Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein. K364R, like several previously described MCAD mutant proteins, appears to be defective in folding. R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity. How the mutations specifically affect the catalytic activity and the folding is further discussed.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.     

Biggest challenges in bioinformatics

The third Heidelberg Unseminars in Bioinformatics (HUB) was held on 18th October 2012, at Heidelberg University, Germany. HUB brought together around 40 bioinformaticians from academia and industry to discuss the ‘Biggest Challenges in Bioinformatics’ in a ‘World Café’ style event.     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

DNA variation at the invertase locus invGE/GF is associated with tuber quality traits in populations of potato breeding clones

Starch and sugar content of potato tubers are quantitative traits, which are models for the candidate gene approach for identifying the molecular basis of quantitative trait loci (QTL) in noninbred plants. Starch and sugar content are also important for the quality of processed products such as potato chips and French fries. A high content of the reducing sugars glucose and fructose results in inferior chip quality. Tuber starch content affects nutritional quality. Functional and genetic models suggest that genes encoding invertases control, among other things, tuber sugar content. The invGE/GF locus on potato chromosome IX consists of duplicated invertase genes invGE and invGF and colocalizes with cold-sweetening QTL Sug9. DNA variation at invGE/GF was analyzed in 188 tetraploid potato cultivars, which have been assessed for chip quality and tuber starch content. Two closely correlated invertase alleles, invGE-f and invGF-d, were associated with better chip quality in three breeding populations. Allele invGF-b was associated with lower tuber starch content. The potato invertase gene invGE is orthologous to the tomato invertase gene Lin5, which is causal for the fruit-sugar-yield QTL Brix9-2-5, suggesting that natural variation of sugar yield in tomato fruits and sugar content of potato tubers is controlled by functional variants of orthologous invertase genes.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Will an Unsupervised Self-Testing Strategy for HIV Work in Health Care Workers of South Africa? A Cross Sectional Pilot Feasibility Study

Background

In South Africa, stigma, discrimination, social visibility and fear of loss of confidentiality impede health facility-based HIV testing. With 50% of adults having ever tested for HIV in their lifetime, private, alternative testing options are urgently needed. Non-invasive, oral self-tests offer a potential for a confidential, unsupervised HIV self-testing option, but global data are limited.

Methods

A pilot cross-sectional study was conducted from January to June 2012 in health care workers based at the University of Cape Town, South Africa. An innovative, unsupervised, self-testing strategy was evaluated for feasibility; defined as completion of self-testing process (i.e., self test conduct, interpretation and linkage). An oral point-of-care HIV test, an Internet and paper-based self-test HIV applications, and mobile phones were synergized to create an unsupervised strategy. Self-tests were additionally confirmed with rapid tests on site and laboratory tests. Of 270 health care workers (18 years and above, of unknown HIV status approached), 251 consented for participation.

Findings

Overall, about 91% participants rated a positive experience with the strategy. Of 251 participants, 126 evaluated the Internet and 125 the paper-based application successfully; completion rate of 99.2%. All sero-positives were linked to treatment (completion rate:100% (95% CI, 66.0–100). About half of sero-negatives were offered counselling on mobile phones; completion rate: 44.6% (95% CI, 38.0–51.0). A majority of participants (78.1%) were females, aged 18–24 years (61.4%). Nine participants were found sero-positive after confirmatory tests (prevalence 3.6% 95% CI, 1.8–6.9). Six of nine positive self-tests were accurately interpreted; sensitivity: 66.7% (95% CI, 30.9–91.0); specificity:100% (95% CI, 98.1–100).

Interpretation

Our unsupervised self-testing strategy was feasible to operationalize in health care workers in South Africa. Linkages were successfully operationalized with mobile phones in all sero-positives and about half of the sero-negatives sought post-test counselling. Controlled trials and implementation research studies are needed before a scale-up is considered.     

Hydroxyethylamine-based inhibitors of BACE1: P1–P3 macrocyclization can improve potency,selectivity, and cell activity

We describe a systematic study of how macrocyclization in the P₁–P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APP_SW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.