Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus)

As part of a programme of comparative measurements of P _d (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be ～6.3 μm. P _d was measured by using an Mn²⁺‐doping 1H NMR (nuclear magnetic resonance) technique. The P _d (cm/s) values were relatively low: ～2.1×10^?3 at 25°C, 2.5×10^?3 at 30°C, 3.4×10^?3 at 37°C and 4.5 at 42°C for the platypus RBCs and ～2.8×10^?3 at 25°C, 3.2×10^?3 at 30°C, 4.5×10^?3 at 37°C and 5.7×10^?3 at 42°C for the crocodile RBCs. In parallel with the low water permeability, the E _a,d (activation energy of water diffusion) was relatively high, ～35 kJ/mol. These results suggest that “conventional” WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.     

Synthesis and analysis of the membrane proximal external region epitopes of HIV‐1

The membrane proximal external region (MPER) of gp41 abuts the viral membrane at the base of HIV‐1 envelope glycoprotein spikes. The MPER is highly conserved and is rich in Trp and other lipophilic residues. The MPER is also required for the infection of host cells by HIV‐1 and is the target of the broadly neutralizing antibodies, 4E10, 2F5, and Z13e1. These neutralizing antibodies are valuable tools for understanding relevant conformations of the MPER and for studying HIV‐1 neutralization, but multiple approaches used to elicit MPER binding antibodies with similar neutralization properties have failed. Here we report our efforts to mimic the MPER using linear as well as constrained peptides. Unnatural amino acids were also introduced into the core epitope of 4E10 to probe requirements of antibody binding. Peptide analogs with C‐terminal Api or Aib residues designed to be helical transmembrane (TM) domain surrogates exhibit enhanced binding to the 4E10 and Z13e1 antibodies. However, we find that placement of constrained amino acids at nonbinding sites within the core epitope significantly reduce binding. These results are relevant to an understanding of native MPER structure on HIV‐1, and form a basis for a chemical synthesis approach to mimic MPER stricture and to construct an MPER‐based vaccine. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Multifaceted deaths orchestrated by mitochondria in neurones

Neurones undergo diverse forms of cell death depending on the nature and severity of the stress. These death outcomes are now classified into various types of programmed cell death, including apoptosis, autophagy and necrosis. Each of these pathways can run in parallel and all have mitochondria as a central feature. Recruitment of mitochondria into cell death signalling involves either (or both) induction of specific death responses through release of apoptogenic proteins into the cytosol, or perturbation in function leading to loss of mitochondrial energisation and ATP synthesis. Cross-talk between these signalling pathways, particularly downstream of mitochondria, determines the resultant pattern of cell death. The differential recruitment of specific death pathways depends on the timing of engagement of mitochondrial signalling. Other influences on programmed cell death pathways occur through stress of the endoplasmic reticulum and the associated ubiquitin-proteasome system normally handling potentially neurotoxic protein aggregates. Based upon contemporary evidence apoptosis is a relatively rare in the mature brain whereas the contribution of programmed necrosis to various neuropathologies has been underestimated. The death outcomes that neurones exhibit during acute or chronic injury or pathological conditions considered here (oxidative stress, hypoxic-ischaemic injury, amyotrophic lateral sclerosis, Parkinson's and Huntington's diseases) fall within a spectrum of the diverse death types across the apoptosis-necrosis continuum. Indeed, dying or dead neurones may simultaneously manifest characteristics of more than one type of death pathway. Understanding neuronal death pathways and their cross-talk not only informs the detailed pathobiology but also suggests novel therapeutic strategies.     

Differences between tuberculosis cases infected with Mycobacterium africanum, West African type 2, relative to Euro-American Mycobacterium tuberculosis: an update

Mycobacterium africanum (MAF) is a common cause of human pulmonary tuberculosis in West Africa. We previously described phenotypic differences between MAF and Mycobacterium tuberculosis (MTB) among 290 patients. In the present analysis, we compared 692 tuberculosis patients infected with the two most common lineages within the (MTB) complex found in the Gambia, namely MAF West African type 2 (39% prevalence) and Euro-American MTB (55% prevalence). We identified additional phenotypic differences between infections with these two organisms. MAF patients were more likely to be older and HIV infected. In addition, they had worse disease on chest X-ray, despite complaining of cough for an equal duration, and were more likely severely malnourished. In this cohort, the prevalence of MAF did not change significantly over a 7-year period.     

Modeling shifts in the rate and pattern of subthalamopallidal network activity during deep brain stimulation

Deep brain stimulation (DBS) of the subthlamic nucleus (STN) represents an effective treatment for medically refractory Parkinson’s disease; however, understanding of its effects on basal ganglia network activity remains limited. We constructed a computational model of the subthalamopallidal network, trained it to fit in vivo recordings from parkinsonian monkeys, and evaluated its response to STN DBS. The network model was created with synaptically connected single compartment biophysical models of STN and pallidal neurons, and stochastically defined inputs driven by cortical beta rhythms. A least mean square error training algorithm was developed to parameterize network connections and minimize error when compared to experimental spike and burst rates in the parkinsonian condition. The output of the trained network was then compared to experimental data not used in the training process. We found that reducing the influence of the cortical beta input on the model generated activity that agreed well with recordings from normal monkeys. Further, during STN DBS in the parkinsonian condition the simulations reproduced the reduction in GPi bursting found in existing experimental data. The model also provided the opportunity to greatly expand analysis of GPi bursting activity, generating three major predictions. First, its reduction was proportional to the volume of STN activated by DBS. Second, GPi bursting decreased in a stimulation frequency dependent manner, saturating at values consistent with clinically therapeutic DBS. And third, ablating STN neurons, reported to generate similar therapeutic outcomes as STN DBS, also reduced GPi bursting. Our theoretical analysis of stimulation induced network activity suggests that regularization of GPi firing is dependent on the volume of STN tissue activated and a threshold level of burst reduction may be necessary for therapeutic effect.     

Assessment of selenium bioavailability from naturally produced high-selenium soy foods in selenium-deficient rats

We assessed the bioavailability of selenium (Se) from a protein isolate and tofu (bean curd) prepared from naturally produced high-Se soybeans. The Se concentrations of the soybeans, the protein isolate and tofu were 5.2 ± 0.2, 11.4 ± 0.1 and 7.4 ± 0.1 mg/kg, respectively. Male weanling Sprague–Dawley rats were depleted of Se by feeding them a 30% Torula yeast-based diet (4.1 μg Se/kg) for 56 days, and then they were replenished with Se for an additional 50 days by feeding them the same diet containing 14, 24 or 30 μg Se/kg from the protein isolate or 13, 23 or 31 μg Se/kg from tofu, respectively. l-Selenomethionine (SeMet) was used as a reference. Selenium bioavailability was determined on the basis of the restoration of Se-dependent enzyme activities and tissue Se concentrations in Se-depleted rats, comparing those responses for the protein isolate and tofu to those for SeMet by using a slope-ratio method. Dietary supplementation with the protein isolate or tofu resulted in linear or log-linear, dose-dependent increases in glutathione peroxidase activities in blood and liver and in thioredoxin reductase activity in liver. Furthermore, supplementation with the protein isolate or tofu resulted in linear or log-linear, dose-dependent increases in the Se concentrations of plasma, liver, muscle and kidneys. These results indicated an overall bioavailability of approximately 101% for Se from the protein isolate and 94% from tofu, relative to SeMet. We conclude that Se from naturally produced high-Se soybeans is highly bioavailable in this model and that high-Se soybeans may be a good dietary source of Se.     

Biological origins of normal-chain hydrocarbons: a pathway model based on cuticular wax analyses of maize silks

Long-chain normal hydrocarbons (e.g. alkanes, alkenes and dienes) are rare biological molecules and their biosynthetic origins are obscure. Detailed analyses of the surface lipids that accumulate on maize silks have revealed that these hydrocarbons constitute a large portion (>90%) of the cuticular waxes that coat this organ, which contrasts with the situation on maize seedling leaves, where the cuticular waxes are primary alcohols and aldehydes. The normal hydrocarbons that occur on silks are part of a homologous series of alkanes, alkenes and dienes of odd-number carbon atoms, ranging between 19 and 33 in number. The alkenes and dienes consist of a homologous series, each of which has double bonds situated at defined positions of the alkyl chains: alkenes have double bonds situated at the sixth, ninth or 12th positions, and dienes have double bonds situated at the sixth and ninth, or ninth and twelfth positions. Finding a homologous series of unsaturated aldehydes and fatty acids suggests that these alkenes and dienes are biosynthesized by a series of parallel pathways of fatty-acid elongation and desaturation reactions, which are followed by sequential reduction and decarbonylation. In addition, the silk cuticular waxes contain metabolically related unsaturated long-chain methylketones, which probably arise via a decarboxylation mechanism. Finally, metabolite profiling analyses of the cuticular waxes of two maize inbred lines (B73 and Mo17), and their genetic hybrids, have provided insights into the genetic control network of these biosynthetic pathways, and that the genetic regulation of these pathways display best-parent heterotic effects.     

GO‐At : in silico prediction of gene function in Arabidopsis thaliana by combining heterogeneous data

Despite recent advances, accurate gene function prediction remains an elusive goal, with very few methods directly applicable to the plant Arabidopsis thaliana. In this study, we present GO‐At (gene ontology prediction in A. thaliana), a method that combines five data types (co‐expression, sequence, phylogenetic profile, interaction and gene neighbourhood) to predict gene function in Arabidopsis. Using a simple, yet powerful two‐step approach, GO‐At first generates a list of genes ranked in descending order of probability of functional association with the query gene. Next, a prediction score is automatically assigned to each function in this list based on the assumption that functions appearing most frequently at the top of the list are most likely to represent the function of the query gene. In this way, the second step provides an effective alternative to simply taking the ‘best hit’ from the first list, and achieves success rates of up to 79%. GO‐At is applicable across all three GO categories: molecular function, biological process and cellular component, and can assign functions at multiple levels of annotation detail. Furthermore, we demonstrate GO‐At’s ability to predict functions of uncharacterized genes by identifying ten putative golgins/Golgi‐associated proteins amongst 8219 genes of previously unknown cellular component and present independent evidence to support our predictions. A web‐based implementation of GO‐At ( http://www.bioinformatics.leeds.ac.uk/goat ) is available, providing a unique resource for plant researchers to make predictions for uncharacterized genes and predict novel functions in Arabidopsis.     

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile as cathepsin S inhibitors

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile analogues were identified as potent and selective cathepsin S inhibitor against both purified enzyme and in human JY cell based cellular assays. This core has a very stable thio-trapping nitrile war-head in comparison with the well reported pyrimidine-2-carbonitrile cysteine cathepsin inhibitors. Compound 47 is also very potent in in vivo mouse spleenic Lip10 accumulation assays.     

The development of benzimidazoles as selective rho kinase inhibitors

Rho Kinase (ROCK) is a serine/threonine kinase whose inhibition could prove beneficial in numerous therapeutic areas. We have developed a promising class of ATP-competitive inhibitors based upon a benzimidazole scaffold, which show excellent potency toward ROCK (IC₅₀ <10 nM). This report details the optimization of selectivity for ROCK over other related kinases such as Protein kinase A (PKA).