Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

Association study of interleukin-4 polymorphisms with paranoid schizophrenia in the Polish population: a critical approach

Changes in immunological system are one of dysfunctions reported in schizophrenia. Some changes based on an imbalance between Th1 and Th2 cytokines results from cytokine gene polymorphisms. Interleukin-4 gene (IL4) is considered as a potential candidate gene in schizophrenia association studies. The aim of the current case-control study was to examine whether the -590C/T (rs2243250) and -33C/T (rs2070874) IL4 gene polymorphisms are implicated in paranoid schizophrenia development in the Polish population. Genotyping of polymorphisms was performed by using PCR-RFLP technique. The genotypes and alleles distribution of both SNPs were analysed in patients (n = 182) and healthy individuals constituted the control group (n = 215). The connection between some clinical variables and studied polymorphisms has been examined as well. We did not revealed any association between the -590C/T and -33C/T polymorphisms and paranoid schizophrenia. In case of both SNPs the homozygous TT genotype was extremely rare. Both polymorphic sites of the IL4 gene were found to be in a very strong linkage disequilibrium. However we did not identify a haplotype predispose to paranoid schizophrenia. No associations were also observed between the clinical course and psychopathology of the disease and the genotypes of both analysed polymorphisms. Our results suggest that the polymorphisms -590C/T in IL4 gene promoter region and -33C/T in the 5'-UTR are not involved in the pathophysiology of paranoid schizophrenia in Polish residents.     

Properties of monocytes generated from haematopoietic CD34+ stem cells from bone marrow of colon cancer patients

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34⁺ stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34⁺ haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14⁺ monocytes were found. They consisted of two subpopulations: CD14⁺⁺CD16⁺ and CD14⁺CD16^?, at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14⁺ monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3⁺ T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu_369–377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8⁺ T lymphocytes (CTL). The CD14⁺⁺CD16⁺ subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34⁺ stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.     

Square-cut: a segmentation algorithm on the basis of a rectangle shape

We present a rectangle-based segmentation algorithm that sets up a graph and performs a graph cut to separate an object from the background. However, graph-based algorithms distribute the graph's nodes uniformly and equidistantly on the image. Then, a smoothness term is added to force the cut to prefer a particular shape. This strategy does not allow the cut to prefer a certain structure, especially when areas of the object are indistinguishable from the background. We solve this problem by referring to a rectangle shape of the object when sampling the graph nodes, i.e., the nodes are distributed non-uniformly and non-equidistantly on the image. This strategy can be useful, when areas of the object are indistinguishable from the background. For evaluation, we focus on vertebrae images from Magnetic Resonance Imaging (MRI) datasets to support the time consuming manual slice-by-slice segmentation performed by physicians. The ground truth of the vertebrae boundaries were manually extracted by two clinical experts (neurological surgeons) with several years of experience in spine surgery and afterwards compared with the automatic segmentation results of the proposed scheme yielding an average Dice Similarity Coefficient (DSC) of 90.97±2.2%.     

PROTON GRADIENT REGULATION5 Is Essential for Proper Acclimation of Arabidopsis Photosystem I to Naturally and Artificially Fluctuating Light Conditions

In nature, plants are challenged by constantly changing light conditions. To reveal the molecular mechanisms behind acclimation to sometimes drastic and frequent changes in light intensity, we grew Arabidopsis thaliana under fluctuating light conditions, in which the low light periods were repeatedly interrupted with high light peaks. Such conditions had only marginal effect on photosystem II but induced damage to photosystem I (PSI), the damage being most severe during the early developmental stages. We showed that PROTON GRADIENT REGULATION5 (PGR5)-dependent regulation of electron transfer and proton motive force is crucial for protection of PSI against photodamage, which occurred particularly during the high light phases of fluctuating light cycles. Contrary to PGR5, the NAD(P)H dehydrogenase complex, which mediates cyclic electron flow around PSI, did not contribute to acclimation of the photosynthetic apparatus, particularly PSI, to rapidly changing light intensities. Likewise, the Arabidopsis pgr5 mutant exhibited a significantly higher mortality rate compared with the wild type under outdoor field conditions. This shows not only that regulation of PSI under natural growth conditions is crucial but also the importance of PGR5 in PSI protection.     

A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland

Until recently Dickeya was regarded as a pathogen not established in Finland. As a result the blackleg symptom observed on potato was often associated with Pectobacterium atrosepticum. The occurrence of Dickeya spp. on potato in Finland was first reported in 2004. Since then the prevalence of Dickeya has been monitored through surveys and routine test of seed lots produced in the country. The results of monitoring of Dickeya spp. in seed lots produced in Finland between the years 2004 and 2008 indicated a steady increase in the incidence of Dickeya spp. The highest incidence was observed in samples from the 2006 growing season where about 37% were positive for Dickeya spp. The summer in 2006 was one of the warmest summers recorded in 100 years in Finland. The majority of infected lots were imported varieties. Since recently heavy blackleg outbreaks have occurred in production fields in the High Grade (HG) zone. A detailed study of these incidents of blackleg outbreaks in North Finland during the years 2008 and 2010 indicated that Dickeya spp. was the major component in the observed blackleg complex. It was detected and isolated from almost all symptomatic plants investigated. Repetitive sequences PCR (REP‐PCR) and Pulsed Field Gel Electrophoresis (PFGE) analysis of strains isolated in Finland showed identical pattern with those isolated recently in other European countries with a proposed name ‘Dickeya solani’. Moreover, the dnaX gene sequence of the representative strains isolated in Finland indicated 100% similarity to the dnaX sequences of ‘D. solani’. The study presents the first report of a detailed analysis of bacteria involved in potato blackleg complex from natural field outbreaks in North Finland HG zone and characterisation of the ‘D. solani’ strains playing the major role in the disease complex.     

Obesity and body fat classification in the metabolic syndrome: Impact on cardiometabolic risk metabotype

Objective:

Obesity is a key factor in the development of the metabolic syndrome (MetS), which is associated with increased cardiometabolic risk. We investigated whether obesity classification by BMI and body fat percentage (BF%) influences cardiometabolic profile and dietary responsiveness in 486 MetS subjects (LIPGENE dietary intervention study).

Design and Methods:

Anthropometric measures, markers of inflammation and glucose metabolism, lipid profiles, adhesion molecules, and hemostatic factors were determined at baseline and after 12 weeks of four dietary interventions (high saturated fat (SFA), high monounsaturated fat (MUFA), and two low fat high complex carbohydrate (LFHCC) diets, one supplemented with long chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFAs)).

Results:

About 39 and 87% of subjects classified as normal and overweight by BMI were obese according to their BF%. Individuals classified as obese by BMI (≥30 kg/m²) and BF% (≥25% (men) and ≥35% (women)) (OO, n = 284) had larger waist and hip measurements, higher BMI and were heavier (P < 0.001) than those classified as nonobese by BMI but obese by BF% (NOO, n = 92). OO individuals displayed a more proinflammatory (higher C reactive protein (CRP) and leptin), prothrombotic (higher plasminogen activator inhibitor‐1 (PAI‐1)), proatherogenic (higher leptin/adiponectin ratio) and more insulin resistant (higher HOMA‐IR) metabolic profile relative to the NOO group (P < 0.001). Interestingly, tumor necrosis factor‐α (TNF‐α) concentrations were lower post‐intervention in NOO individuals compared with OO subjects (P < 0.001).

Conclusions:

In conclusion, assessing BF% and BMI as part of a metabotype may help to identify individuals at greater cardiometabolic risk than BMI alone.