G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Profile of micronucleus frequencies and nuclear abnormalities in different species of electric fishes (Gymnotiformes) from the Eastern Amazon

The frequency of spontaneous micronucleus (MN) formation in fish species needs to be determined to evaluate their usefulness for genotoxic biomonitoring. The definition of a good bioindicator takes into account the current knowledge of its metabolic traits as well as other factors including its feeding behavior and relationship to the environment. In this study, we compared the basal frequencies of micronucleated erythrocytes and nuclear abnormalities (NA) among different species of the fish Order Gymnotiformes (Rhamphichthys marmoratus, Steatogenys elegans, Sternopygus macrurus, Parapteronotus hasemani, Gymnotus mamiraua, Gymnotus arapaima, Brachyhypopomus beebei, Brachyhypopomus n. sp. BENN) sampled in several localities of the Eastern Amazon. A baseline of MN and NA frequency in these fish was determined, enabling the identification of potentially useful species as models for genotoxicity studies. Only one impacted sample collected at a site in the River Caripetuba showed a significant number of NAs, which may be due to the release of wastewater by neighbouring mining industries and by the burnt fuel released by the small boats used by a local community. Our results may provide support for further studies in areas of the Eastern Amazon affected by mining, deforestation and other anthropogenic activities.     

Angiogenic factors in women ten years after severe very early onset preeclampsia

Background

Women with a history of mainly severe and early onset preeclampsia have an increased risk of future cardiovascular disease. During these complicated pregnancies increased levels of anti-angiogenic factors can be found. We hypothesize that women with a history of severe very early onset preeclampsia still have increased levels of these biomarkers years after this pregnancy, resulting in increased risk for cardiovascular disease.

Methods

Twenty women with severe early onset preeclampsia before 24 weeks'' gestation, who delivered between 1993–2003 in a tertiary referral centre and twenty matched controls with uncomplicated pregnancies and healthy term infants, were addressed for participation in the study. Venous plasma samples were analyzed for basic fibroblast growth factor (bFGF), placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF), E- and P-selectin, soluble intercellular adhesion molecule-3 (sICAM-3) and thrombomodulin by ELISA.

Results

Sixteen case subjects and 18 control subjects consented participation. The median time interval index pregnancy to study was 9.4 and 9.7 years for cases and controls, respectively. Median levels for cases-controls (p-value) were not different; bFGF: 17.43–11.11 pg/mL (0.33), sFlt-1: 102.98–101.92 pg/ml (0.84), PLGF: 3.57–4.20 pg/mL (0.38), VEGF: 64.05–45.72 pg/mL (0.73), E-selectin: 5.11–4.68 ng/mL (0.20), P-selectin: 85.35–71.69 ng/mL (0.69), sICAM-3: 0.42–0.63 ng/mL (0.41) and Thrombomodulin: 0.92–0.93 ng/mL (0.59).

Conclusion

There were no differences in angiogenic biomarkers between women with a history of severe early onset preeclampsia versus uncomplicated pregnancy almost 10 years later, suggesting that these angiogenic factors will not contribute to the early detection of women at risk for future cardiovascular disease.     

Effects of age and physical performance capacity on distribution and composition of high-density lipoprotein subfractions in men

The influences of age and maximal aerobic capacity (VO2max) on serum lipoproteins with special regard to the concentration, composition and distribution of high density lipoprotein (HDL) subfractions were investigated in 51 healthy males of different characteristics: younger than 35 years, untrained (n = 14, mean age 28.2 years, SD 6.0; VO2max, 47.9 ml.kg-1.min-1, SD 5.8) and trained (n = 11, mean age 27.9 years, SD 4.3; VO2max, 61.1 ml.kg-1.min-1, SD 5.1), older than 50 years untrained (n = 14, mean age 58.9 years, SD 5.9, VO2max, 29.3 ml.kg-1.min-1, SD 5.3) and trained (n = 12, mean age 59.3 years, SD 7.2, VO2max, 45.7 ml.kg-1.min-1, SD 7.7). The fasting-state serum concentrations of total cholesterol, tri-acylglycerol and lipoprotein-cholesterol were measured. The HDL-subfractions were separated by density (rho) gradient ultracentrifugation. Concentrations of cholesterol, cholesterylester, tri-acylglycerol, phospholipids, apolipoprotein (apo) A-I and A-II were measured in the subfractions HDL2b: rho = 1.063-1.100 g.ml-1; HDL2al: rho = 1.00-1.110 g.ml-1; HDL2a2: rho = 1.110-1.150 g.ml-1; HDL3: rho = 1.150-1.210 g.ml-1. Elderly untrained subjects showed increased serum concentrations of total-, very low- and low density lipoprotein-cholesterol and elevated tri-acylglycerol levels. The HDL-cholesterol concentration was decreased, due to reduced concentrations of HDL2-subfractions. Significant changes in the composition of HDL2-subfractions were found in elderly untrained subjects. The HDL2-subfractions had more protein, a decreased apoA-I:A-II ratio and less phospholipids in comparison to HDL2-subfractions from younger untrained and trained, and elderly trained subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro and Sensory Evaluation of Capsaicin-Loaded Nanoformulations

Capsaicin has known health beneficial and therapeutic properties. It is also able to enhance the permeability of drugs across epithelial tissues. Unfortunately, due to its pungency the oral administration of capsaicin is limited. To this end, we assessed the effect of nanoencapsulation of capsaicin, under the hypothesis that this would reduce its pungency. Core-shell nanocapsules with an oily core and stabilized with phospholipids were used. This system was used with or without chitosan coating. In this work, we investigated the in vitro release behavior of capsaicin-loaded formulations in different physiological media (including simulated saliva fluid). We also evaluated the influence of encapsulation of capsaicin on the cell viability of buccal cells (TR146). To study the changes in pungency after encapsulation we carried out a sensory analysis with a trained panel of 24 students. The in vitro release study showed that the systems discharged capsaicin slowly in a monotonic manner and that the chitosan coating had an effect on the release profile. The cytotoxic response of TR146 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, was reduced following its encapsulation. The sensory study revealed that a chitosan coating results in a lower threshold of perception of the formulation. The nanoencapsulation of capsaicin resulted in attenuation of the sensation of pungency significantly. However, the presence of a chitosan shell around the nanoformulations did not mask the pungency, when compared with uncoated systems.     

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA⁻ cell lines are shown.     

A simple rapid immunoassay for S-adenosylhomocysteine in plasma

The measurement of plasma S-adenosylhomocysteine is a more sensitive indicator of the risk for vascular disease than is plasma homocysteine. Because the level of S-adenosylhomocysteine is normally in the nanomolar range, it has been difficult to measure and necessitated the development of complex fluorometric and mass-spectrophotometric methods. We have now adapted an existing immunoassay used for the measurement of homocysteine to the measurement of S-adenosylhomocysteine in plasma. This assay is sensitive down to the level of less than 0.1 pmol, and there is no interference by S-adenosylmethionine. The assay is carried out in microplates, allows the measurement of 12 samples per plate and can easily be carried out in a 4-h period. The method is applicable to plasma samples having S-adenosylhomocysteine concentrations ranging from 10 to 150 nM without dilution. The mean value for 16 normal subjects by this method was 18.9±1.4 nM (S.E.M.), compared with 17.8±1.4 nM obtained by a previously described method using two high-performance liquid chromatography columns with fluorescence derivatization. Mean values for seven cirrhotic patients were 46.5±3.3 nM by this new method compared with 44.6±5.3 by the former method. The ease and speed of this method should allow the widespread measurement of this important metabolite in laboratories without access to sophisticated equipment.     

Diarrhea,Stimulation and Growth Predict Neurodevelopment in Young North Indian Children

Background and Objective

Infants and young children in low to middle-income countries are at risk for adverse neurodevelopment due to multiple risk factors. In this study, we sought to identify stimulation and learning opportunities, growth, and burden of respiratory infections and diarrhea as predictors for neurodevelopment.

Methods

We visited 422 North Indian children 6 to 30 months old weekly for six months. Childhood illnesses were assessed biweekly. At end study, we assessed neurodevelopment using the Ages and Stages Questionnaire 3^rd ed. (ASQ-3) and gathered information on stimulation and learning opportunities. We identified predictors for ASQ-3 scores in multiple linear and logistic regression models.

Results

We were able to explain 30.5% of the variation in the total ASQ-3 score by the identified predictors. When adjusting for child characteristics and annual family income, stimulation and learning opportunities explained most of the variation by 25.1%. Height for age (standardized beta: 0.12, p<.05) and weight for height z-scores (std. beta: 0.09, p<.05) were positively associated with the total ASQ-3 score, while number of days with diarrhea was negatively associated with these scores (std. beta: -0.13, p<0.01).

Conclusion

Our results support the importance of early child stimulation and general nutrition for child development. Our study also suggests that diarrhea is an additional risk factor for adverse neurodevelopment in vulnerable children.