Human MSH2 (hMSH2) protein controls ATP processing by hMSH2-hMSH6

The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ～8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.

     

A NPxY-independent beta5 integrin activation signal regulates phagocytosis of apoptotic cells

Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the β chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (β5) integrin cDNA was expressed in αv positive, β5 and β3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, β5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing β5 mutant (Y750A) abrogated adhesion and β5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of β5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a β5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 μm diameter microspheres developed as apoptotic cell mimetics. The critical sequences in β5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of β5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the β5 cytoplasmic tail for adhesion and phagocytosis.     

Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology

The effect of microwave irradiation on the simultaneous extraction and transesterification (in situ transesterification) of dry algal biomass to biodiesel was investigated. A high degree of oil/lipid extraction from dry algal biomass and an efficient conversion of the oils/lipids to biodiesel were demonstrated in a set of well-designed experimental runs. A response surface methodology (RSM) was used to analyze the influence of the process variables (dry algae to methanol (wt/vol) ratio, catalyst concentration, and reaction time) on the fatty acid methyl ester conversion. Based on the experimental results and RSM analysis, the optimal conditions for this process were determined as: dry algae to methanol (wt/vol) ratio of around 1:12, catalyst concentration about 2 wt.%, and reaction time of 4 min. The algal biodiesel samples were analyzed with GC-MS and thin layer chromatography (TLC) methods. Transmission electron microscopy (TEM) images of the algal biomass samples before and after the extraction/transesterification reaction are also presented.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.     

Demographic and clinical variables affecting mid‐ to late‐life trajectories of plasma ceramide and dihydroceramide species

It has been increasingly recognized at the basic science level that perturbations in ceramide metabolism are associated with the development and progression of many age‐related diseases. However, the translation of this work to the clinic has lagged behind. Understanding the factors longitudinally associated with plasma ceramides and dihydroceramides (DHCer) at the population level and how these lipid levels change with age, and by sex, is important for the clinical development of future therapeutics and biomarkers focused on ceramide metabolism. We, therefore, examined factors cross‐sectionally and longitudinally associated with plasma concentrations of ceramides and DHCer among Baltimore Longitudinal Study of Aging participants (n = 992; 3960 total samples), aged 55 years and older, with plasma at a mean of 4.1 visits (range 2–6). Quantitative analyses were performed on a high‐performance liquid chromatography‐coupled electrospray ionization tandem mass spectrometer. Linear mixed models were used to assess the relationships between plasma ceramide and DHCer species and demographics, diseases, medications, and lifestyle factors. Women had higher plasma concentrations of most ceramide and DHCer species and showed steeper trajectories of age‐related increases compared to men. Ceramides and DHCer were more associated with waist–hip ratio than body mass index. Plasma cholesterol and triglycerides, prediabetes, and diabetes were associated with ceramides and DHCer, but the relationship showed specificity to the acyl chain length and saturation. These results demonstrate the importance of examining the individual species of ceramides and DHCer, and of establishing whether intra‐individual age‐ and sex‐specific changes occur in synchrony to disease onset and progression.     

Pharmacokinetic study of 11-Keto beta-Boswellic acid.

INTRODUCTION: Boswellia serrata has been used in traditional medicine for treatment of inflammatory diseases since antiquity. However human kinetic studies are lacking for this. Hence to better elucidate its effects in humans and determine its optimal dosing, this study was planned. MATERIAL AND METHODS: Twelve healthy adult men volunteers were given capsule Wok Vel containing 333 mg of Boswellia Serrata Extract, orally, after a seven days washout period. Venous blood samples were drawn through indwelling canula from each volunteer prior to drug administration and at 30, 60, 120, 150, 180, 210, 240, 300, 360, 480, 600, 720, 840 minutes after drug administration. Plasma obtained after centrifuge was analyzed to measure concentration of 11-Keto beta-Boswellic Acid (KBA) by HPLC. Various kinetic parameters were then calculated from the plasma concentrations. RESULTS: The results are expressed as mean +/- Standard Error of Mean. The peak plasma levels (2.72 x 10(-3) +/- 0.18 micromoles/ml) of BSE were reached at 4.5 +/- 0.55 h. The concentration declined with a mean elimination half life of 5.97 +/- 0.95 h. The apparent volume of distribution averaged 142.87 +/- 22.78 L and the plasma clearance was 296.10 +/- 24.09 ml/min. The AUC(0-infinity) was 27.33 x 10(-3) +/- 1.99 micromoles/ml h. CONCLUSION: Elimination half life of nearly six hours suggests that the drug needs to be given orally at the interval of six hours. The plasma concentration will attain the steady state after approximately 30 hours. BSE is a safe drug and well tolerated on oral administration. No adverse effects were seen with this drug when administered as single dose in 333 mg.