Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

Founder effects and stochastic dispersal at the continental scale of the fungal pathogen of bananas Mycosphaerella fijiensis

The worldwide destructive epidemic of the fungus Mycosphaerella fijiensis on banana started recently, spreading from South-East Asia. The founder effects detected in the global population structure of M. fijiensis reflected rare migration events among continents through movements of infected plant material. The main objective of this work was to infer gene flow and dispersal processes of M. fijiensis at the continental scale from population structure analysis in recently invaded regions. Samples of isolates were collected from banana plantations in 13 countries in Latin America and the Caribbean and in Africa. The isolates were analysed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellite molecular markers. The results indicate that a high level of genetic diversity was maintained at the plantation and the plant scales. The loci were at gametic equilibrium in most of the samples analysed, supporting the hypothesis of the existence of random-mating populations of M. fijiensis, even at the plant scale. A low level of gene diversity was observed in some populations from the Africa and Latin America-Caribbean regions. Nearly half the populations analysed showed a significant deviation from mutation-drift equilibrium with gene diversity excess. Finally, a high level of genetic differentiation was detected between populations from Africa (FST = 0.19) and from the Latin America-Caribbean region (FST = 0.30). These results show that founder effects accompanied the recent invasion of M. fijiensis in both regions, suggesting stochastic spread of the disease at the continental scale. This spread might be caused by either the limited dispersal of ascospores or by movements of infected plant material.     

Environmental and intraperitoneal ethanol influences morphine antinociceptive effect in mice

The ability of acute environmental or intraperitoneal (i.p.) ethanol to influence morphine antinociceptive effect was studied in mice. In order to induce tolerance to morphine analgesia, mice received daily injections of 10 mg/Kg morphine over a period of 10 days. Mice were divided into three groups: i.p. ethanol (E), environmental ethanol (E*), and control saline (M). During the induction of tolerance these groups were treated identically except on days 1 and 11. On these days, 10 minutes prior to morphine injection, mice received either i.p. ethanol (1g/Kg), environmental ethanol (a bottle of 10% ethanol placed next to the animals cage during the experiments), or an equivalent volume of saline. Analgesia was assessed using a standard hot plate protocol and dose-response cumulative curves for morphine analgesia were obtained on days 1 and 11. On day 1, both the i.p. and environmental administration of ethanol showed similar morphine-potentiation effects [Mean Effective Dose: ED50 (M1)=4.5 mg/kg; ED50 (E1)=2.4 mg/kg; ED50 (E*1)=2.1 mg/kg]. On day 11, control group mice showed a reduction of morphine analgesia at test [ED50 (M11)=14.1 mg/kg]. Mice receiving i.p. and environmental ethanol again showed a leftward shift in dose-response cumulative curves for morphine antinociception with respect to controls [ED50 (E11)=9.1 mg/kg; ED50 (E*11)=4.7 mg/kg]. I.p. ethanol administration at non-antinociceptive doses enhances the morphine antinociception effect similarly in tolerant and non-tolerant (naive) mice. The presence of environmental ethanol can also induce a similar pattern of increase in morphine antinociception effect.     

Comparative genomic analysis reveals significant enrichment of mobile genetic elements and genes encoding surface structure-proteins in hospital-associated clonal complex 2 Enterococcus faecalis

Background  

Enterococci rank among the leading causes of nosocomial infections. The failure to identify pathogen-specific genes in Enterococcus faecalis has led to a hypothesis where the virulence of different strains may be linked to strain-specific genes, and where the combined endeavor of the different gene-sets result in the ability to cause infection. Population structure studies by multilocus sequence typing have defined distinct clonal complexes (CC) of E. faecalis enriched in hospitalized patients (CC2, CC9, CC28 and CC40).     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Dual neofunctionalization of a rapidly evolving aquaporin-1 paralog resulted in constrained and relaxed traits controlling channel function during meiosis resumption in teleosts

The preovulatory hydration of teleost oocytes is a unique process among vertebrates. The hydration mechanism is most pronounced in marine acanthomorph teleosts that spawn pelagic (floating) eggs; however, the molecular pathway for water influx remains poorly understood. Recently, we revealed that whole-genome duplication (WGD) resulted in teleosts harboring the largest repertoire of molecular water channels in the vertebrate lineage and that a duplicated aquaporin-1 paralog is implicated in the oocyte hydration process. However, the origin and function of the aquaporin-1 paralogs remain equivocal. By integrating the molecular phylogeny with synteny and structural analyses, we show here that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication rather than WGD and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut, a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. We demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. We further show that the rapid structural divergence of the C-terminal regulatory domain causes ex vivo loss of function of halibut Aqp1ab when expressed in amphibian oocytes but not in zebrafish or native oocytes. However, by using chimeric constructs of halibut Aqp1aa and -1ab and antisera specifically raised against the C-terminus of Aqp1ab, we found that this cytoplasmic domain regulates in vivo trafficking to the microvillar portion of the oocyte plasma membrane when intraoocytic osmotic pressure is at a maximum. Interestingly, by coinjecting polyA(+) mRNA from postvitellogenic halibut follicles, ex vivo intracellular trafficking of Aqp1ab is rescued in amphibian oocytes. These data reveal that the physiological role of Aqp1ab during meiosis resumption is conserved in teleosts, but the remarkable degeneracy of the cytoplasmic domain has resulted in alternative regulation of the trafficking mechanism.     

The risk for depression in patients with ankylosing spondylitis: a population-based cohort study

Introduction

Depression is frequent in ankylosing spondylitis (AS) patients. However, epidemiological data about the potential increase in risk are lacking. This study compares the rate of doctor-diagnosed depression in a well defined cohort of AS patients to the general population seeking care.

Methods

The Skåne Healthcare Register comprises healthcare data of each resident in Region Skåne, Sweden (population 1.2 million), including ICD-10 diagnoses. Using physician coded consultation data from years 1999 to 2011, we calculated depression consultation rates for all AS patients. We obtained standardized depression-rate ratios by dividing the observed depression rate in AS patients by the expected rate based on the corresponding age- and sex-specific rates of depression in the general population seeking care. A ratio >1 equals a higher rate of depression among AS patients.

Results

The AS cohort consisted of 1738 subjects (65% men) with a mean age of 54 years. The reference population consisted of 967,012 subjects. During the 13-year observation period 10% (n = 172) of the AS cohort had a doctor-diagnosed depression compared to 6% (n = 105) to be expected. The standardized estimate of depression-rate ratio was 1.81 (95% confidence interval 1.44 to 2.24) in women men and 1.49 (1.20 to 1.89) in men.

Conclusions

The rate of doctor-diagnosed depression is increased about 80% in female and 50% in male AS patients. Future challenges are to timely identify and treat the AS patients who suffer from depression.     

A novel liver specific isoform of the rat LAR transcript is expressed as a truncated isoform encoded from a 5'UTR located within intron 11

Background  

The leukocyte common antigen related receptor (LAR) protein has been shown to modulate the signal transduction of a number of different growth factors, including insulin and insulin-like growth factor 1. Splice variants exhibit differing roles and are expressed according to tissue type and developmental stage.