A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Sleeping under the Ocean: Despite Total Isolation,Nuclear Submariners Maintain Their Sleep and Wake Patterns throughout Their Under Sea Mission

BackgroundTo assess the effects of isolation, inadequate exposure to light and specific shift work on the subjective and objective measurements of sleep and alertness of submariners.PurposeA strictly controlled randomized crossover study with the polysomnography recorded twice during the mission.MethodsSetting: Shift and night work with prolonged (70 days) social isolation from the real world (with no phone or Internet contact with families or friends during a routine mission aboard the “Téméraire” French Strategic Submarine with Ballistic Nuclear missiles (SSBN). Participants: 19 submariners working on a 24-hour shift for three days in a row schedule. Interventions: The participants attended two polysomnographic (PSG) recordings of night sleep on Day 21 (D21) and Day 51 (D51) of the 70-day patrol; urine cortisol levels were also taken after sleep, and subjective assessments of sleep, sleepiness, mood and anxiety on D21 and D51. The light and temperature on board were also recorded.ResultsPSG analyses showed that sleep did not significantly vary in length (total sleep time) or in quality between D21 and D51. The mariners reported the same subjective sleep, sleepiness, anxiety or mood (except for a slightly worse score for confusion on D51). Blood cortisol levels did not vary significantly.ConclusionsThese results show that humans living in an isolated environment for more than two months with this specific shift schedule do not suffer from any significant effects on sleep, sleepiness and confusion between D21 and D51, when they follow an organized regular shift pattern with controlled light and temperature.     

Enhanced pelvic responses to stressors in female CRF-overexpressing mice

Acute stress affects gut functions through the activation of corticotropin-releasing factor (CRF) receptors. The impact of acute stress on pelvic viscera in the context of chronic stress is not well characterized. We investigated the colonic, urinary, and locomotor responses monitored as fecal pellet output (FPO), urine voiding, and ambulatory activity, respectively, in female and male CRF-overexpressing (CRF-OE) mice, a chronic stress model, and their wild-type littermates (WTL). Female CRF-OE mice, compared with WTL, had enhanced FPO to 2-min handling (150%) and 60-min novel environment (155%) but displayed a similar response to a 60-min partial restraint stress. Female CRF-OE mice, compared with WTL, also had a significantly increased number of urine spots (7.3 +/- 1.4 vs. 1.3 +/- 0.8 spots/h) and lower locomotor activity (246.8 +/- 47.8 vs. 388.2 +/- 31.9 entries/h) to a novel environment. Male CRF-OE mice and WTL both responded to a novel environment but failed to show differences between them in colonic and locomotor responses. Male WTL, compared with female WTL, had higher FPO (113%). In female CRF-OE mice, the CRF(1)/CRF(2) receptor antagonist astressin B and the selective CRF(2) receptor agonist mouse urocortin 2 (injected peripherally) prevented the enhanced defecation without affecting urine or locomotor responses to novel environment. RT-PCR showed that CRF(1) and CRF(2) receptors are expressed in the mouse colonic tissues. The data show that chronic stress, due to continuous central CRF overdrive, renders female CRF-OE mice to have enhanced pelvic and altered behavioral responses to superimposed mild stressors and that CRF(1)-initiated colonic response is counteracted by selective activation of CRF(2) receptor.     

The ancestor of the <Emphasis Type="Italic">Paulinella</Emphasis> chromatophore obtained a carboxysomal operon by horizontal gene transfer from a <Emphasis Type="Italic">Nitrococcus</Emphasis>-like γ-proteobacterium

Background  

Paulinella chromatophora is a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin that are closely related to free-living Prochlorococcus and Synechococcus species (PS-clade). Members of the PS-clade of cyanobacteria contain a proteobacterial form 1A RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) that was acquired by horizontal gene transfer (HGT) of a carboxysomal operon. In rDNA-phylogenies, the Paulinella chromatophore diverged basal to the PS-clade, raising the question whether the HGT occurred before or after the split of the chromatophore ancestor.     

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.     

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.