unc-94 encodes a tropomodulin in Caenorhabditis elegans

unc-94 is one of about 40 genes in Caenorhabditis elegans that, when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform a; sf20 has a stop codon in exon 5, which is shared by both isoform a and isoform b. The use of promoter-green fluorescent protein constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles and in spermatheca and intestinal epithelial cells. By Western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild-type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell-to-cell boundaries.     

The levels of melatonin and its metabolites in conditioned corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) and cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seeds during storage

The efficiency of pre-sowing conditioning/priming methods used to apply melatonin into seeds was verified: osmopriming in the case of dicot Cucumis sativus and hydropriming of monocot Zea mays seeds. Both priming techniques were selected experimentally as optimal for the studied plant species. Four different seed variants were compared: control non-treated ones, and seeds conditioned with water or with 50 or 500 μM melatonin water solutions. The HPLC–MS quantitative and qualitative analyses were used to determine the content of melatonin and of its potential metabolites in the seeds during 1 year following the conditioning. The control seeds and those conditioned with water contained small amount of endogenous melatonin in both species. However, the level of this indoleamine increased markedly in cucumber and corn seeds primed with exogenous melatonin and it was always correlated with the concentration of melatonin applied. It was noted that melatonin was metabolized during seed storage by its gradual oxidation, thus it protects dry seeds against oxidative stress, prevents potential injuries and significantly increases seeds quality. Interestingly, in the control and water-primed seeds, seasonal fluctuations of endogenous melatonin concentration were noted and significant increase in this indoleamine in the winter month was observed. This suggests that in seeds endogenous melatonin could play a crucial role in seasonal rhythms independently of environmental conditions.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Thermal and hydrodynamic modelling of active catheters for interventional radiology

Interventional radiologists desire to improve their operating tools such as catheters. Active catheters in which the tip is moved using shape memory alloy actuators activated using the Joule effect present a promising approach for easier navigation in the small vessels. However, the increase in temperature caused by this Joule effect must be controlled in order to prevent damage to blood cells and tissues. This paper is devoted to the simulation and experimental validation of a fluid-thermal model of an active catheter prototype. Comparisons between computer-predicted and experimentally measured temperatures are presented for both experiments in air and water at 37°C. Good agreement between the computational and experimental results is found, demonstrating the validity of the developed computer model. These comparisons enable us to highlight some important issues in the modelling process and to determine the optimal current for the activation of the catheter.     

Signal-transduction networks and the regulation of muscle protein degradation

Protein degradation in muscle functions in maintaining normal physiological homeostasis and adapting to new homeostatic states, and is required for muscle wasting or atrophy in various pathological states. The interplay between protein synthesis and degradation to maintain homeostasis is complex and responds to a variety of autocrine and intercellular signals from neuronal inputs, hormones, cytokines, growth factors and other regulatory molecules. The intracellular events that connect extracellular signals to the molecular control of protein degradation are incompletely understood, but likely involve interacting signal-transduction networks rather than isolated pathways. We review some examples of signal-transduction systems that regulate protein degradation, including effectors of proteolysis inducing factor (PIF), insulin and insulin-like growth factor (IGF) and their receptors, and fibroblast growth factor (FGF) and its receptors.     

Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV.     

The heterogeneity of ion channels in chromaffin granule membranes

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic ⁸⁶Rb⁺ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.     

Mitochondrial potassium channels: from pharmacology to function

Mitochondrial potassium channels, such as ATP-regulated or large conductance Ca2+ -activated and voltage gated channels were implicated in cytoprotective phenomenon in different tissues. Basic effects of these channels activity include changes in mitochondrial matrix volume, mitochondrial respiration and membrane potential, and generation of reactive oxygen species. In this paper, we describe the pharmacological properties of mitochondrial potassium channels and their modulation by channel inhibitors and potassium channel openers. We also discuss potential side effects of these substances.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

The use of <Emphasis Type="Italic">spa</Emphasis> and phage typing for characterization of clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in the University Clinical Center in Gdańsk,Poland

The emergence of spa types and spa–clonal complexes (CC) among clinical methicillin-resistant Staphylococcus aureus isolates collected from the University Clinical Center in Gdańsk between 2008 and 2009 were investigated. Phage typing was used as the initial screening in the study. The basic set of phages and the additional set of phages were used. Most of the isolates (56 %) belonged to the phage group III. With the additional set of phages, eight types were found, with predominant one MR8 (50 %). Sixteen distinct spa types were observed. The most frequent were t003 (22 %), t151 (16 %), and t008 (12 %). The spa types were clustered into two spa-CC and eight singletons. The predominant CC010 (50 %) consisted of six types, with the most common t003 (36.7 %) and t151(26.7 %), and in 80 % was identified as staphylococcal chromosomal casette mec (SCCmec) type II. The second cluster has no founder (12 %) with only two spa types: t037 belonging to SCCmec type III and t029. In the most frequent singleton, spa type t008 alone was clustered in 12 % of the isolates. All singletons correspond to SCCmec type IV. The CC010 was distributed in most of the hospital wards, corresponded to Multilocus sequence typing type ST5/ST225 and was constantly present throughout the observed period. The isolates of CC010 generally belonged to the phage group III, and most of them (53.3 %) were resistant to erythromycin, clindamycin, and ciprofloxacin. The concordance between spa-clone and phage type was very high, but the same phage type MR8 was observed within different spa types of the predominant clone.