinations, artemether–lumefantrine (A/L) or atovaquone-proguanil (A/P). In the first cohort (n = 6) where volunteers received ∼360 BSP, none reached the target parasitemia of 1,000 before the day designated for antimalarial treatment (day 6). In the second and third cohorts, 13 volunteers received 1,800 BSP, with all reaching the target parasitemia before receiving treatment (A/L, n = 6; A/P, n = 7) The study demonstrated safety in the 19 volunteers tested, and a significant difference in the clearance kinetics of parasitemia between the drugs in the 13 evaluable subjects, with mean parasite reduction ratios of 759 for A/L and 17 for A/P (95% CI 120–4786 and 7–40 respectively; p<0.01).

Conclusions

This system offers a flexible and safe approach to testing the in vivo activity of novel antimalarials.

Trial Registration:

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01055002","term_id":"NCT01055002"}}NCT01055002     

Analysis of the gut microbiota in the old order amish and its relation to the metabolic syndrome

Obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. To explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the Old Order Amish sect, a culturally homogeneous Caucasian population of Central European ancestry. We characterized the gut microbiota in 310 subjects by deep pyrosequencing of bar-coded PCR amplicons from the V1-V3 region of the 16S rRNA gene. Three communities of interacting bacteria were identified in the gut microbiota, analogous to previously identified gut enterotypes. Neither BMI nor any metabolic syndrome trait was associated with a particular gut community. Network analysis identified twenty-two bacterial species and four OTUs that were either positively or inversely correlated with metabolic syndrome traits, suggesting that certain members of the gut microbiota may play a role in these metabolic derangements.     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

The nucleolar channel system of human endometrium is related to endoplasmic reticulum and R-rings

The nucleolar channel system (NCS) is a well-established ultrastructural hallmark of the postovulation endometrium. Its transient presence has been associated with human fertility. Nevertheless, the biogenesis, composition, and function of these intranuclear membrane cisternae are unknown. Membrane systems with a striking ultrastructural resemblance to the NCS, termed R-rings, are induced in nuclei of tissue culture cells by overexpression of the central repeat domain of the nucleolar protein Nopp140. Here we provide a first molecular characterization of the NCS and compare the biogenesis of these two enigmatic organelles. Like the R-rings, the NCS consists of endoplasmic reticulum harboring the marker glucose-6-phosphatase. R-ring formation initiates at the nuclear envelope, apparently by a calcium-mediated Nopp140-membrane interaction, as supported by the calcium-binding ability of Nopp140, the inhibition of R-ring formation by calcium chelators, and the concentration of Nopp140 and complexed calcium in R-rings. Although biogenesis of the NCS may initiate similarly, the reduced presence of complexed calcium and Nopp140 suggests the involvement of additional factors.     

Evidence-based management of coronary artery disease in the elderly--current perspectives

Cardiovascular disease accounts for significant morbidity and mortality in the elderly. The clinical trial data available to guide therapy in this growing population subset are relatively limited. This review will focus on treatment approaches and recommendations obtained from subgroup analyses of elderly patients from major clinical trials for the management of chronic stable angina, acute coronary syndromes (unstable angina and non-ST-segment elevation myocardial infarction), and coronary revascularization. Recent advances in the treatment of stable angina have shown that use of angiotensin-converting enzyme inhibitors and lipid-lowering therapy as adjunctive measures show benefit in the elderly by reducing the occurrence of death, nonfatal myocardial infarction, and unstable angina. However, if patients experience disabling or unstable anginal symptoms despite effective medical therapy, coronary revascularization must be considered. Several clinical trials have shown a significant reduction in major adverse cardiac events when using intravenous glycoprotein receptor antagonists periprocedurally during percutaneous revascularization approaches in elderly patients with unstable angina or non-ST-segment elevation myocardial infarction, especially when these measures are performed as soon as possible. However, the success of myocardial revascularization by a percutaneous or surgical approach is highly dependent on the patient's associated comorbidities, especially in patients over age 80 years.     

A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos

To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.     

Nitric oxide and promotion of cardiac myocyte apoptosis

The removal of damaged, superfluous or energy-starved cells is essential for biological homeostasis, and occurs in every tissue type. Programmed cell death occurs through several closely regulated signal pathways, including apoptosis, in which cell components are broken down and packaged into small membrane-bound fragments that are then removed by neighbouring cells or phagocytes. This process is activated in the cardiac myocyte in response to a variety of stresses, including oxidative and nitrosative stress, and involves mitochondria-derived signals. Loss of cardiac myocytes through apoptosis has been shown to induce cardiomyopathy in a variety of gene-targeted animal models. Because cardiac myocytes have strictly limited ability to regenerate, sustained programmed cell death is likely to contribute to the development and progression of heart failure in a variety of myocardial diseases. At the same time, the cardiac myocyte possesses a number of mechanisms for defence against short-term haemodynamic and oxidative stresses. Our laboratory has recently examined the role of nitric oxide (NO) as a regulator of the programmed death of cardiac myocytes, and the potential contribution of NO and NO-dependent signalling to the loss of myocytes in heart failure. We will review the role of c-Jun N-terminal kinase in response to oxidative and nitrosative stress, and summarise evidence for its role as a cytoprotective mechanism. We will also review evidence implicating NO in the pathophysiology of heart failure, in the context of the extensive and sometimes contradictory body of research on NO and cell survival. (Mol Cell Biochem 263: 35–53, 2004)