Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

The awr gene family encodes a novel class of Ralstonia solanacearum type III effectors displaying virulence and avirulence activities

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophanarginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (¿awr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts.

Smoking cessation and the incidence of pre-diabetes and type 2 diabetes: a cohort study.

AIMS: Smoking cessation has been suggested to increase the short-term risk of type 2 diabetes mellitus (T2DM). This study aimed at assessing the association between smoking cessation and incidence of T2DM and impaired fasting glucose (IFG). METHODS: Data from participants in the CoLaus study, Switzerland, aged 35-75 at baseline and followed for 5.5years were used. Participants were classified as smokers, recent (≤5years), long-term (>5years) quitters, and non-smokers at baseline. Outcomes were IFG (fasting serum glucose (FSG) 5.6-6.99mmol/l) and T2DM (FSG ≥7.0mmol/l and/or treatment) at follow up. RESULTS: 3,166 participants (63% women) had normal baseline FSG, of whom 26.7% were smokers, 6.5% recent quitters, and 23.5% long-term quitters. During follow-up 1,311 participants (41.4%) developed IFG (33.6% women, 54.7% men) and 47 (1.5%) developed T2DM (1.1% women, 2.1% men). Former smokers did not have statistically significant increased odds of IFG compared with smokers after adjustment for age, education, physical activity, hypercholesterolemia, hypertension and alcohol intake, with OR of 1.29 [95% confidence interval 0.94-1.76] for recent quitters and 1.03 [0.84-1.27] for long-term quitters. Former smokers did not have significant increased odds of T2DM compared with smokers with multivariable-adjusted OR of 1.53 [0.58-4.00] for recent quitters and 0.64 [0.27-1.48] for long-term quitters. Adjustment for body-mass index and waist circumference attenuated the association between recent quitting and IFG (OR 1.07 [0.78-1.48]) and T2DM (OR 1.28 [0.48-3.40]. CONCLUSION: In this middle-aged population, smoking cessation was not associated with an increased risk of IFG or T2DM.

Cell type-specific expression and localization of cytochrome P450 isoforms in tridimensional aggregating rat brain cell cultures.

Within the Predict-IV FP7 project a strategy for measurement of in vitro biokinetics was developed, requiring the characterization of the cellular model used, especially regarding biotransformation, which frequently depends on cytochrome P450 (CYP) activity. The extrahepatic in situ CYP-mediated metabolism is especially relevant in target organ toxicity. In this study, the constitutive mRNA levels and protein localization of different CYP isoforms were investigated in 3D aggregating brain cell cultures. CYP1A1, CYP2B1/B2, CYP2D2/4, CYP2E1 and CYP3A were expressed; CYP1A1 and 2B1 represented almost 80% of the total mRNA content. Double-immunolabeling revealed their presence in astrocytes, in neurons, and to a minor extent in oligodendrocytes, confirming the cell-specific localization of CYPs in the brain. These results together with the recently reported formation of an amiodarone metabolite following repeated exposure suggest that this cell culture system possesses some metabolic potential, most likely contributing to its high performance in neurotoxicological studies and support the use of this model in studying brain neurotoxicity involving mechanisms of toxication/detoxication.

Chronic kidney disease in type 2 diabetic patients followed-up by primary care physicians in Switzerland: prevalence and prescription of antidiabetic drugs.

QUESTION UNDER STUDY: The aim of this study was to assess the prevalence of chronic kidney disease (CKD) among type 2 diabetic patients in primary care settings in Switzerland, and to analyse the prescription of antidiabetic drugs in CKD according to the prevailing recommendations. METHODS: In this cross-sectional study, each participating physician was asked to introduce anonymously in a web database the data from up to 15 consecutive diabetic patients attending her/his office between December 2013 and June 2014. Demographic, clinical and biochemical data were analysed. CKD was classified with the KDIGO nomenclature based on estimated glomerular filtration rate (eGFR) and urinary albumin/creatinine ratio. RESULTS: A total of 1 359 patients (mean age 66.5 ± 12.4 years) were included by 109 primary care physicians. CKD stages 3a, 3b and 4 were present in 13.9%, 6.1%, and 2.4% of patients, respectively. Only 30.6% of patients had an entry for urinary albumin/creatinine ratio. Among them, 35.6% were in CKD stage A2, and 4.1% in stage A3. Despite prevailing limitations, metformin and sulfonylureas were prescribed in 53.9% and 16.5%, respectively, of patients with advanced CKD (eGFR <30 ml/min). More than a third of patients were on a dipeptidyl-peptidase-4 inhibitor across all CKD stages. Insulin use increased progressively from 26.8% in CKD stage 1-2 to 50% in stage 4. CONCLUSIONS: CKD is frequent in patients with type 2 diabetes attending Swiss primary care practices, with CKD stage 3 and 4 affecting 22.4% of cases. This emphasizes the importance of routine screening of diabetic nephropathy based on both eGFR and urinary albumin/creatinine ratio, the latter being largely underused by primary care physicians. A careful individual drug risk/benefit balance assessment is mandatory to avoid the frequently observed inappropriate prescription of antidiabetic drugs in CKD patients.

Smoking cessation and the incidence of pre-diabetes and type 2 diabetes : a cohort study

Le tabagisme est associé à un risque augmenté de développer un diabète de type 2. Arrêter de fumer devrait donc diminuer le risqué de diabète. Seulement, les études concernant le risque métabolique à l'arrêt du tabac sont discordantes. Par ailleurs, les effets métaboliques du tabac et de l'arrêt du tabac diffèrent probablement selon le sexe, avec notamment un effet différent du tabac sur la santé des femmes, et une prise pondérale plus importante à l'arrêt que chez les hommes. Notre étude vise à évaluer le risque métabolique à l'arrêt du tabac, chez les femmes et les homes séparément. Nous avons utilisé les données de l'étude de cohorte prospective CoLaus, qui évalue différents facteurs de risque cardiovasculaire chez des sujets choisis de manière aléatoire, dans la population Lausannoise entre 35 et 75 ans, suivis sur 5.5 ans en moyenne. Parmi ceux avec une glycémie à jeun normale au départ, nous avons divisé les participants en quatre groupes selon leur statut tabagique : non fumeurs, personnes ayant arrêté de fumer depuis plus de 5 ans, celles ayant arrêté depuis moins de 5 ans, et fumeurs actifs. Nous avons mesuré les incidences de glycémie à jeun altérée (5.6-6.99 mmol/l) et de diabète (glycémie à jeun ≥ 7 mmol/l et/ou traitement pour le diabète) durant le période de suivi, stratifiées par sexe. Puis le risque d'incidence de glycémie altérée et de diabète a été calculé avec trois niveaux d'ajustement pour les facteurs confondants pour un risque métabolique. Nous avons inclus 3166 participants, dont 63% de femmes. Au total, 26.3% étaient fumeurs, 6.5% ex-fumeurs depuis moins de 5 ans et 23.5% ex-fumeurs depuis plus de 5 ans. Durant le suivi, 1311 (41.4%) personnes ont développé une glycémie à jeun altérée (33.6% des femmes, 54.7% des homes), et 47 (1.5%) ont développé un diabète (1.1% des femmes, 2.1% des hommes). Les personnes ayant arrêté de fumer n'avait pas de risque significativement plus élevé de développer une glycémie à jeun altérée ou un diabète que les fumeurs, après ajustement pour l'âge, l'éducation, l'hypercholestérolémie, la prise d'alcool, l'activité physique, la prise de poids, le BMI initial et le BMI d'arrivée dans les différents modèles d'ajustement. L'analyse de l'interaction du sexe avec ces résultats est également négative. Les analyses de sensibilité ont montré que l'exclusion des personnes ayant changé de statut tabagique durant le suivi ne changeait pas ces résultats. Nous avons refait les analyses en incluant les participants ayant une glycémie altérée au début du suivi, mais le risque d'incidence de diabète n'est pas plus élevé chez les ex-fumeurs que chez les fumeurs non plus dans cette population. Sur demande d'un reviewer, nous avons également refait les analyses avec la glycémie en continue (valeurs de base et valeurs à 5.5 ans), et la glycémie moyenne n'était pas différente par groupe de tabagisme. En conclusion, dans cette population européenne d'âge moyen, avec une prévalence basse d'obésité et une prise de poids modérée durant le suivi, nous n'avons pas trouvé de risque significativement plus élevé de développer un diabète en arrêtant de fumer, et ce pour les deux sexes. L'arrêt du tabac doit donc être encouragé chez toutes les fumeuses et tous les fumeurs.

Cooperation in R&D, firm size and type of partnership: Evidence for the Spanish automotive industry

This paper aims to analyse cooperation in R&D in the automobile industry in Spain. It first examines to what extent firms cooperate with external actors in the field of technological innovation, and if so, with what type of cooperation partner, paying special attention to the differentiation according to the size of the firms. Second, it aims to study how the firm’s size may affect not only the decision of cooperating but also with which type of partner, while controlling for other determinants that have been considered in the literature as main drivers of collaborative activities in R&D. We use data provided by the Technological Innovation Panel in the 2006-2008 period for firms in the automotive sector. We estimate a bivariate probit model that takes into account the two types of cooperation mostly present in the automotive industry, vertical and institutional, explicitly considering the interdependencies that may arise in the simultaneous choice of both.

Lifecourse socioeconomic status and type 2 diabetes : the role of chronic inflammation in the English Longitudinal Study of Ageing.

We examined the association between lifecourse socioeconomic status (SES) and the risk of type 2 diabetes at older ages, ascertaining the extent to which adult lifestyle factors and systemic inflammation explain this relationship. Data were drawn from the English Longitudinal Study of Ageing (ELSA) which, established in 2002, is a representative cohort study of ≥50-year olds individuals living in England. SES indicators were paternal social class, participants' education, participants' wealth, and a lifecourse socioeconomic index. Inflammatory markers (C-reactive protein and fibrinogen) and lifestyle factors were measured repeatedly; diabetes incidence (new cases) was monitored over 7.5 years of follow-up. Of the 6218 individuals free from diabetes at baseline (44% women, mean aged 66 years), 423 developed diabetes during follow-up. Relative to the most advantaged people, those in the lowest lifecourse SES group experienced more than double the risk of diabetes (hazard ratio 2.59; 95% Confidence Interval (CI) = 1.81-3.71). Lifestyle factors explained 52% (95%CI:30-85) and inflammatory markers 22% (95%CI:13-37) of this gradient. Similar results were apparent with the separate SES indicators. In a general population sample, socioeconomic inequalities in the risk of type 2 diabetes extend to older ages and appear to partially originate from socioeconomic variations in modifiable factors which include lifestyle and inflammation.

Cooperation in R&D, firm size and type of partnership: Evidence for the Spanish automotive industry

This paper aims to analyse cooperation in R&D in the automobile industry in Spain. It first examines to what extent firms cooperate with external actors in the field of technological innovation, and if so, with what type of cooperation partner, paying special attention to the differentiation according to the size of the firms. Second, it aims to study how the firm’s size may affect not only the decision of cooperating but also with which type of partner, while controlling for other determinants that have been considered in the literature as main drivers of collaborative activities in R&D. We use data provided by the Technological Innovation Panel in the 2006-2008 period for firms in the automotive sector. We estimate a bivariate probit model that takes into account the two types of cooperation mostly present in the automotive industry, vertical and institutional, explicitly considering the interdependencies that may arise in the simultaneous choice of both.

Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Microfinance and credit rationing in Ghana: Does the microfinance type matter?

This study sets out to examine the extent to which access to credit and credit rationing are influenced by the microfinance type based on the major factors determining micro, small and medium enterprises’ access to credit from microfinance institutions in the era of financial liberalization. The data for the study were gleaned from the microfinance institutions’ credit and loan records consisting of the various pieces of information provided by the borrowers in the application process. Our results are puzzling and show that credit rationing is not influenced by the microfinance types but by the individual microfinance institutions. Keywords: Microfinance, Ghana, Credit Rationing. JEL codes: G21