In human leukemia cells ephrin-B-induced invasive activity is supported by Lck and is associated with reassembling of lipid raft signaling complexes

Proteins of the ephrin-B group operate in nonlymphoid cells through the control of their migration and attachment, and are crucial for the development of the vascular, lymphatic, and nervous systems. Ephrin-B activity is deregulated in various nonlymphoid malignancies; however, their precise role in cancer has only started to be addressed. We show here that ephrin-B1, a member of the ephrin-B group, is expressed in pediatric T-cell leukemias, including leukemia cell line Jurkat. Treatment of Jurkat cells with ephrin-B-stimulating EphB3 enhances ephrin-B1 phosphorylation and induces its relocalization into lipid rafts. These events are mediated by the T lineage-specific kinase, Lck, as ephrin-B1 phosphorylation and lipid raft association are blocked in the Lck-deficient clone of Jurkat, JCAM1.6. Ephrin-B1 also induces colocalization of the CrkL and Rac1 cytoskeleton regulators and initiates in leukemic cells a strong repulsive response. The absence of Lck blocks ephrin-B1-induced signaling and repulsion, confirming the essential role for Lck in ephrin-B1-mediated responses. This shows a new role for ephrin-B1 in the regulation of leukemic cells through the Lck-dependent Rac1 colocalization with its signaling partner, CrkL, in lipid rafts. In agreement with its repulsive action, ephrin-B1 seems to support metastatic properties of leukemic cells, as suppression of ephrin-B1 signaling inhibits their invasiveness. Because ephrin-B1-activating EphB proteins are ubiquitously expressed, our findings suggest that ephrin-B1 is likely to play an important role in the regulation of malignant T lymphocytes through the control of lipid-raft-associated signaling, adhesion, and invasive activity, and therefore may represent a novel target for cancer treatment.     

Estrogen and progesterone concentrations in bovine milk during the estrous cycle

Estrogen and progesterone concentrations in milk during the estrous cycle were estimated in 18 normally cycling Holstein dairy cows. The estrogen and progesterone concentrations in milk during the estrous cycle followed the pattern described for them in blood in the corresponding period. During most of the estrous cycle, estrogen concentration remained at approximately 200 pg/ml and reached a proestrous peak of 360 +/- 127 pg/ml on day 19. The progesterone concentration in milk during the estrous cycle increased to a peak on day 13 (45.5 +/- 6.6 ng/ml) and thereafter declined towards estrus. Estrus detection/prediction based on milk progesterone concentrations appears feasible in view of the significant differences in milk progesterone concentrations between the early luteal (post-ovulatory), luteal and rapid follicular growth periods of the estrous cycle.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase-8-Z-DEVD-cho complex

Caspase-8 is an initiator enzyme in the Fas-mediated pathway of which the downstream executioner caspase-3 is a physiological target. Caspases are cysteine proteases that are specific for substrates with an aspartic acid residue at the P(1) position and have an optimal recognition motif that incorporates four amino acid residues N-terminal to the cleavage site. Caspase-8 has been classified as a group III caspase member because it shows a preference for a small hydrophobic residue at the P(4) substrate position. We report the X-ray crystallographic structure of caspase-8 in complex with benzyloxycarbonyl-Asp-Glu-Val-Asp-aldehyde (Z-DEVD), a specific group II caspase inhibitor. The structure shows that the inhibitor interacts favourably with the enzyme in subsite S(4). Kinetic data reveal that Z-DEVD (K(i) 2 nM) is an almost equally potent inhibitor of caspase-8 as the specific group III inhibitor Boc-IETD-aldehyde (K(i) 1 nM). In view of this finding, the original classification of caspases into three specificity groups needs to be modified, at least for caspase-8, which tolerates small hydrophobic residues as well as the acidic residue Asp in subsite S(4). We propose that the subsite S(3) must be considered as an important specificity-determining factor.     

Mycobacterial Antigen Driven Activation of CD14++CD16? Monocytes Is a Predictor of Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14⁺⁺CD16⁻ monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment.     

Does Exercise Improve Glycaemic Control in Type 1 Diabetes? A Systematic Review and Meta-Analysis

Objective

Whilst regular exercise is advocated for people with type 1 diabetes, the benefits of this therapy are poorly delineated. Our objective was to review the evidence for a glycaemic benefit of exercise in type 1 diabetes.

Research Design and Methods

Electronic database searches were carried out in MEDLINE, Embase, Cochrane’s Controlled Trials Register and SPORTDiscus. In addition, we searched for as yet unpublished but completed trials. Glycaemic benefit was defined as an improvement in glycosylated haemoglobin (HbA1c). Both randomised and non-randomised controlled trials were included.

Results

Thirteen studies were identified in the systematic review. Meta-analysis of twelve of these (including 452 patients) demonstrated an HbA1c reduction but this was not statistically significant (standardised mean difference (SMD) −0.25; 95% CI, −0.59 to 0.09).

Conclusions

This meta-analysis does not reveal evidence for a glycaemic benefit of exercise as measured by HbA1c. Reasons for this finding could include increased calorie intake, insulin dose reductions around the time of exercise or lack of power. We also suggest that HbA1c may not be a sensitive indicator of glycaemic control, and that improvement in glycaemic variability may not be reflected in this measure. Exercise does however have other proven benefits in type 1 diabetes, and remains an important part of its management.     

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene

Key message

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ).

Abstract

Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T₁ generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T₀ lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.     

Chikungunya virus outbreak in Kerala, India, 2007: a seroprevalence study

India was affected by a major outbreak of chikungunya fever caused by Chikungunya virus (CHIKV) during 2006-2007. Kerala was the worst affected state during 2007 with a contribution of 55.8% suspected cases in the country. However, except for clinically reported case records, no systematic information is available on infection status of CHIKV in the region. Hence, we carried out a post-epidemic survey to estimate seroprevalence status [immunoglobulin G (IgG)] in the community using commercially available indirect immunofluorescence test. This methodology had been reported to be highly specific and sensitive for CHIKV infection. The study area selected was the worst affected mid-highlands region of Kerala which harbour vast area of rubber plantations. The study evidenced 68% of the population to be seropositive for CHIKV IgG. Males were found more affected than females (χ2 = 9.86; p = 0.002). Among males, prevalence was significantly higher in the age classes 21-30 (χ2 = 5.46; p = 0.019) and 31-40 (χ2 = 5.84; p = 0.016) years. This may be due to high occupational risk of the male population engaged in plantation activities exposed to infective bites of Aedes albopictus. The current study provides an insight into the magnitude of CHIKV outbreak in Kerala.     

Attitudes and Barriers to Exercise in Adults with Type 1 Diabetes (T1DM) and How Best to Address Them: A Qualitative Study

Background

Regular physical activity has recognised health benefits for people with T1DM. However a significant proportion of them do not undertake the recommended levels of activity. Whilst questionnaire-based studies have examined barriers to exercise in people with T1DM, a formal qualitative analysis of these barriers has not been undertaken. Our aims were to explore attitudes, barriers and facilitators to exercise in patients with T1DM.

Methodology

A purposeful sample of long standing T1DM patients were invited to participate in this qualitative study. Twenty-six adults were interviewed using a semi-structured interview schedule to determine their level of exercise and barriers to initiation and maintenance of an exercise programme.

Principal findings

Six main barriers to exercise were identified: lack of time and work related factors; access to facilities; lack of motivation; embarrassment and body image; weather; and diabetes specific barriers (low levels of knowledge about managing diabetes and its complications around exercise). Four motivators to exercise were identified: physical benefits from exercise; improvements in body image; enjoyment and the social interaction of exercising at gym or in groups. Three facilitators to exercise were identified: free or reduced admission to gyms and pools, help with time management, and advice and encouragement around managing diabetes for exercise.

Significance

Many of the barriers to exercise in people with T1DM are shared with the non-diabetic population. The primary difference is the requirement for education about the effect of exercise on diabetes control and its complications. There was a preference for support to be given on a one to one basis rather than in a group environment. This suggests that with the addition of the above educational requirements, one to one techniques that have been successful in increasing activity in patients with other chronic disease and the general public should be successful in increasing activity in patients with T1DM.     

Amphetamine‐induced release of dopamine in primate prefrontal cortex and striatum: striking differences in magnitude and timecourse

The psychostimulant amphetamine (AMPH) is frequently used to increase catecholamine levels in attention disorders and positron emission tomography imaging studies. Despite the fact that most radiotracers for positron emission tomography studies are characterized in non‐human primates (NHPs), data on regional differences of the effect of AMPH in NHPs are very limited. This study examined the impact of AMPH on extracellular dopamine (DA) levels in the medial prefrontal cortex and the caudate of NHPs using microdialysis. In addition to differences in magnitude, we observed striking differences in the temporal profile of extracellular DA levels between these regions that can likely be attributed to differences in the regulation of dopamine uptake and biosynthesis. The present data suggest that cortical DA levels may remain elevated longer than in the caudate which may contribute to the clinical profile of the actions of AMPH.