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.     

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.

     

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.     

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.     

Nitric oxide modulates capillary formation at the endothelial cell-tumor cell interface

Nitric oxide synthase expression has been documented in lung tumors, but a potential role for nitric oxide (NO) in induction of capillary formation remains to be elucidated. The purpose of this report was to characterize the direct effects of NO at the level of the tumor-endothelium interface with respect to angiogenesis. A Transwell two-compartment culture system, human endothelial cells (EC), and two human non-small cell lung cancer (CA) lines that constitutively produce NO were used to simulate the EC-tumor cell interface. Both histological types of lung CA, squamous and adenocarcinoma, induced baseline capillary formation by EC within 3 days. This process was inhibited by NO in the microenvironment because decreasing NO production with 100 microM aminoguanidine (AG) significantly increased capillary formation, whereas coincubation with 100 microM AG plus 400 microM L-arginine returned angiogenesis to baseline values. We demonstrate further that NO may exert its inhibitory effects by influencing matrix metalloproteinase expression/activity and tyrosine phosphorylation of proteins in the sprouting tips of nascent capillaries.     

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.     

Structural and functional analysis of caspase active sites

Amino acid sequences of caspases 1, 3, 7, and 8 were aligned with their published three-dimensional (3D) structures. The resultant alignment was used as a template to compare the primary structures of caspases 2, 4-6, and 9-11 to build 3D homology models. The structural models were subsequently refined and validated using structure-activity relationship data obtained from an array of substrate-like inhibitors. All caspases were shown to have identical S1 and catalytic dyad architecture but diverse S2-S4 structures. S2 pockets of these 11 caspases can be briefly categorized into two groups: Csp3, -6, and -7 as one and Csp1, -2, -4, -5, -8, -9, -10, and -11 as the other. S2 pockets of Csp3, -6, and -7 are smaller than those of the other eight caspases, and are limited to binding small P2 residues such as Ala and Val. At the S3 site, the presence of a conserved Arg in all caspases suggests that Glu would be a universally preferred P3 residue. Csp8 and Csp9 have an additional Arg in this pocket that can further enhance the binding of a P3 Glu, whereas Csp2 has a Glu adjacent to the conserved Arg. As such, Csp2 is the only caspase that can accommodate both positively and negatively charged P3. At S4, Csp1, -4, -5, and -11 are closely related with respect to their structures and binder preferences; all have a large hydrophobic pocket and prefer large hydrophobic residues such as Trp. S4 of Csp2, -3, and -7 represents an opposite group with a conformation that is highly specific in binding an Asp. The S4 structures of Csp6, -8, -9, and -10 appear to be hybrids of the two extremes, and have little specificity for any P4. Information revealed from this work provides a guide for designing potent caspase inhibitors with desirable specificity.     

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.     

The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis.

BACKGROUND: In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported. RESULTS: We report here the crystal structure of a complex of the activated human caspase-8 (proteolytic domain) with the irreversible peptidic inhibitor Z-Glu-Val-Asp-dichloromethylketone at 2.8 A resolution. This is the first structure of a representative of the long prodomain initiator caspases and of the group III substrate specificity class. The overall protein architecture resembles the caspase-1 and caspase-3 folds, but shows distinct structural differences in regions forming the active site. In particular, differences observed in subsites S(3), S(4) and the loops involved in inhibitor interactions explain the preference of caspase-8 for substrates with the sequence (Leu/Val)-Glu-X-Asp. CONCLUSIONS: The structural differences could be correlated with the observed substrate specificities of caspase-1, caspase-3 and caspase-8, as determined from kinetic experiments. This information will help us to understand the role of the various caspases in the propagation of the apoptotic signal. The information gained from this investigation should be useful for the design of specific inhibitors.