A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.     

Weight regain after sustained weight reduction is accompanied by suppressed oxidation of dietary fat and adipocyte hyperplasia

A dual-tracer approach (dietary 14C-palmitate and intraperitoneal 3H-H2O) was used to assess the trafficking of dietary fat and net retention of carbon in triglyceride depots during the first 24 h of weight regain. Obesity-prone male Wistar rats were allowed to mature under obesogenic conditions for 16 wk. One group was switched to ad libitum feeding of a low-fat diet for 10 wk (Obese group). The remaining rats were switched to an energy-restricted, low-fat diet for 10 wk that reduced body weight by 14% and were then assessed in energy balance (Reduced group), with free access to the low-fat diet (Relapse-Day1 group), or with a provision that induced a minor imbalance (+10 kcal) equivalent to that observed in obese rats (Gap-Matched group). Fat oxidation remained at a high, steady rate throughout the day in Obese rats, but was suppressed in Reduced, Gap-Matched, and Relapse-Day1 rats though 9, 18, and 24 h, respectively. The same caloric excess in Obese and Gap-Matched rats led to less fat oxidation over the day and greater trafficking of dietary fat to visceral depots in the latter. In addition to trafficking nutrients to storage, Relapse-Day1 rats had more small, presumably new, adipocytes at the end of 24 h. Dietary fat oxidation at 24 h was related to the phosphorylation of skeletal muscle acetyl-CoA carboxylase and fatty acid availability. These observations provide evidence of adaptations in the oxidation and trafficking of dietary fat that extend beyond the energy imbalance, which facilitate rapid, efficient regain during the relapse to obesity.     

Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum

A solid-phase extract from Protoceratium reticulatum was partitioned between water and butanol and the two fractions purified on an alumina column. Fractionation was monitored by ELISA and LC–MS. Results indicate that while almost all yessotoxin (1) was extracted into butanol, large amounts of yessotoxin analogs remained in the aqueous extract along with lesser amounts in the butanolic extract. NMR analysis of selected fractions from reverse-phase chromatography of the extracts confirmed the presence of yessotoxin analogs, although structure determinations were not possible due to the complexity of the mixtures. Analysis of fractions with LC–MS³ and neutral-loss LC–MS/MS indicated the presence of more than 90 yessotoxin analogs, although structures for most of these have not yet been determined. These analogs provide a mechanism to rationalise the discrepancy between ELISA and LC–MS analyses of algae and shellfish.     

KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin–RNA interacting proteins (CRIPs) conserved from fission yeast to man

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.     

Regulation of carnitine status in ruminants and efficacy of carnitine supplementation on performance and health aspects of ruminant livestock: a review

Carnitine has long been known to play a critical role for energy metabolism. Due to this, a large number of studies have been carried out to investigate the potential of supplemental carnitine in improving performance of livestock animals including ruminants, with however largely inconsistent results. An important issue that has to be considered when using carnitine as a feed additive is that the efficacy of supplemental carnitine is probably dependent on the animal’s carnitine status, which is affected by endogenous carnitine synthesis, carnitine uptake from the gastrointestinal tract and carnitine excretion. The present review aims to summarise the current knowledge of the regulation of carnitine status and carnitine homeostasis in ruminants, and comprehensively evaluate the efficacy of carnitine supplementation on performance and/or health in ruminant livestock by comparing the outcomes of studies with carnitine supplementation in dairy cattle, growing and finishing cattle and sheep. While most of the studies show that supplemental carnitine, even in ruminally unprotected form, is bioavailable in ruminants, its effect on either milk or growth performance is largely disappointing. However, supplemental carnitine appears to be a useful strategy to offer protection against ammonia toxicity caused by consumption of high levels of non-protein N or forages with high levels of soluble N both, in cattle and sheep.     

Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.     

Proteomic Characterization of Caenorhabditis elegans Larval Development

The nematode Caenorhabditis elegans is widely used as a model organism to study cell and developmental biology. Quantitative proteomics of C. elegans is still in its infancy and, so far, most studies have been performed on adult worm samples. Here, we used quantitative mass spectrometry to characterize protein level changes across the four larval developmental stages (L1–L4) of C. elegans. In total, we identified 4130 proteins, and quantified 1541 proteins that were present across all four stages in three biological replicates from independent experiments. Using hierarchical clustering and functional ontological analyses, we identified 21 clusters containing proteins with similar protein profiles across the four stages, and highlighted the most overrepresented biological functions in each of these protein clusters. In addition, we used the dataset to identify putative larval stage‐specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides system‐wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the C. elegans research community. MS data were deposited in ProteomeXchange ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the primary accession identifier PXD006676.