Non-invasive analysis of gallbladder bile composition in cynomolgus monkeys using in vivo 1H magnetic resonance spectroscopy

The purpose of the present study was (i) to establish a modality for non-invasively probing bile composition in cynomolgus monkeys and (ii) to ascertain the variability in biliary metabolism by repeatedly assessing gallbladder bile in situ. Localised in vivo (1)H magnetic resonance spectroscopy (MRS) provided high-resolution spectra of gallbladder bile that allowed for the first time different species of bile acids, their taurine and glycine conjugates, and phospholipids to be identified and quantified in situ. A combined cross-sectional and longitudinal study of bile composition was conducted over 4 weeks in monkeys kept under standardised nutritional conditions. All biles were composed of the same major constituents. Bile acids contributed 267+/-47 micromol/ml whereof cholate, deoxycholate and chenodeoxycholate were the most abundant primary bile acids. Bile acid conjugation reached an extent of 100%. However, the actual quantitative contributions of different bile constituents varied distinctly. Correlation analysis revealed that intra-individual variability (r=0.77+/-0.03) was significantly (p<0.01) smaller than inter-individual variability (r=0.68+/-0.01), thus purporting the notion that bile composition is a hallmark of individual metabolism. Extension of quantitative bile analysis by in vivo (1)H-MRS to pathological states will provide a rapid and non-invasive modality for monitoring an important, yet elusive compartment of cholesterol and lipid metabolism.     

Microcytosis in ank/ank mice and the role of ANKH in promoting erythroid differentiation

Progressive ankylosis (Ank and the human homolog, ANKH) is a transmembrane protein which regulates transport of inorganic pyrophosphate (PPi). ank/ank mice with a mutated ank gene, have calcification and bone ankylosis of the affected joints. In the course of studying these mutant mice, we found that they have microcytosis. These mutant mice have lower mean red blood cell volume (MCV) and lower hemoglobin content in red cells (mean corpuscular hemoglobin, MCH) than normal mice. Using quantitative real-time PCR analysis, we showed that Ank was expressed in the E/Meg bipotent precursor, BFU-E, CFU-E, but there was no Ank expression in the hemoglobinizing erythroblasts. Stable ANKH transfectants in K562 cells highly expressed two immature erythroid cell markers, E-cadherin and endoglin. Enhanced Erythropoietin (Epo) expression and downregulation of SHP-1 were detected in these transfectants. Consequently, the autocrine Epo-EpoR signaling pathway was activated, as evidenced by higher p-Tyr JAK2, p-Tyr EpoR and p-Tyr STAT5B in the ANKH transfectants. Our results revealed a novel function of ANKH in the promotion of early erythroid differentiation in K562 cells. We also showed that ank/ank mice have lower serum levels of Epo than the normal littermates, and this is the likely cause of microcytosis in these mutant mice.     

Cortical actin filament organization in developing and functioning stomatal complexes of Zea mays and Triticum turgidum

Cortical actin filament (AF) organization was studied in detail in developing stomatal complexes of the grasses Zea mays and Triticum turgidum. AF arrays during the whole stomatal complex development are dynamic, partly following the pattern of cortical microtubule (MT) organization. They also exhibit particular patterns of organization, spatially and temporarily restricted. Among AF arrays, the radial ones that underlie young guard cell (GC) periclinal walls, those that line the bulbous GC ends and the AF ring at the junction between subsidiary cells (SCs) and GCs are described here for the first time. Although many similarities in cortical AF organization exist among the stomatal cells of both plants studied, considerable differences have also been observed between them. Our data reveal that the expanding areas of stomatal cell walls are lined by distinct cortical AF aggregations that probably protect the plasmalemma against mechanical stresses. Experimental AF disruption does not seem to affect detectably stomatal cell morphogenesis. Moreover, the structural and experimental data of this study revealed that, in contrast to the elliptical stomata, in the dumbbell-shaped ones the AFs and MTs seem not to be involved in the mechanism of opening and closing of the stomatal pore.     

Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation

We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.     

Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.     

Cephalopods in neuroscience: regulations,research and the 3Rs

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of “live cephalopods” became regulated within the European Union by Directive 2010/63/EU on the “Protection of Animals used for Scientific Purposes”, giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce “guidelines” and the potential contribution of neuroscience research to cephalopod welfare.     

Genome Sequence and Analysis of Buzura suppressaria Nucleopolyhedrovirus: A Group II Alphabaculovirus

The genome of Buzura suppressaria nucleopolyhedrovirus (BusuNPV) was sequenced by 454 pyrosequencing technology. The size of the genome is 120,420 bp with 36.8% G+C content. It contains 127 hypothetical open reading frames (ORFs) covering 90.7% of the genome and includes the 37 conserved baculovirus core genes, 84 genes found in other baculoviruses, and 6 unique ORFs. No typical baculoviral homologous repeats (hrs) were present but the genome contained a region of repeated sequences. Gene Parity Plots revealed a 28.8 kb region conserved among the alpha- and beta-baculoviruses. Overall comparisons of BusuNPV to other baculoviruses point to a distinct species in group II Alphabaculovirus.     

Histamine modulates the cellular stress response in yeast

The cellular stress response is a universal protective reaction to adverse environmental or microenvironmental conditions, such as heat and drugs, associated in part with the highly conserved heat shock proteins (HSPs). Histamine is a key inflammatory mediator derived from l-histidine that governs vital cellular processes beyond inflammation, while recent evidence implies additional actions in both prokaryotes and eukaryotes. This study explored the possible role of histamine in the heat shock response in yeast, an established experimental model for the pharmacological investigation of the cellular stress response. The response was evaluated by determining growth and viability of post-logarithmic phase grown yeast cultures after heat shock at 53°C for 30 min. Thermal preconditioning at 37°C for 2 h served as a positive control. The effect of histamine was investigated following long-term administration through the post-logarithmic phase of growth or short-term administration for 2 h prior to heat shock. Short-term treatment with 1 mM histamine resulted in de novo protein synthesis-dependent acquisition of thermotolerance, while lower doses or long-term administration of histamine failed to induce the heat-resistant phenotype. Preliminary investigation of HSP104, HSP70 and HSP60 expression by western blotting showed an increase of these proteins after thermal preconditioning. However, a differential HSP and tubulin expression appeared to underlie the response of yeast cells to histamine. In conclusion, histamine was capable of inducing the adaptive phenotype, while the contribution of HSPs and tubulin and the potential implications remain largely elusive.