Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement

A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.     

Mapping of major and modifying genes for high oleic acid content in safflower

Oils with high oleic acid content are in great demand because they have optimal properties for food and non-food uses. Two different levels of high oleic acid content (>75 and >84%) have been reported in safflower (Carthamus tinctorius L.). The trait is mainly controlled by partially recessive alleles at a major gene Ol, but the highest levels have been attributed to modifying genes. The objectives of this research were to map the Ol locus and modifying genes involved in oleic acid content of safflower seeds and to determine the nature of Ol through a candidate gene approach. Two F2 mapping populations from the nuclear male-sterile line CL-1 and the high oleic acid lines CR-6 (>75% oleic acid) and CR-9 (>84%) were developed and phenotyped for oleic acid content at the F2 and F3 seed level. A genetic linkage map comprising 15 linkage groups and 116 random amplified polymorphic DNA, simple sequence repeat (SSR), and sequence-characterized amplified regions marker loci was constructed for the CL-1?×?CR-9 population. The Ol gene was mapped to linkage group (LG) T3 tightly linked to the SSR marker ct365, which was confirmed in the CL-1?×?CR-6 population. Additionally, a quantitative trait locus with a minor effect on increasing oleic acid content was identified on LG T2. The candidate gene approach indicated that an oleoyl-phosphatidylcholine desaturase FAD2-1 locus underlies the Ol gene. Both the genetic information and the markers developed in this research will contribute to marker-assisted selection for high oleic acid content in safflower.     

Biochemical screening of Lactobacilli in midgut of wild Aedes aegypti mosquitoes. A step toward paratransgenesis candidate

This study was conducted to isolate and identify lactobacilli from larval and adult midgut of wild Aedes aegypti (Ae. aegypti) to find candidate bacteria for paratransgenic control. Characterization of the bacterial symbionts was done using Gram staining, motility test, catalase test, and biochemical tests, among others, and the morphological features were confirmed using a standard scheme that simplifies the identification of lactic acid bacteria. A total of 174 strains were isolated and identified, 135 strains from larval midgut and 39 strains from adult midgut (mean ± SE, 4.00 ± 0.72; P = 0.00). The isolated species were confirmed to be Lactobacillus fermentum, L. casei, L. acidophilus, L. viridescens, L. brevis and L. gasseri. It can be concluded that Ae. aegypti has the potential of harboring the cultivable bacterial symbionts. In conclusion, the isolated species were nominated for paratransgenic control, particularly L. fermentum, being found in large numbers from both larval and adulxt midgut.     

Involvement of neuroleptic drugs in selenium deficiency and sudden death of cardiac origin: study and human post-mortem examination

The involvement of psychotropic drugs in sudden deaths has been highlighted. The objective of this work was to establish a link between selenium levels in heart tissue, psychotropic treatment and sudden death. Selenium levels were measured by electrothermal atomic absorption spectroscopy post-mortem in heart, brain and liver. Histological examination evidenced dilated cardiomyopathy in 45% of cases, left ventricular hypertrophy in 36%, and ischemic coronaropathy in 18%. A significant reduction of myocardial selenium levels compared to controls was seen in patients treated with neuroleptic drugs or meprobamate. No changes in brain or liver selenium levels were seen. These results suggest that selenium deficiency can facilitate sudden death in patients on psychotropic drugs. The reduced activity of glutathione peroxidase due to selenium deficiency can result in augmented oxidative stress in myocardial cells and myocardiopathy leading to sudden death.     

Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils

Background

Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.

Objective

In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.

Methods

Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.

Results

The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.

Conclusions

Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.     

Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.     

Cyclin-Dependent Kinase Five Mediates Activation of Lung Xanthine Oxidoreductase in Response to Hypoxia

Background

Xanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown.

Methods and Results

Using an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif.

Conclusions and Significance

These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury.     

Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS

Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.     

Anti-obesity potential of almond (Prunus dulcis) in experimental animals under cafeteria and atherogenic diets

Background & objectivesNatural dietary supplements are progressively getting famous to supplant synthetic substances particularly in chronic morbidities. The aim of this study was to evaluate the anti-obesity potential of almond on the normal, Cafeteria, and Atherogenic diets.Materials and methodsParameters such as change in body weight, body temperature, lipid profile, organ weights, and fat pad weights were assessed. Central Nervous System related studies (Despair Swim test and Elevated Plus maze test) were also performed to comprehend the effect of the diets, and almond on the brain. All of the experimental animals were randomly assigned to one of three diet categoriesregular, cafeteria, or atherogenic, and fed those diets for 40 days. Each diet had the control group, standard drug group and three almond groups (low dose: 50; medium dose: 100 and high dose: 200 mg/kg body weight). Body weight was recorded every alternate day. On 40th day, body temperature was measured. On day 41, lipid parameters, organ weights, fat pad weights and the CNS parameters were evaluated. ANOVA followed by Duncans Multiple Range Test were used for statistical analysis.ResultsTreatment of animals with either a low or high dose of almond as well as a standard herb prevented a rise in body weight significantly (p = 0.01) in all three diet groups. When a regular diet was replaced with a cafeteria and atherogenic diet, the serum levels of triglycerides and LDL increased significantly, while HDL levels decreased significantly. Overall, almond preparation reduced lipid parameters, organ weights, fat-pad weights, and stabilized CNS parameters substantially.Interpretation & conclusionThe almond high dose was the most effective of all the almond preparations. Our study suggests that chronic administration of almond independently reduces the body weight in experimental animals.