Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

Improved pregnancy and birth rates with routine application of nonsurgical embryo transfer

Nonsurgical embryo transfer (NSET) of blastocysts to pseudopregnant female recipients provides many benefits over surgical implantation with less distress for the mice, no anesthesia or analgesia required and a considerable reduction in implantation time per mouse. Although a disposable device to perform NSET is on the market since 2009, it is not generally used in transgenic facilities, most likely because surgical implantation is efficient and inexpensive. Here, we report that with several refinements to the original protocol, the NSET method becomes very attractive and outperforms the traditional surgical transfer on basis of pregnancy rate, birth rate and implantation-related discomfort. Furthermore, repeated use of the same NSET device on several recipient females reduces the costs to a reasonable level. The data presented covers all embryo transfers over the last 5 years at the transgenic facility of the Netherlands Cancer Institute, of which the last 2 years were performed exclusively with NSET.     

The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-like motif

The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. Here we have identified the serine protease Omi/HtrA2 as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis.     

A Regional Initiative to Reduce Skin Infections amongst Aboriginal Children Living in Remote Communities of the Northern Territory,Australia

Background

Linked to extreme rates of chronic heart and kidney disease, pyoderma is endemic amongst Aboriginal children in Australia''s Northern Territory (NT). Many of those with pyoderma will also have scabies. We report the results of a community-based collaboration within the East Arnhem Region, which aimed to reduce the prevalence of both skin infections in Aboriginal children.

Methodology/Principal Findings

Commencing September 2004, we conducted an ecological study that included active surveillance for skin infections amongst children aged <15 years in five remote East Arnhem communities over a three year period. Screening was undertaken by trained local community workers, usually accompanied by another project team member, using a standard data collection form. Skin infections were diagnosed clinically with the aid of a pictorial flip chart developed for the purpose. Topical 5% permethrin was provided for age-eligible children and all household contacts whenever scabies was diagnosed, whilst those with pyoderma were referred to the clinic for treatment in accordance with current guidelines. In addition, annual mass scabies treatment (5% permethrin cream) was offered to all community residents in accordance with current guidelines but was not directly observed. Pyoderma and scabies prevalence per month was determined from 6038 skin assessments conducted on 2329 children. Pyoderma prevalence dropped from 46.7% at baseline to a median of 32.4% (IQR 28.9%–41.0%) during the follow-up period – an absolute reduction of 14.7% (IQR 4.7%–16.8%). Compared to the first 18 months of observation, there was an absolute reduction in pyoderma prevalence of 18 cases per 100 children (95%CI −21.0, −16.1, p≤0.001) over the last 18 months. Treatment uptake increased over the same period (absolute difference 13.4%, 95%CI 3.3, 23.6). While scabies prevalence was unchanged, the prevalence of infected scabies (that is with superimposed pyoderma) decreased from 3.7% (95%CI 2.4, 4.9) to 1.5% (95%CI 0.7, 2.2), a relative reduction of 59%.

Conclusion

Although pyoderma prevalence remained unacceptably high, there was a substantial reduction overall with improvements in treatment uptake a critical factor. More acceptable alternatives, such as cotrimoxazole for pyoderma and ivermectin as a community-wide scabicide, warrant further investigation in these settings. We are encouraged by progress made through this work, where local action was led by local community members and primary health care providers with external training and support.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00884728","term_id":"NCT00884728"}}NCT00884728     

Cholesterol and Clioquinol modulation of A beta(1-42) interaction with phospholipid bilayers and metals

The beta-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-beta peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, A beta(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state 31P and 2H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in beta-sheet, were observed using specifically 13C-labelled A beta(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely 15N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric beta-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

Increased gene expression of plasminogen activators and inhibitors in left ventricular hypertrophy

In the early stages of left ventricular hypertrophy (LVH) acute adaptive changes occur in the coronary vasculature as it remodels. Plasminogen activators (PAs) and inhibitors (PAIs) have the potential effects of proteolytic degradation that is relevant to tissue remodeling and angiogenesis. Our study focused on the possible roles of PAI-1, PAI-2, uPA and tPA in myocyte hypertrophy and angiogenesis in the early and late stages of pressure overload induced left ventricular hypertrophy (LVH). We divided seventeen adult swine, weighing 24.2 ± 6.5 kg, into four groups: control, sham-operated, early LVH and late heart failure LVH group. At surgery we placed a fixed constrictor on the ascending aorta immediately above the aortic valve. This increased LV systolic pressure from 133 ± 15 to 193 ± 24 mm Hg after the surgery. We subdivided the early group into groups of 3 animals each that we euthanized at 8, 24 and 72 h after operation and obtained heart samples for analysis. In the late heart failure group individual animals were euthanized at 55, 59, 62 and 72 days after the detection of congestive heart failure. We also obtained tissue samples from the control and sham-operated swine. Sections for histologic analysis were fixed in 10% buffered formalin. We isolated RNA, size fractionated it using 1% formaldehyde-agarose gel electrophoresis and then did Northern blots. The mRNAs from both PAI-1 and PAI-2 showed a remarkable increase at 8 and 24 h after acute aortic constriction and returned to control by 72 h. Regional differences showed that most of the increases were in the endocardium. Three animals in the late heart failure LVH group were determined to be in congestive heart failure at about 2 months after the onset of aortic constriction. In these animals PAI-1 and PAI-2 were increased in both the left and right ventricles but remained low in an animal of the same elevation in aortic pressure seen by the LV who did not have congestive failure. These data suggest that PA and PAI gene expressions change before morphologic changes occur in the early stages of developing LVH. Also at the time of onset of congestive heart failure this increased expression reappears. PAs and PA inhibitors mRNA levels vary in the different regions of the heart reflecting changing wall stresses. Thus, the PAs and PA inhibitors may play an important role in angiogenesis that occurs during the early stages of LVH. The increased expression in the late stage of LVH may reflect further changes in wall stresses since these animals also showed overt clinical signs of heart failure.     

Genetic model of selective COX2 inhibition reveals novel heterodimer signaling

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.     

Life history of the silvertip shark Carcharhinus albimarginatus from Papua New Guinea

Coral Reefs - Growth and maturity of the silvertip shark Carcharhinus albimarginatus from Papua New Guinea were estimated to form the basis of future population assessments. Samples were collected...