Characterization of IS 1110, a highly mobiie genetic element from Mycobacterium avium

A highly mobile Insertion sequence designated IS 1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20′) were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS 1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS 1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited In M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.     

Can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride?

To address the question can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride, we have characterised the selectivity of lipases from a wide range of oilseeds with diverse fatty acid compositions. For this study, a novel hydrolysis assay using a fully randomised oil, was developed. From some seed sources (e.g. Cinnamomum camphora), lipases show high preference for particular fatty acids, whilst from others (e.g. Brassica napus, Theobroma cacao80% saturated or 'unusual' fatty acids may contain lipases which exhibit selectivity. It therefore follows that since the majority of seeds are composed of unsaturated fatty acids, that highly selective lipases will be unusual in nature. However lipases from some species of the Cuphea genera show exceptionally high preference for particular fatty acids. For example, lipase from seeds of Cuphea procumbans has over 20-fold selectivity for C10:0.     

Structural variations in copper(I) iodide coordination polymers of sulfide and disulfide containing flexible 3-substituted pyridine ligands

The three-substituted dipyridyl ligand bis(3-pyridylmethyl)sulfide (L¹) was prepared by the reaction of 3-(chloromethyl)pyridine hydrochloride with thioacetamide under basic conditions. L¹ was reacted with CuI to give complexes with 1:2 and 1:1 molar ratios. Crystal structures of [(CuI)₂(L¹)]_∞ (1) and [CuI(L¹)]_∞ (2) were determined. In complex 1 the CuI species formed a one-dimensional staircase polymer to which L¹ was bound in a side-by-side fashion with π-π interactions between the ligands on each side. Complex 2 consisted of a one-dimensional ribbon polymer of metallomacrocycles formed from two L¹ ligands bridging Cu₂I₂ dimers which were fused within the macrocyclic ring. The analogous disulfide ligand bis(3-pyridylmethyl)disulfide (L²) was prepared by oxidation of the corresponding thiol 3-(sulfanylmethyl)pyridine. L² was reacted with CuI in 1:2 and 1:1 molar ratios and products isolated but only the 1:1 product was able to be crystallised. The crystal structure of [CuI(L²)]_∞ (3) consisted of a one-dimensional ribbon polymer of metallomacrocycles formed from two L² ligands linked through Cu₂I₂ dimers. The difference in the metallomacrocycle linking between the related structures 2 and 3 was attributed to the difference in ligand conformation.     

Attitudes toward and uptake of H1N1 vaccine among health care workers during the 2009 H1N1 pandemic

Background

Though recommended by many and mandated by some, influenza vaccination rates among health care workers, even in pandemics, remain below optimal levels. The objective of this study was to assess vaccination uptake, attitudes, and distinguishing characteristics (including doctor-nurse differences) of health care workers who did and did not receive the pandemic H1N1 influenza vaccine in late 2009.

Methodology/Principal Findings

In early 2010 we mailed a self-administered survey to 800 physicians and 800 nurses currently licensed and practicing in Minnesota. 1,073 individuals responded (cooperation rate: 69%). 85% and 62% of Minnesota physicians and nurses, respectively, reported being vaccinated. Accurately estimating the risk of vaccine side effects (OR 2.0; 95% CI 1.5–2.7), agreeing with a professional obligation to be vaccinated (OR 10.1; 95% CI 7.1–14.2), an ethical obligation to follow public health authorities'' recommendations (OR 9.9; 95% CI 6.6–14.9), and laws mandating pandemic vaccination (OR 3.1; 95% CI 2.3–4.1) were all independently associated with receiving the H1N1 influenza vaccine.

Conclusions/Significance

While a majority of health care workers in one midwestern state reported receiving the pandemic H1N1 vaccine, physicians and nurses differed significantly in vaccination uptake. Several key attitudes and perceptions may influence health care workers'' decisions regarding vaccination. These data inform how states might optimally enlist health care workers'' support in achieving vaccination goals during a pandemic.     

Immunological detection of NADH-specific enoyl-ACP reductase from rape seed (Brassica napus)--induction, relationship of alpha and beta polypeptides, mRNA translation and interaction with ACP

An antibody has been raised against rape seed enoyl-ACP reductase. This recognizes both the alpha and beta polypeptides of the enzyme. Immunoblotting of fresh seed demonstrates that beta is not present in seed material, and that it is produced by proteolysis during isolation. It is thus deduced that rape seed enoyl reductase is an alpha 4 homotetramer. Leaf material from both rape and Arabidopsis have an enoyl reductase with a similar electrophoretic mobility to the rape seed enzyme when analyzed on SDS-PAGE. Quantitative immunoassay has demonstrated that the enzyme continually increases during lipid deposition, indicating that an increase in this enzyme is required to sustain high levels of lipid biosynthesis. In vitro translation experiments show that the enzyme is nuclear coded and synthesized as a precursor form. Immunogold electron microscopy has demonstrated that enoyl reductase is located in plastids. It is shown that ACP-Sepharose may be used as a matrix in the purification of enoyl-ACP reductase.     

Enhancement of seed phytosterol levels by expression of an N-terminal truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase

Dietary intake of phytosterols (plant sterols) has been shown to be effective in reducing blood cholesterol levels, thereby reducing the risk of cardiovascular disease. Phytosterols are most commonly sourced from vegetable oils, where they are present as minor components. We report here the generation of transgenic tobacco seeds substantially enhanced in phytosterol content by the expression of a modified form of one of the key sterol biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The constitutive expression of an N-terminal truncated Hevea brasiliensis HMGR (t-HMGR), lacking the membrane binding domain, enhanced seed HMGR activities by 11-fold, leading to increases in total seed sterol of 2.4-fold. Seed-specific expression of t-HMGR enhanced total seed sterol levels by 3.2-fold, to 1.36% dry weight or 3.25% of oil. 4-desmethylsterols were increased by 2.2-fold, whilst certain sterol biosynthetic intermediates, in particular cycloartenol and 24-ethylidene lophenol, also accumulated. The additional sterol in seed tissue was present in the form of fatty acid esters. Constitutive expression of t-HMGR increased leaf phytosterol sterol levels by 10-fold, representing 1.8% dry weight, and the sterol was sequestered, in acyl ester form, as cytoplasmic 'oil droplets'. These studies establish HMGR as a key enzyme controlling overall flux into the sterol biosynthesis pathway in seed tissue, but the accumulation of certain intermediates suggests additional slow steps in the pathway. The expression of an N-truncated HMGR activity has generated novel phytosterol-enriched raw materials that may provide the basis of new sourcing opportunities for this important class of cholesterol-lowering actives.     

Adenosine receptor signalling in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer’s disease. The adenosine A₁ and A_2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic β-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer’s disease and the therapeutic potential of A₁ and A_2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response.     

Neuregulin-dependent protein synthesis in C2C12 myotubes and rat diaphragm muscle

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [³H]phenylalanine into the protein pool) were investigated in differentiated C₂C₁₂ myotubes and rat diaphragm muscle (DIAm). In C₂C₁₂ myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased 30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [³H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K. Akt; ErbB; heregulin; protein biosynthesis; skeletal muscle     

Reduced ribosomal protein s6 phosphorylation after progressive resistance exercise in growing adolescent rats

The purpose of this study was to investigate moderate intensity progressive resistance exercise (PRE) in growing adolescent rats and its effect on muscle hypertrophy (defined as an increase in fiber cross-sectional area [CSA]). We hypothesized that in adolescent animals moderate intensity PRE would increase (a) fiber CSA; (b) myosin heavy chain (MyHC) content; and (c) expression and phosphorylation of cell signaling molecules involved in translational regulation, compared with that in age-matched sedentary (SED) controls. In the PRE group, 3-week-old male rats were trained to climb a vertical ladder as a mode of PRE training such that by 10 weeks all animals in the PRE group had progressed to carry an additional 80% of their body weight per climb. In agreement with our hypotheses, we observed that 10 weeks of moderate PRE in adolescent animals was sufficient to increase the CSA of muscle fibers and increase MyHC content. The average muscle fiber CSA increased by >10%, and the total MyHC content increased by 35% (p < 0.05) in the PRE group compared with that in the SED animals. Concurrently, we investigated sustained changes in the expression and phosphorylation of key signaling molecules that are previously identified regulators of hypertrophy in adult animal models. Contrary to our hypotheses, expression and phosphorylation of the translational regulators mammalian target of rapamycin and Akt were not increased in the PRE group. In addition, we observed that the ratio of phosphorylated-to-unphosphorylated ribosomal protein S6 (rpS6) was reduced over sixfold in PRE animals (p < 0.05) and that total rpS6 protein levels were unchanged between PRE and SED animals (p > 0.05). We conclude that moderate intensity PRE is sufficient to induce muscle hypertrophy in adolescent animals, whereas the signaling mechanisms associated with muscle hypertrophy may differ between growing adolescents and adults.