Analysis of glycated insulin by MALDI-TOF mass spectrometry

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.     

Activation of an alternative, rec12 (spo11)-independent pathway of fission yeast meiotic recombination in the absence of a DNA flap endonuclease

Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Delta mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Delta rec12Delta strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

IL-10 is up regulated in early and transitional stages in vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense

IL-10 has been suggested as a possible parameter for human African trypanosomiasis stage determination. However, conclusive experimental studies have not been carried out to evaluate this, which is a prerequisite before a potential test can be validated in humans for diagnostic purposes. We used the vervet monkey model of trypanosomiasis to scrutinize IL-10 in blood and cerebrospinal fluid (CSF). Five adult males were experimentally infected with T. b. rhodesiense. The infected animals became anemic and exhibited weight loss. Parasitemia was patent after 3 days and fluctuated around 3.7 × 10⁷ trypanosomes/ml throughout the experimental period. The total CSF white cell counts increased from pre-infection means around 3 cells/μl to a peak of 30 cells/μl, 42 days post-infection (DPI). IL-10 was not detectable (< 2 pg/ml) in serum prior to infection. IL-10 serum concentrations increased to 273 pg/ml 10 DPI coinciding with the first peak of parasitemia. Thereafter the levels declined to a mean value of 77 pg/ml 34 DPI followed by a significant rise to a second peak of 304 pg/ml (p < 0.008) 42 DPI. There was no detectable IL-10 in CSF. IL-10 synthesis is thus stimulated both in the early and transitional stages of experimental trypanosomiasis. That IL-10 is produced in early stage disease is an interesting finding unlikely to be detected in humans where it is difficult to determine the exact time of infection. The IL-10 peak observed on day 42 of infection might indicate onset of parasite neuroinvasion coinciding with a peak in white blood cell counts in the blood and CSF.     

CbgA, a protein involved in cortex formation and stress resistance in Myxococcus xanthus spores

CbgA plays a role in cortex formation and the acquisition of a subset of stress resistance properties in Myxococcus xanthus spores. The cbgA mutant produces spores with thin or no cortex layers, and these spores are more sensitive to heat and sodium dodecyl sulfate than their wild-type counterparts.     

Measurements of DNA-loop formation via Cre-mediated recombination

The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.     

Piperine suppresses cerebral ischemia–reperfusion-induced inflammation through the repression of COX-2, NOS-2, and NF-κB in middle cerebral artery occlusion rat model

The pathophysiological mechanisms leading to neuronal injury in middle cerebral artery occlusion (MCAO) model of cerebral stroke are complex and multifactorial that form the bases of behavioral deficits and inflammation mediated damage. The present study demonstrates the effect of piperine pretreatment (10 mg/kg b wt, once daily p.o. for 15 days) on cerebral ischemia-induced inflammation in male Wistar rats. The right middle cerebral artery was occluded for 2 h followed by reperfusion for 22 h. A maximum infarct volume (57.80 %) was observed in ischemic MCAO group. However, piperine administration prior to ischemia showed a significant reduction in infarct volume (28.29 %; p < 0.05) and neuronal loss (12.72 %; p < 0.01). As a result of piperine pretreatment, a significant improvement in behavioral outputs of MCAO rats (p < 0.05-0.01) was observed. Piperine successfully reduced the level of proinflammatory cytokines IL-1β, IL-6 and TNF-α, in ischemic group (p < 0.01). Ischemic group brain has shown edematous morphology with vacuolated architecture and pyknotic nuclei in H & E staining which was successfully ameliorated by piperine administration. Moreover, piperine also succeeded in lowering the expression of COX-2, NOS-2, and NF-κB (p < 0.01). Both cytosolic and nuclear NF-κB were down-regulated in ischemic group pre-administered with piperine (p < 0.01). The present study suggests that piperine is able to salvage the ischemic penumbral zone neurons by virtue of its anti-inflammatory property, thereby limiting ischemic cell death.     

Antibodies to influenza A virus in wild birds across Mongolia, 2006-2009

Wild waterbirds sampled July 2006-September 2009 in Mongolia were tested for antibodies to avian influenza (AI) virus with the use of a commercially available blocking enzyme-linked immunosorbent assay. Antibodies were detected in 25% (572/2,282) of tested birds representing 26 species, and all antibody-positive samples were from 12 species in the orders Anseriformes and Charadriiformes. The highest antibody prevalence was in Ruddy Shelducks (Tadorna ferruginea; 61.7%; n=261; 95% confidence interval [CI] 55.8-67.6%), Whooper Swans (Cygnus cygnus; 38.4%; n=242; 95% CI 32.3-44.5%), Swan Geese (Anser cygnoides; 15%; n=127; 95% CI 8.6-21.4%), Bar-headed Geese (Anser indicus; 13%; n=738; 95% CI 10.3-15.1%), and Mongolian Gulls (Larus mongolicus; 3.9%; n=255; 95% CI 1.3-6.5%). There was no significant temporal or spatial variation in the presence of antibodies in the sampled species. However, Bar-headed Geese and Mongolian Gulls showed spatial variation in antibody prevalence in 2007 and 2008, respectively. Our study provides insights into the hatch year waterbirds' exposure to AI virus at their natal and molting sites in Mongolia.