MutS�� and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington’s disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSβ (MSH2–MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (∼30–40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSβ subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSβ, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSβ in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSβ, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.     

Production of ψ-linolenic acid by the fungus Mucor rouxii on cheap nitrogen and carbon sources

Summary The production of -linolenic acid (GLA) by the fungus Mucor rouxii CBS 416.77 was studied on low budget nitrogen and carbon sources, i.e. rape meal, cocos expeller and two types of yeast extract (nitrogen sources), and starch, starch hydrolysate, beet molasses and cocos expeller (carbon sources). As references, Difco yeast extract and glucose were used. In flask cultivations the three yeast extracts were fully interchangeable, while the Difco yeast extract (the most expensive of those tested) gave a higher productivity of GLA in fermentor cultures (14 mg·l^–1·h^–1). The yield of lipids and GLA were increased in the order yeast extract < rape meal < cocos expeller. Thus the amount of lipid increased from 0.56 to 2.8 g·l^–1, and that of GLA from 0.15 to 0.33 g·l^–1. Use of beet molasses or cocos expeller as carbon sources gave poor growth. Starch and starch hydrolysate resulted in better productivity of GLA than glucose (4.7 and 4.9 compared to 3.4 mg·l^–1·h^–1). Offsprint requests to: A.-M. Lindberg     

A scalable life cycle inventory of an electrical automotive traction machine—Part I: design and composition

Purpose

A scalable life cycle inventory (LCI) model of a permanent magnet electrical machine, containing both design and production data, has been established. The purpose is to contribute with new and easy-to-use data for LCA of electric vehicles by providing a scalable mass estimation and manufacturing inventory for a typical electrical automotive traction machine. The aim of this article (part I of two publications) is to present the machine design, the model structure, and an evaluation of the models’ mass estimations.

Methods

Data for design and production of electrical machines has been compiled from books, scientific papers, benchmarking literature, expert interviews, various specifications, factory records, and a factory site visit. For the design part, one small and one large reference machine were constructed in a software tool, which linked the machines’ maximum ability to deliver torque to the mass of its electromagnetically active parts. Additional data for remaining parts was then gathered separately to make the design complete. The two datasets were combined into one model, which calculates the mass of all motor subparts from an input of maximum power and torque. The range of the model is 20–200 kW and 48–477 Nm. The validity of the model was evaluated through comparison with seven permanent magnet electrical traction machines from established brands.

Results and discussion

The LCI model was successfully implemented to calculate the mass content of 20 different materials in the motor. The models’ mass estimations deviate up to 21% from the examples of real motors, which still falls within expectations for a good result, considering a noticeable variability in design, even for the same machine type and similar requirements. The model results form a rough and reasonable median in comparison to the pattern created by all data points. Also, the reference motors were assessed for performance, showing that the electromagnetic efficiency reaches 96–97%.

Conclusions

The LCI model relies on thorough design data collection and fundamental electromagnetic theory. The selected design has a high efficiency, and the motor is suitable for electric propulsion of vehicles. Furthermore, the LCI model generates representative mass estimations when compared with recently published data for electrical traction machines. Hence, for permanent magnet-type machines, the LCI model may be used as a generic component estimation for LCA of electric vehicles, when specific data is lacking.

     

Optimization of selenium status by a single intraperitoneal injection of Se in Se-deficient rat: possible application to burned patient treatment

In order to investigate the efficiency of a single selenium (Se) administration in restoring selenium status, Se and antioxidant enzymes were studied in an animal model of Se depletion. In Se-depleted animals receiving or not a single parenteral administration of Se, plasma, red blood cell (RBC), and tissue Se levels were measured concurrently with glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities. The oxidative stress was assessed by thiobarbituric acid-reactive species (TBARs), total thiol groups, glutathione, and tocopherol measurements. Our study showed that Se depletion with alterations in the antioxidant defense system (Se and GPx activity decreases) led to an increase of lipid peroxidation, a decrease of the plasma vitamin E level, and SOD activation. Sodium selenite injection resulted after 24 h in an optimal plasma Se level and a reactivation of GPx activity. In liver, brain, and kidney, Se levels in injected animals were higher than those in reference animals. However, this single administration of Se failed to decrease free radical damage induced by Se depletion. Therefore, in burned patients who exhibit an altered Se status despite a daily usually restricted Se supplementation, the early administration of a consistent Se amount to improve the GPx activity should be of great interest in preventing the impairment of the antioxidant status.     

Prominin-1/CD133, a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer

Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (hE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that hE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowmans capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, hE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SPP 1109, CO 298/2-1 to D.C., Hu 275/7-1 to W.B.H.; SPP 1111, Hu 275/8-2 to W.B.H.) and the Fonds der Chemischen Industrie (to W.B.H.)     

Sophorose lipid production from lipidic precursors: Predictive evaluation of industrial substrates

Summary Sophorose lipids stand out as biosurfactants with a wide potential for industrial application and which can be produced in good yield from glucose and a lipidic cosubstrate.Candida bombicola CBS 6009 (ATCC 22214) was used in the present study. The influence of the lipidic cosubstrate on various aspects of production performance of these glycolipids (final concentration, yield) and on product composition (in particular, the structure of the hydroxy fatty acid vegetable and animal oils, markedly influenced product composition. In terms of production performance, the best substrates were oils or esters rich in C18:0 and C18:1 fatty acids. Optimal overall performance was obtained with esters (340 g L^–1 sophorose lipids with rapeseed esters). Conclusions drawn from the results allow predictive evaluation of lipidic industrial substrates.     

Nucleotide Variability and Translation Efficiency of the 5′ Untranslated Region of Hepatitis A Virus: Update from Clinical Isolates Associated with Mild and Severe Hepatitis

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% ± 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.Hepatitis A virus (HAV) is a nonenveloped RNA virus of the Picornaviridae family. The viral genome consists of an approximately 7,500-nucleotide (nt)-long, positive-stranded RNA divided in three parts: a 5′ untranslated region (5′ UTR), a single open reading frame that encodes both structural and nonstructural proteins, and a 3′ UTR with a short poly(A) tail. By sequencing of the VP1-2A junction and the VP1 gene, 3 genotypes (I, II, and III) divided into A and B subtypes have been described in humans (7, 27). HAV is the main cause of acute viral hepatitis worldwide. The majority of cases follow a benign course, but some may be present with fulminant forms, characterized by acute liver failure (factor V levels of <50% and encephalopathy). HAV-induced liver disease appears to result primarily from immunologic mechanisms, chiefly on the basis of in vitro studies. Most HAV strains have no detectable cytopathic effect in cell culture and no apparent effect on cell growth or metabolism (16), and HAV-infected cells are lysed by cytotoxic T cells isolated from the liver of acutely infected patients (30, 31). Clinical studies have suggested that host factors such as age and underlying liver disease were involved in the severity of liver diseases (32, 33) and that the host immune response also played a role in the fulminant forms of hepatitis A, as evidenced by markedly low viral loads (26).Nevertheless, the existence of viral determinants of hepatitis A severity is suggested by both experimental and clinical studies. Indeed, mutations within the VP1-2A and 2C genes have been shown to enhance virulence in tamarins (9). It has also been suggested that 5′ UTR mutations associated with viral adaptation to cell culture were also responsible for viral attenuation in vivo (15). The 5′ UTR of HAV is about 735 nucleotides long and is considered the most conserved region of the genome. The 5′ UTR is involved in genome replication and translation initiation. Folding predictions and biochemical probing showed that this region forms a highly ordered secondary structure containing a pyrimidine-rich tract (PRT) and an internal ribosomal entry site (IRES) with 10 to 12 AUG triplets upstream of the initiator codon (18). The IRES allows the initiation of the cap-independent translation of the viral genome. Most knowledge of HAV IRES activity is derived from studies of the HM-175 reference strain and its cell culture-adapted variants (4, 5, 36). These experiments have shown that HAV presents the lowest IRES-dependent translation initiation activity among picornaviruses both in reticulocyte lysates and in a variety of cell lines, including the human hepatoma cell line HepG2 (type III IRES) (3, 6). These features have been attributed to a lower affinity of the HAV 5′ UTR for translation factors (6). The hypothesis that the slow growth of HAV in cell culture could be related to this inefficient translation is supported by the emergence of 5′ UTR mutations in cell culture-adapted variants with enhanced viral replication (8). The finding that these mutations were associated with viral attenuation in vivo supports the hypothesis of viral determinants of virulence in the 5′ UTR (15). Among the few clinical studies which have addressed this question, Fujiwara et al., by comparing full-length HAV genomes obtained from Japanese patients with benign or fulminant hepatitis, found less nucleotide variation in the 5′ UTRs from patients with fulminant hepatitis (12, 13) and suggested that two IRES mutations (G324A and C372G/T) might influence the course of HAV infection (14).The aim of the present study was to further examine the genetic variability of 5′ UTR sequences from field isolates, to assess the potential impact of nucleotide variations on IRES activity by using validated techniques, and to search for a relationship with disease severity by comparing isolates obtained from patients with benign or fulminant forms of hepatitis A.     

The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.     

Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis.

In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns). In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol. Our data clearly show that the CDP-diacylglycerol-independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously. Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 microm CMP. Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis. All of the PtdIns metabolizing activities require free manganese ions. EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.