Developmental changes in gut microbiota and enzyme activity predict obesity risk in rats arising from reduced nests

The aim of the study was to assess the impact of preweaning overnutrition upon the ontogeny of intestinal microbiota, alkaline phosphatase activity (AP) and parameters of growth and obesity in male Sprague-Dawley rats. We tested whether intestinal characteristics acquired in suckling pups could programme the development of enhanced fat deposition during normalized nutrition beyond weaning. Postnatal nutrition was manipulated by adjusting the number of pups in the nest to 4 (small litters--SL) and 10 (normal litters--NL). In the postweaning period both groups were fed with a standard diet. The jejunal and colonic Lactobacillus/Enterococcus (LAB) and the Bacteroides/Prevotella (BAC) were determined using the FISH technique, and the jejunal AP activity was assayed histochemically. At 15 and 20 days of age the SL pups became heavier, displayed increased adiposity accompanied by significantly higher LAB and lower numbers of BAC and with higher AP activity in comparison with rats nursed in NL nests. These differences persisted to day 40 and withdrawal of the previous causal dietary influence did not prevent the post-weaning fat accretion. These results reveal the significance of early nutritional imprint upon the gut microbial/functional development and allow better understanding of their involvement in the control of obesity.     

Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC₅₀ <100 μM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K_i value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.     

Posttranslational modification of 6-phosphofructo-1-kinase as an important feature of cancer metabolism

Background

Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved.

Methodology/Principal Findings

In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux.

Conclusions/Significance

Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.     

Dynamics of responses in compatible potato-Potato virus Y interaction are modulated by salicylic acid

To investigate the dynamics of the potato-Potato virus Y (PVY) compatible interaction in relation to salicylic acid-controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVY(NTN), the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid-analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.     

Cholesterol conjugated oligonucleotide and LNA: A comparison of cellular and nuclear uptake by Hep2 cells enhanced by Streptolysin-O

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported.We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5′ end. All ONs were 3′ labeled with fluorescent Cy5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization.The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 μM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours. (Mol Cell Biochem 276: 61–69, 2005)