Standardized Aronia melanocarpa extract regulates redox status in patients receiving hemodialysis with anemia

Molecular and Cellular Biochemistry - The aim of our study was to investigate the effects of one-month consumption of polyphenol-rich standardized Aronia melanocarpa extract (SAE) on redox status...     

Protein changes and proteolytic degradation in red and white clover plants subjected to waterlogging

Red (Trifolium pratense L., cv. “Start”) and white clover varieties (Trifolium repens L., cv. “Debut” and cv. “Haifa”) were waterlogged for 14 days and subsequently recovered for the period of 21 days. Physiological and biochemical responses of the clover varieties were distinctive, which suggested different sensitivity toward flooding. The comparative study of morphological and biochemical parameters such as stem length, leaflet area, dry weight, protein content, protein pattern and proteolytic degradation revealed prominent changes under waterlogging conditions. Protease activity in the stressed plants increased significantly, especially in red clover cv. “Start”, which exhibited eightfold higher azocaseinolytic activity compared to the control. Changes in the protein profiles were detected by SDS-PAGE electrophoresis. The specific response of some proteins (Rubisco, Rubisco-binding protein, Rubisco activase, ClpA and ClpP protease subunits) toward the applied stress was assessed by immunoblotting. The results characterized the red clover cultivar “Start” as the most sensitive toward waterlogging, expressing reduced levels of Rubisco large and small subunits, high content of ClpP protease subunits and increased activity of protease isoforms.     

AKT isoforms modulate Th1‐like Treg generation and function in human autoimmune disease

Foxp3⁺ regulatory T cells (Tregs) exhibit plasticity, which dictates their function. Secretion of the inflammatory cytokine IFNγ, together with the acquisition of a T helper 1 (Th1)‐like effector phenotype as observed in cancer, infection, and autoimmune diseases, is associated with loss of Treg suppressor function through an unknown mechanism. Here, we describe the signaling events driving the generation of human Th1‐Tregs. Using a genome‐wide gene expression approach and pathway analysis, we identify the PI3K/AKT/Foxo1/3 signaling cascade as the major pathway involved in IFNγ secretion by human Tregs. Furthermore, we describe the opposing roles of AKT isoforms in Th1‐Treg generation ex vivo. Finally, we employ multiple sclerosis as an in vivo model with increased but functionally defective Th1‐Tregs. We show that the PI3K/AKT/Foxo1/3 pathway is activated in ex vivo‐isolated Tregs from untreated relapsing–remitting MS patients and that blockade of the pathway inhibits IFNγ secretion and restores the immune suppressive function of Tregs. These data define a fundamental pathway regulating the function of human Tregs and suggest a novel treatment paradigm for autoimmune diseases.     

Biological peculiarities of someYersinia species: strain-dependent virulence and strain-dependent stress proteins

A pair ofYersinia enterocolitica serotype O∶3 strains — incubated in growth medium with 10% NaCl and 2 mM glycin betaine at 4 °C, were used to study the plasmid role in the infection of BALB/c mice. The isogenic plasmid-bearing strain, but not its plasmidless derivative, caused enteric infection and histological changes in intestines, stomach and liver of the mice. Two strainsY. enterocolitica andYersinia pseudotuberculosis were incubated at different temperatures (4 and 25 °C) in media, supplemented with different concentrations of NaCl. Two concentrations of betaine as osmoprotector were tested. The initial strains and their substrains were characterised by protein profiles using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results show that in salinity conditions, the presence of osmoprotectant (betaine) as well as the temperature of cultivation plays a great role in the expression of some bacterial proteins. The manner in which the different strains answer to the stress situations is specific for each of them.     

Progression of optic neuritis to multiple sclerosis in the County of Split-Dalmatia, Croatia

The aim of this study was to determine the incidence of monosymptomatic optic neuritis (MON) and progression of MON to multiple sclerosis (MS) from the Mediterranean region of southern Europe in the County of Split-Dalmatia, Croatia during the 11 years period from 1991 to 2001. This study was made retrospectively on the 87 cases (59 female, aged 25.9 +/- 11.3 and 28 male aged 29.9 +/- 9.2) of MON, which were treated at the Department of Ophthalmology and Department of Neurology, Split, University Hospital, from January 1991 to December 2001. In each case the diagnosis was confirmed by a chart review and cases were ascribed to the data of admittance at hospital. The annual incidence of MON was 1.9 per 100,000 (95% CI, 0.4-3.5). The incidence among males was 1.2 (95% CI, 0-2.9) cases / 100,000 per year and 2.5 (95% CI, 0.1-4.9) among females. A significant seasonal variations in the incidence of MON was not found (chi2 = 6.81, p = 0.08). MS developed in 20 of 87 patients (22.9%) and median time was 25 (SE 8) months, (95% CI, 9-41) after the MON onset. After two years 12.6% of patients with MON developed MS, 20.6% after 5 years and 22.9% after 10 years. MS was slightly but not significantly more frequent in women than in men (chi2 = 0.72, p = 0.3). In conclusion, the progression of MON to MS in the County of Split-Dalmatia, Croatia was at a relatively moderate frequency.     

Articular chondrocyte aging and endothelin-1

OBJECTIVE: To compare in cell culture endothelin-1 (ET-1) production, receptor density, and effect on macromolecular synthesis by articular chondrocytes (AC). METHODS: AC were isolated from 1-month and 18-month old rats and cultured as monolayers. They were incubated with ET-1 without or with iNOS inhibitors, nitro-L-arginine methyl ester (L-NAME) or guanylate cyclase inhibitor, LY83583 and then [3H]thymidine, 35SO4 and [3H]proline incorporations were measured. The density and affinity for 125I-ET-1 of binding sites, and receptor isotypes were determined. The cells were also treated with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha), and then ET-1 productions measured. As well, the cells were challenged with NOC-5 (nitric oxide donor) or ET-1 and then ET-1 and NO respectively were measured. RESULTS: A concentration-dependent stimulation of DNA, PG, collagen and NO synthesis was obtained when cells were incubated with ET-1 for 24-h. Eighteen-month old chondrocytes incorporated per microg DNA more [3H]thymidine, 35SO4 and [3H]proline but less NO when challenged with ET-1 than the 1-month old cells. However, strong inhibition of this initial stimulation was seen after 48-h. L-NAME and LY83583 enhanced basal-, and ET-1-induced initial stimulation and completely suppressed late (at 48-h to 72-h) ET-1-induced inhibition, suggesting NO was responsible for this inhibitory effect. Eighteen-month old chondrocytes expressed per mug DNA more high affinity receptors of predominantly ET(A) subtype. They also produced more ET-1 but less NO under basal conditions and more ET-1 when challenged with IL-1beta and TNF-alpha. NOC-5 inhibited the production of ET-1. CONCLUSIONS: Eighteen-month old chondrocytes produce more ET-1, possess more ET-1-specific receptors, and increase more DNA, PG and collagen synthesis when challenged during 24-h with ET-1. NO, which suppresses ET-1 production and the production of which is increased by ET-1, seems to account for the late ET-1-induced inhibition of macromolecular synthesis. The possible implication of ET-1 in aging as related to osteoarthritis is discussed.     

Retroviral display of urokinase-binding domain fused to amphotropic envelope protein

Tumors frequently express urokinase (uPA) receptor (uPAR). To investigate whether uPAR can efficiently target cancerous cells using amphotropic retroviral vectors, we generated a retrovirus displaying the amino-terminal fragment (ATF) of uPA as an N-terminal extension of viral envelope protein. We also made use of a "two-step strategy" by inserting a uPA cleavage site between the ATF moiety and the envelope. We measured the ability of ATF-bearing chimeric envelopes to infect huPAR-overexpressing Madin-Darby canine kidney (MDCK) and control MDCK II cells. The ATF-viruses infected both MDCK cell lines with an equivalent efficiency, suggesting that the chimeric viruses were not sequestered by uPAR and infect cells preferentially via the Pit-2 receptor. The addition of a uPA cleavage site increased the infection level of huPAR-MDCK cells by 2-fold when uPA was present in the infection medium. Surprisingly, ATF-env viruses infected huPAR-MDCK cells 5.5-fold more efficiently in the presence of exogenous uPA. This stimulatory effect of uPA on infection of huPAR-MDCK cells by the ATF-env virus was completely abolished by methyl-beta-cyclodextrin, suggesting that this effect involves the caveolar endocytosis pathway.     

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf–MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)–ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.     

IL12RB2 Gene Is Associated with the Age of Type 1 Diabetes Onset in Croatian Family Trios

Background

Common complex diseases are influenced by both genetic and environmental factors. Many genetic factors overlap between various autoimmune diseases. The aim of the present study is to determine whether four genetic variants known to be risk variants for several autoimmune diseases could be associated with an increased susceptibility to type 1 diabetes mellitus.

Methods and Findings

We genotyped four genetic variants (rs2358817, rs1049550, rs6679356, rs9865818) within VTCN1, ANXA11, IL12RB2 and LPP genes respectively, in 265 T1DM family trios in Croatian population. We did not detect association of these polymorphisms with T1DM. However, quantitative transmission disequilibrium test (QTDT, orthogonal model) revealed a significant association between the age of onset of T1DM and IL12RB2 rs6679356 variant. An earlier onset of T1DM was associated with the rs6679356 minor dominant allele C (p = 0.005). The association remained significant even after the Bonferroni correction for multiple testing and permutation.

Conclusions

Variants originally associated with juvenile idiopathic arthritis (VTCN1 gene), sarcoidosis (ANXA11 gene), primary biliary cirrhosis (IL12RB2 gene) and celiac disease (LPP gene) were not associated with type 1 diabetes in our dataset. Nevertheless, association of IL12RB2 rs6679356 polymorphism with the age of T1DM onset suggests that this gene plays a role in defining the time of disease onset.