The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

Nestor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation

Background: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Nestor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure.

Results: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope.

Conclusions: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

ADP can induce aggregation of human platelets via both P2Y(1) and P-2T receptors

1 In the present study we have investigated the roles of P2Y(1) and P-2T receptor subtypes in adenosine 5'-diphosphate (ADP)-induced aggregation of human platelets in heparinized platelet rich plasma.

Neuro developmental status at 1 year predicts neuropsychiatric outcome at 14-15 years of age in very preterm infants

Background: Neurodevelopmental and behavioural problems have been repeatedly reported in very preterm. survivors, often showing themselves later in childhood as poor school performance. Early identification of problems would mean that appropriate remedial therapy can be implemented. We have previously shown that neurodevelopmental status at 1 year was predictive of outcome at 8 years in a cohort of preterm. infants. The aim of this paper was to see if neurodevelopmental outcome in adolescence could be predicted by assessment by 1 year in the same cohort of pretem infants. Study design: Prospective cohort study. Subjects: 150 adolescents, born before 33 weeks gestation. Outcome measures: Neurological examination, developmental quotient, vision and hearing by 1 year. At 14-15 years, neurological examination, school performance questionnaire, Schonnell test of reading age, a premorbid adjustment score, Rutter behavioural score and for those born from 1981, cognitive tests (WISC-R). Results: A highly significant relationship existed between neurological status by 1 year and the need for extra educational provision, overall neurodevelopmental status, cognitive function in those that had their IQs measured and premorbid adjustment score of prepsychotic symptoms in adolescence. However, status at 1 year was not predictive of adolescent reading age or behavioural score. Conclusions: Neurodevelopmental assessment at 1 year ispredictive of school performance and outcome in the adolescent period. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Influences of phosphorus starvation on OsACR2.1 expression and arsenic metabolism in rice seedlings

The effects of phosphorus (P) status on arsenate reductase gene (OsACR2.1) expression, arsenate reductase activity, hydrogen peroxide (H(2)O(2)) content, and arsenic (As) species in rice seedlings which were exposed to arsenate after -P or +P pretreatments were investigated in a series of hydroponic experiments. OsACR2.1 expression increased significantly with decreasing internal P concentrations; more than 2-fold and 10-fold increases were found after P starvation for 30 h and 14 days, respectively. OsACR2.1 expression exhibited a significant positive correlation with internal root H(2)O(2) accumulation, which increased upon P starvation or exposure to H(2)O(2) without P starvation. Characterization of internal and effluxed As species showed the predominant form of As was arsenate in P-starved rice root, which contrasted with the +P pretreated plants. Additionally, more As was effluxed from P-starved rice roots than from non-starved roots. In summary, an interesting relationship was observed between P-starvation induced H(2)O(2) and OsACR2.1 gene expression. However, the up-regulation of OsACR2.1 did not increase arsenate reduction in P-starved rice seedlings when exposed to arsenate.

1-D constitutive model for evolution of stress-induced R-phase and localized Lüders-like stress-induced martensitic transformation of super-elastic NiTi wires

NiTi alloys have been widely used in the applications for micro-electro-mechanical-systems (MEMS), which often involve some precise and complex motion control. However, when using the NiTi alloys in MEMS application, the main problem to be considered is the degradation of functional property during cycling loading. This also stresses the importance of accurate prediction of the functional behavior of NiTi alloys. In the last two decades, a large number of constitutive models have been proposed to achieve the task. A portion of them focused on the deformation behavior of NiTi alloys under cyclic loading, which is a practical and non-negligible situation. Despite of the scale of modeling studies of the field in NiTi alloys, two experimental observations under uniaxial tension loading have not received proper attentions. First, a deviation from linearity well before the stress-induced martensitic transformation (SIMT) has not been modeled. Recent experiments confirmed that it is caused by the formation of stress-induced R phase. Second, the influence of the well-known localized Lüders-like SIMT on the macroscopic behavior of NiTi alloys, in particular the residual strain during cyclic loading, has not been addressed. In response, we develop a 1-D phenomenological constitutive model for NiTi alloys with two novel features: the formation of stress-induced R phase and the explicit modeling of the localized Lüders-like SIMT. The derived constitutive relations are simple and at the same time sufficient to describe the behavior of NiTi alloys. The accumulation of residual strain caused by R phase under different loading schemes is accurately described by the proposed model. Also, the residual strain caused by irreversible SIMT at different maximum loading strain under cyclic tension loading in individual samples can be explained by and fitted into a single equation in the proposed model. These results show that the proposed model successfully captures the behavior of R phase and the essence of localized SIMT.

Structural and magnetic properties of Ni1-xZnxFe2O4 (x=0, 0.5 and 1) nanopowders prepared by sol-gel method

A series of nanostructured Ni-Zn ferrites Ni1-xZnxFe2O4 (x=0, 0.5 and 1) with a grain size from 24 to 65 nm have been prepared with a sol-gel method. The effect of composition and sintering temperature on morphology, magnetic properties, Curie temperature, specific heating rate at 295 kHz and hysteresis loss have been studied. The highest coercivity of 50 and 40 Oe, were obtained for NiFe2O4 and Ni0.5Zn0.5Fe2O4 samples with the grain size of 35 and 29 nm, respectively. The coercivity of Ni and Ni-Zn mixed ferrites decreased with temperature. The Bloch exponent was 1.5 for all samples. As the grain size increased, the Curie temperature of NiFe2O4 increased from 849 to 859 K. The highest saturation magnetization of 70 emu/g at 298 K and the highest specific heating rate of 1.6 K/s under radiofrequency heating at 295 kHz were observed over NiFe2O4 calcined at 1073 K. Both the magnitude of the hysteresis loss and the temperature dependence of the loss are influenced by the sintering temperature and composition.

Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment

Background We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor alpha (TNF-alpha) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD).

Design Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab.

Results Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-gamma-secreting Th1 cells and IFN-alpha-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab.

Conclusions New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL23 antibody therapy is a highly effective therapy for these lesions.

Distinct methylation patterns in genes that affect mitochondrial function are associated with kidney disease in blood-derived DNA from individuals with Type 1 diabetes

AIMS: Epigenetic modifications, such as DNA methylation, can influence the risk of developing kidney disease. We studied methylation profiles in genes related to mitochondrial function to assess whether differences in these epigenetic features were associated with diabetic kidney disease in people with Type 1 diabetes.

METHODS: A case-control association study was undertaken (n = 196 individuals with diabetic kidney disease vs. n = 246 individuals without renal disease). Participants were White and diagnosed with Type 1 diabetes before 31 years of age. Genes that encode mitochondrial proteins (n = 780) were downloaded from mitoproteome. org. DNA methylation profiles from blood-derived DNA were generated using the Illumina Infinium HumanMethylation450 (262 samples) and Illumina Infinium HumanMethylation27 (192 samples) arrays. Beta values (β) were calculated and quality control was conducted, including evaluating blind duplicate DNA samples.

RESULTS: Fifty-four Cytosine-phosphate-Guanine probes across 51 unique genes were significantly associated (P ≤ 10(-8) ) with diabetic kidney disease across both the 450K and the 27K methylation arrays. A subanalysis, employing the 450K array, identified 755 Cytosine-phosphate-Guanine probes in 374 genes that were significantly associated (P ≤ 10(-8) ) with end-stage renal disease. Forty-six of the top-ranked variants for diabetic kidney disease were also identified as being differentially methylated in individuals with end-stage renal disease. The largest change in methylation (Δβ = 0.2) was observed for cg03169527 in the TAMM41 gene, chromosome 3p25.2. Three genes, PMPCB, TSFM and AUH, were observed with differential methylation at multiple Cytosine-phosphate-Guanine sites each (P < 10(-12) ).

CONCLUSIONS: Differential methylation in genes that influence mitochondrial function are associated with kidney disease in individuals with Type 1 diabetes. 

Seasonal variation in month of diagnosis in children with type 1 diabetes registered in 23 European centers during 1989-2008: little short-term influence of sunshine hours or average temperature

BACKGROUND: The month of diagnosis in childhood type 1 diabetes shows seasonal variation.

OBJECTIVE: We describe the pattern and investigate if year-to-year irregularities are associated with meteorological factors using data from 50 000 children diagnosed under the age of 15 yr in 23 population-based European registries during 1989-2008.

METHODS: Tests for seasonal variation in monthly counts aggregated over the 20 yr period were performed. Time series regression was used to investigate if sunshine hour and average temperature data were predictive of the 240 monthly diagnosis counts after taking account of seasonality and long term trends.

RESULTS: Significant sinusoidal pattern was evident in all but two small centers with peaks in November to February and relative amplitudes ranging from ±11 to ±38% (median ±17%). However, most centers showed significant departures from a sinusoidal pattern. Pooling results over centers, there was significant seasonal variation in each age-group at diagnosis, with least seasonal variation in those under 5 yr. Boys showed greater seasonal variation than girls, particularly those aged 10-14 yr. There were no differences in seasonal pattern between four 5-yr sub-periods. Departures from the sinusoidal trend in monthly diagnoses in the period were significantly associated with deviations from the norm in average temperature (0.8% reduction in diagnoses per 1 °C excess) but not with sunshine hours.

CONCLUSIONS: Seasonality was consistently apparent throughout the period in all age-groups and both sexes, but girls and the under 5 s showed less marked variation. Neither sunshine hour nor average temperature data contributed in any substantial way to explaining departures from the sinusoidal pattern.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

Data on carotid intima-media thickness and lipoprotein subclasses in type 1 diabetes from the Diabetes Control and Complications Trial and the Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC)

Type 1 diabetes (T1DM) is associated with increased risk of macrovascular complications. We examined longitudinal associations of serum conventional lipids and nuclear magnetic resonance (NMR)-determined lipoprotein subclasses with carotid intima-media thickness (IMT) in adults with T1DM (n=455) enrolled in the Diabetes Control and Complications Trial (DCCT). Data on serum lipids and lipoproteins were collected at DCCT baseline (1983-89) and were correlated with common and internal carotid IMT determined by ultrasonography during the observational follow-up of the DCCT, the Epidemiology of Diabetes Interventions and Complications (EDIC) study, at EDIC 'Year 1' (199-1996) and EDIC 'Year 6' (1998-2000). This article contains data on the associations of DCCT baseline lipoprotein profiles (NMR-based VLDL & chylomicrons, IDL/LDL and HDL subclasses and 'conventional' total, LDL-, HDL-, non-HDL-cholesterol and triglycerides) with carotid IMT at EDIC Years 1 and 6, stratified by gender. The data are supplemental to our original research article describing detailed associations of DCCT baseline lipids and lipoprotein profiles with EDIC Year 12 carotid IMT (Basu et al. in press) [1].