Deregulation of Microcephalin and ASPM Expression Are Correlated with Epithelial Ovarian Cancer Progression

Mutations in the MCPH1 (Microcephalin) and ASPM (abnormal spindle-like microcephaly associated) genes cause primary microcephaly. Both are centrosomal associated proteins involved in mitosis. Microcephalin plays an important role in DNA damage response and ASPM is required for correct division of proliferative neuro-epithelial cells of the developing brain. Reduced MCPH1 mRNA expression and ASPM mRNA over-expression have been implicated in the development of human carcinomas. Epithelial ovarian cancer (EOC) is characterised by highly aneuploid tumours. Previously we have reported low Microcephalin and high ASPM protein levels and associations with clinico-pathological parameters in malignant cells from ascitic fluids. To confirm these previous findings on a larger scale Microcephalin and ASPM expression levels and localisations were evaluated by immunohistochemistry in two cohorts; a training set of 25 samples and a validation set of 322 EOC tissue samples. Results were correlated to the associated histopathological data. In normal ovarian tissues the Microcephalin nuclear staining pattern was consistently strong. In the cancer tissues, we identified low nuclear Microcephalin expression in high grade and advanced stage tumours (p<0.0001 and p = 0.0438 respectively). ASPM had moderate to high nuclear and low to moderate cytoplasmic expression in normal tissue. Cytoplasmic ASPM expression decreased with tumour grade and stage in the serous subtype of EOC (p = 0.023 and p = 0.011 respectively). Cytoplasmic ASPM increased with tumour stage in the endometrioid subtype (p = 0.023). Increasing tumour invasiveness (T3) and lymph node involvement (N1) also correlated with a decrease in cytoplasmic ASPM in EOC (p = 0.02 and p = 0.04 respectively). We have validated previous findings of deregulated expression of Microcephalin and ASPM in EOC by confirming associations for low nuclear Microcephalin levels and high cytoplasmic ASPM levels in a larger scale tumour tissue study. Microcephalin and ASPM may prove useful biomarkers in EOC.     

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture — primary culture cells markedly differ from fourth-passage cells

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase⁺/74% Thy-1/CD90⁺ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.     

Ectomycorrhizal Colonization and Diversity in Relation to Tree Biomass and Nutrition in a Plantation of Transgenic Poplars with Modified Lignin Biosynthesis

Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana. The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R² = 97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations.     

The DivJ,CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen‐fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.     

A Case-Control Study of the Protective Effect of Alcohol,Coffee, and Cigarette Consumption on Parkinson Disease Risk: Time-Since-Cessation Modifies the Effect of Tobacco Smoking

The aim of this study was to investigate the possible reduced risk of Parkinson Disease (PD) due to coffee, alcohol, and/or cigarette consumption. In addition, we explored the potential effect modification by intensity, duration and time-since-cessation of smoking on the association between cumulative pack-years of cigarette smoking (total smoking) and PD risk. Data of a hospital based case-control study was used including 444 PD patients, diagnosed between 2006 and 2011, and 876 matched controls from 5 hospitals in the Netherlands. A novel modeling method was applied to derive unbiased estimates of the potential modifying effects of smoking intensity, duration, and time-since-cessation by conditioning on total exposure. We observed no reduced risk of PD by alcohol consumption and only a weak inverse association between coffee consumption and PD risk. However, a strong inverse association of total smoking with PD risk was observed (OR = 0.27 (95%CI: 0.18–0.42) for never smokers versus highest quartile of tobacco use). The observed protective effect of total smoking was significantly modified by time-since-cessation with a diminishing protective effect after cessation of smoking. No effect modification by intensity or duration of smoking was observed indicating that both intensity and duration have an equal contribution to the reduced PD risk. Understanding the dynamics of the protective effect of smoking on PD risk aids in understanding PD etiology and may contribute to strategies for prevention and treatment.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Atrial natriuretic peptide reduces cyclosporin toxicity in renal cells: role of cGMP and heme oxygenase-1.

Using cultured proximal renal tubular epithelial cells (LLC-PK1), the present study investigates the effect of atrial natriuretic peptide (ANP) on cytotoxicity induced by cyclosporin A (CsA). Preincubation with ANP (1-100 nM) protected LLC-PK1 cells from CsA-induced toxicity in a concentration-dependent manner. A cytoprotective effect comparable to ANP was observed when preincubating the cells with 8-bromo cGMP (1-100 microM) or the antioxidant heme oxygenase (HO) metabolite bilirubin (0.1-10 microM). ANP or cGMP produced increases in HO-1 protein levels at concentrations that were also effective in cellular protection. Moreover, incubation with ANP or 8-bromo cGMP led to increased HO activity, i.e., formation of bilirubin in the cell lysate (up to 3-fold over basal). Tin protoporphyrin-IX (SnPP; 19 microM), an inhibitor of HO activity, completely abolished ANP-induced cytoprotection. Our results demonstrate that HO-1 is a cellular target of ANP and cGMP in renal cells. HO-1 induction and ensuing formation of antioxidant metabolites may be a novel pathway by which ANP protects from CsA-dependent nephrotoxicity and preserves renal function.