Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.     

Expression of genes for cytokines and cytokine-related functions in leukocytes infected with Herpes simplex virus: comparison between resistant and susceptible mouse strains

Cytokines and chemokines play an important role in the first line of defence against viral infections. Moreover, these groups of proteins also contribute significantly to regulation of the acquired immune response. Therefore, knowledge of the expression of cytokines, chemokines and factors involved in their action may provide information about the immune reaction responsible for elimination of viral infections and for immune-mediated pathology. Using cDNA arrays, we have evaluated the expression of cytokines and genes related to cytokine function in resting murine peritoneal cells and in inflammatory macrophages infected with Herpes simplex virus (HSV)-1 and -2. To allow comparison, the experiments were performed using both the resistant mouse strain C57BL/6 and the susceptible strain BALB/c. The work identified a group of genes that is differentially expressed during HSV infection of cells from the two strains. Another group of genes was affected by HSV-1 but not HSV-2 infection and vice versa. Further analysis of these genes may provide new information about host defense against viral infections and could also lead to identification of the molecular basis for the pathological differences between infections with HSV-1 and -2.     

Microtubule release from the centrosome in migrating cells

In migrating cells, force production relies essentially on a polarized actomyosin system, whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule (MT) and the actin filament networks (Goode, B.L., D.G. Drubin, and G. Barnes. 2000. Curr. Opin. Cell Biol., 12:63-71). Targeting and capture of MT plus ends at the cell periphery has been described, but whether or not the minus ends of these MTs are anchored at the centrosome is not known. Here, we show that release of short MTs from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired. We further show that MT release, but not MT nucleation or polymerization dynamics, is abolished by overexpression of the centrosomal MT-anchoring protein ninein. In addition, a dramatic inhibition of cell migration was observed; but, contrary to cells treated by drugs inhibiting MT dynamics, polarized membrane ruffling activity was not affected in ninein overexpressing cells. We thus propose that the balance between MT minus-end capture and release from the centrosome is critical for efficient cell migration.     

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.     

Probiotic bacteria: safety, functional and technological properties

During the past two decades probiotic (health promoting) micro-organisms have been increasingly included in various types of food products, especially in fermented milks. Several aspects, including safety, functional and technological characteristics, have to be taken into consideration in the selection process of probiotic micro-organisms. Safety aspects include specifications such as origin (healthy human GI-tract), non-pathogenicity and antibiotic resistance characteristics. Functional aspects include viability and persistence in the GI-tract, immunomodulation, antagonistic and antimutagenic properties. Before probiotic strains, chosen on the basis of their good safety and functional characteristics, can benefit the consumer, they must first be able to be manufactured under industrial conditions. Furthermore, they have to survive and retain their functionality during storage, and also in the foods into which they are incorporated without producing off-flavours. Factors related to the technological and sensory aspects of probiotic food production are of utmost importance since only by satisfying the demands of the consumer can the food industry succeed in promoting the consumption of functional probiotic products in the future.     

Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

Background  

The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Design of a partial PPARdelta agonist

Structure based ligand design was used in order to design a partial agonist for the PPARdelta receptor. The maximum activation in the transactivation assay was reduced from 87% to 39%. The crystal structure of the ligand binding domain of the PPARdelta receptor in complex with compound 2 was determined in order to understand the structural changes which gave rise to the decrease in maximum activation.     

Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.Mitochondria are the primary energy-generating systems in eukaryotes. They play a crucial role in oxidative metabolism, including carbohydrate metabolism, fatty acid oxidation, and urea cycle, as well as in calcium signaling and apoptosis (1, 2). Mitochondrial dysfunction is centrally involved in a number of human pathologies, such as type 2 diabetes, Parkinson disease, and cancer (3). The most prevalent form of cellular protein post-translational modifications (PTMs),1 reversible phosphorylation (4–6), is emerging as a central mechanism in the regulation of mitochondrial functions (7, 8). The steadily increasing numbers of reported mitochondrial kinases, phosphatases, and phosphoproteins imply an important role of protein phosphorylation in different mitochondrial processes (9–11).Mass spectrometry (MS)-based proteome analysis is a powerful tool for global profiling of proteins and their PTMs, including protein phosphorylation (12, 13). A variety of proteomics techniques have been developed for specific enrichment of phosphorylated proteins and peptides and for phosphopeptide-specific data acquisition techniques at the MS level (14). Enrichment methods based on affinity chromatography, such as titanium dioxide (TiO₂) (15–17), zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) (18), immobilized metal affinity chromatography (IMAC) (19, 20), and ion exchange chromatography (strong anion exchange and strong cation exchange) (21, 22), have shown high efficiencies for enrichment of phosphopeptides (14). Recently, we demonstrated that calcium phosphate precipitation (CPP) is highly effective for enriching phosphopeptides (23). It is now generally accepted that no single method is comprehensive, but combinations of different enrichment methods produce distinct overlapping phosphopeptide data sets to enhance the overall results in phosphoproteome analysis (24, 25). Phosphopeptide sequencing by mass spectrometry has seen tremendous advances during the last decade (26). For example, MS/MS product ion scanning, multistage activation, and precursor ion scanning are effective methods for identifying serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylated peptides (14, 26).A “complete” mammalian mitochondrial proteome was reported by Mootha and co-workers (27) and included 1098 proteins. The mitochondrial phosphoproteome has been characterized in a series of studies, including yeast, mouse and rat liver, porcine heart, and plants (19, 28–31). To date, the largest data set by Deng et al. (30) identified 228 different phosphoproteins and 447 phosphorylation sites in rat liver mitochondria. However, the in vivo phosphoproteome of human mitochondria has not been determined. A comprehensive mitochondrial phosphoproteome is warranted for further elucidation of the largely unknown mechanisms by which protein phosphorylation modulates diverse mitochondrial functions.The percutaneous muscle biopsy technique is an important tool in the diagnosis and management of human muscle disorders and has been widely used to investigate metabolism and various cellular and molecular processes in normal and abnormal human muscle, in particular the molecular mechanism underlying insulin resistance in obesity and type 2 diabetes (32). Skeletal muscle is rich in mitochondria and hence a good source for a comprehensive proteomics and functional analysis of mitochondria (32, 33).The major aim of the present study was to obtain a comprehensive overview of site-specific phosphorylation of mitochondrial proteins in functionally intact mitochondria isolated from human skeletal muscle. Combining an efficient protocol for isolation of skeletal muscle mitochondria with several different state-of-the-art phosphopeptide enrichment methods and high performance LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, many of which have not been reported before. We characterized this mitochondrial phosphoproteome by using bioinformatics tools to classify functional groups and functions, including kinase substrate motifs.     

Age-dependent role for CCR5 in antiviral host defense against herpes simplex virus type 2

Elimination of viral infections is dependent on rapid recruitment and activation of leukocytes with antiviral activities to infected areas. Chemokines constitute a class of cytokines that have regulatory effects on leukocyte migration and activity. In this study we have studied the role of CC chemokine receptor 1 (CCR1) and CCR5 in host defense during a generalized herpes simplex virus type 2 (HSV-2) infection. Whereas both 4- and 8-week-old CCR1(-/-) mice resembled wild-type mice (C57BL/6) with respect to defense against the infection, significantly higher virus titers were seen in the livers and brains of 4-week-old CCR5(-/-) mice. At the age of 8 weeks, CCR5(-/-) were indistinguishable from wild-type mice and cleared the infection from liver and spleen. Although 4-week-old CCR5(-/-) mice were able to recruit natural killer (NK) cells to the site of infection, these cells had reduced cytotoxic activity compared to NK cells from wild-type mice. This was not due to lower production of alpha/beta interferon or interleukin-12, two well-described activators of cytotoxic activity in NK cells. We also noted that the spleens of young CCR5(-/-) mice did not increase in size during infection as did the spleens of wild-type and CCR1(-/-) mice. This observation was accompanied by impaired proliferation of CCR5(-/-) splenocytes (SCs) ex vivo. Moreover, migration of CD8(+) T cells to the liver in response to infection was impaired in CCR5(-/-) mice, and adoptive transfer of SCs from CCR5(-/-) mice infected for 6 days into newly infected wild-type mice did not improve antiviral activity in the liver, in contrast to what was seen in mice receiving immune SCs from wild-type mice. Altogether, this study shows that CCR5 plays an age-dependent role in host defense against HSV-2 by supporting both the innate and adaptive immune response.