Mitotic partitioning of endosomes and lysosomes

BACKGROUND: Some of the mechanisms underlying cell division and partitioning of the cellular components into the daughter cells are well known. Within the endomembrane system, there is a general cessation of membrane traffic, including endocytosis and endosome fusion, at the onset of mitosis. However, the fate of endosomes and lysosomes during mitosis has been less well studied. RESULTS: Using video and confocal microscopy of living cells, we show here that endosomes and lysosomes remain intact and separate during mitosis. The segregation into daughter cells takes place by coordinated movements, and during cytokinesis, these organelles accumulate in the vicinity of the microtubule organization center. However, partitioning into daughter cells is not more accurate than a calculated stochastic distribution, despite the apparent order to the process. CONCLUSION: We conclude that partitioning of endosomes and lysosomes is an ordered, yet imprecise, process, and that the organelle copy number is maintained by the daughter cells.     

Rab7a modulates ER stress and ER morphology

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called “ER matrices” together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22?N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells.     

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.     

Long-term repeatability of force, endurance time and muscle activity during isometric contractions.

We determined the repeatability and correlations between force, endurance and muscle activity during isometric contractions over three years. Twenty-six subjects, with and without complaints of the shoulder and neck, performed standardized maximal and submaximal shoulder-abduction contractions and wrist extension-contractions at yearly intervals from 1997 to 1999. Peak forces developed during maximal contraction and the endurance times of submaximal contractions during shoulder abduction and wrist extension were measured. Electromyography (EMG) of muscle activity was recorded bilaterally from the upper trapezius, middle deltoid, and forearm extensor muscles. Root mean square EMG amplitudes were calculated. We found statistically significant associations between peak forces developed during wrist extension and shoulder abduction, and between endurance times of submaximal wrist extension and shoulder abduction. No statistically significant changes in peak force and EMG(peak) were found over the measurement years. The responses were not statistically significantly influenced by gender, or neck and shoulder pain. However, we observed considerable intra-individual variation in the inter-year measurements particularly for the responses to submaximal contraction. Such large variations represent a challenge when attempting to use the responses to interpret the effects of therapies.     

The Norwegian naturalistic treatment study of depression in general practice (NORDEP)-I: randomised double blind study

ObjectiveTo evaluate the efficacy of emotional support and counselling combined with placebo or antidepressants with single or dual mechanism of action in the treatment of depression in primary care.DesignRandomised double blind study.SettingSeveral locations in Norway.Subjects372 patients with depression.Results Intention to treat analyses showed 47% remission in patients randomised to placebo compared with 61% remission in patients randomised to sertraline (odds ratio 0.56, 95% confidence interval 0.33 to 0.96) and 54% in patients randomised to mianserin (0.75, 0.44 to 1.27). Women responded better than men (1.86, 1.17 to 2.96). Subgroup analyses showed that subjects with recurrent depression (n=273) responded more frequently to sertraline than to placebo (0.43, 0.23 to 0.82) than those having their first episode of depression (1.18, 0.39 to 3.61). Statistically significant interactions between type of drug treatment and history of depression were not shown by logistic regression.Conclusion The combination of active drug and simple psychological treatment (counselling, emotional support, and close follow up over a 24 week period) was more effective than simple psychological treatment alone, in particular for those with recurrent depression. Overall, women may benefit more than men. If confirmed in future studies, the findings should lead to more differentiated treatment guidelines for depression in primary care.

Key messages

• The effectiveness of simple psychological treatment and active drug provided by general practitioners is comparable to treatment results reported by psychiatrists and clinical psychologists
• Treatment benefits women more than men
• There may be differences in response to treatment depending on the nature of depression
• A 6 month treatment period is necessary to evaluate effectiveness of treatments for depression in general practice
• The development of more differentiated treatment guidelines for depression in primary care is needed

     

Genetic association between human chitinases and lung function in COPD

Two primary chitinases have been identified in humans--acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host's immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to chronic obstructive pulmonary disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the caucasian LHS population, the baseline forced expiratory volume in one second (FEV(1)) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV(1) and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV(1). Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups.     

Transcriptional regulation of IL-17A and other inflammatory markers during the development of soybean meal-induced enteropathy in the distal intestine of Atlantic salmon (Salmo salar L.)

Progression of soybean meal (SBM)-induced enteropathy in Atlantic salmon (Salmo salar L.) distal intestine (DI) was studied to investigate pathophysiological mechanisms and immune responses. Seawater-adapted salmon were fed an extracted SBM-containing diet (200gkg(-1)) from day 1-21 and compared with fish fed a fishmeal-based diet (day 0). Histological evaluation of the DI revealed signs of inflammation from day 5, which progressively increased in severity and affected more fish with increasing SBM exposure time. The expression profiles of 16 genes were analyzed by quantitative PCR. The pro-inflammatory cytokines interleukin 17A (IL-17A), IL-1β, interferon α (IFNα) and IFNγ, as well as IL-17A receptor, T-cell receptor γ (TCRγ), cluster of differentiation 4α (CD4α), CD8β, transforming growth factor β (TGFβ), trypsin, protease-activated receptor 2 (PAR2) and myeloid differentiation primary response gene 88 (MyD88) were significantly up-regulated during early and/or late inflammation stages, whereas interferon-γ-inducible lysosomal thiol reductase (GILT) was downregulated. Up-regulation of TCRγ from day seven suggests proliferation of intraepithelial γδ T cells. IL-17A, up-regulated by 218-fold during early inflammation, indicates involvement of T helper 17 cells in the pathogenesis of the SBM-induced inflammatory response.     

Nanoparticle Mediated P-Glycoprotein Silencing for Improved Drug Delivery across the Blood-Brain Barrier: A siRNA-Chitosan Approach

The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.     

Comparative Genomics to Delineate Pathogenic Potential in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) from Patients with and without Haemolytic Uremic Syndrome (HUS) in Norway

Shiga toxin-producing Escherichia coli (STEC) cause infections in humans ranging from asymptomatic carriage to bloody diarrhoea and haemolytic uremic syndrome (HUS). Here we present whole genome comparison of Norwegian non-O157 STEC strains with the aim to distinguish between strains with the potential to cause HUS and less virulent strains. Whole genome sequencing and comparisons were performed across 95 non-O157 STEC strains. Twenty-three of these were classified as HUS-associated, including strains from patients with HUS (n = 19) and persons with an epidemiological link to a HUS-case (n = 4). Genomic comparison revealed considerable heterogeneity in gene content across the 95 STEC strains. A clear difference in gene profile was observed between strains with and without the Locus of Enterocyte Effacement (LEE) pathogenicity island. Phylogenetic analysis of the core genome showed high degree of diversity among the STEC strains, but all HUS-associated STEC strains were distributed in two distinct clusters within phylogroup B1. However, non-HUS strains were also found in these clusters. A number of accessory genes were found to be significantly overrepresented among HUS-associated STEC, but none of them were unique to this group of strains, suggesting that different sets of genes may contribute to the pathogenic potential in different phylogenetic STEC lineages. In this study we were not able to clearly distinguish between HUS-associated and non-HUS non-O157 STEC by extensive genome comparisons. Our results indicate that STECs from different phylogenetic backgrounds have independently acquired virulence genes that determine pathogenic potential, and that the content of such genes is overlapping between HUS-associated and non-HUS strains.     

Characterization of mitochondrial ribosomal RNA genes in gadiformes: sequence variations,secondary structural features,and phylogenetic implications

Secondary structure features of mitochondrial ribosomal RNAs (mt-rRNAs) of bony fishes were investigated by a DNA sequence alignment approach. The small subunit (SSU) and large subunit (LSU) mt-rRNA genes were found to contain several additional variable regions compared to their mammalian counterparts. Fish mt-LSU rRNA genes were found to be longer than the mammalians due to increased length of some of the variable regions. The 5' and 3' ends of Atlantic cod mt-rRNAs were precisely mapped. The 3' ends of mt-SSU rRNAs were found to be homogenous and mono-adenylated, whereas that of the mt-LSU rRNAs were heterogenous and oligo-adenylated. The 5' ends of mt-SSU rRNAs appeared to be heterogenous, corresponding to the presumed first and second positions of the gene. Sequences of the central domain and the D-domain of the mt-SSU and mt-LSU rRNA genes, respectively, were determined and characterized for 11 gadiform species (representing the families Gadidae, Lotidae, Ranicipitidae, Merlucciidae, Phycidae, and Macrouridae) and one Lophiidae species. Detailed secondary structure models of the RNA regions are presented for the Atlantic cod (Gadus morhua) and Roundnose grenadier (Coryphaeonides rupestris). Saturation plots revealed that DNA nucleotide positions corresponding to unpaired RNA regions become saturated with transitions at sequence divergence levels about 0.15. Phylogenetic analyses revealed some aspects of gadiform relationships. Gadidae was identified as the most derived of the gadiform families. Lotidae was found to be the family closest related to Gadidae, and Ranicipitidae was also recognized as a derived gadiform taxon.