Meiosis resumption, calcium-sensitive period, and PLC-beta1 relocation into the nucleus in the mouse oocyte

We aimed to determine whether the breakdown of the germinal vesicle of the mouse oocyte and the nuclear import of phospholipase C-beta1 were calcium-dependent. We chelated Ca2+ ions with BAPTA-dextran at different times after the release of the oocyte from the ovarian follicle, i.e. after meiosis resumption has started, and we studied the effects on the kinetics of germinal vesicle breakdown, and on the migration of phospholipase C-beta1. We discriminate between two key-periods of calcium-sensitivity during the process of meiosis resumption. During the first hour, changes in the cytosolic Ca2+ especially promoted the migration of phospholipase C-beta1 into the nucleus, whereas changes in the nuclear concentration of Ca2+ were not implicated. Moreover, at this time, the cytosolic calcium pathway is PLC-beta1-dependent. By contrast, during the second hour following the onset of meiosis resumption, and thus just previous GVBD, the PLC-beta1-dependent Ca2+ signals in both cellular compartments were equally necessary for the resumption of meiosis. This particular period of the meiotic process corresponds to the moment when the phospholipase C-beta1 has strongly migrated into the nucleus. Our results highlight also the role played by the nucleus during the second key-period in the control of the GVBD via a Ca2+-dependent pathway.     

Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes

Insulin receptor substrate (IRS) has been suggested as a molecular target of free fatty acids (FFAs) for insulin resistance. However, the signaling pathways by which FFAs lead to the inhibition of IRS function remain to be established. In this study, we explored the FFA-signaling pathway that contributes to serine phosphorylation and degradation of IRS-1 in adipocytes and in dietary obese mice. Linoleic acid, an FFA used in this study, resulted in a reduction in insulin-induced glucose uptake in 3T3-L1 adipocytes. This mimics insulin resistance induced by high-fat diet in C57BL/6J mice. The reduction in glucose uptake is associated with a decrease in IRS-1, but not IRS-2 or GLUT4 protein abundance. Decrease in IRS-1 protein was proceeded by IRS-1 (serine 307) phosphorylation that was catalyzed by serine kinases inhibitor kappaB kinase (IKK) and c-JUN NH2-terminal kinase (JNK). IKK and JNK were activated by linoleic acid and inhibition of the two kinases led to prevention of IRS-1 reduction. We demonstrate that protein kinase C (PKC) theta is expressed in adipocytes. In 3T3-L1 adipocytes and fat tissue, PKCtheta was activated by fatty acids as indicated by its phosphorylation status, and by its protein level, respectively. Activation of PKCtheta contributes to IKK and JNK activation as inhibition of PKCtheta by calphostin C blocked activation of the latter kinases. Inhibition of either PKCtheta or IKK plus JNK by chemical inhibitors resulted in protection of IRS-1 function and insulin sensitivity in 3T3-L1 adipocytes. These data suggest that: 1) activation of PKCtheta contributes to IKK and JNK activation by FFAs; 2) IKK and JNK mediate PKCtheta signals for IRS-1 serine phosphorylation and degradation; and 3) this molecular mechanism may be responsible for insulin resistance associated with hyperlipidemia.     

Familial deafness,congenital heart defects,and posterior embryotoxon caused by cysteine substitution in the first epidermal-growth-factor-like domain of jagged 1

In the present study, we report a kindred with hearing loss, congenital heart defects, and posterior embryotoxon, segregating as autosomal dominant traits. Six of seven available affected patients manifested mild-to-severe combined hearing loss, predominantly affecting middle frequencies. Two patients were diagnosed with vestibular pathology. All patients had congenital heart defects, including tetralogy of Fallot, ventricular septal defect, or isolated peripheral pulmonic stenosis. No individual in this family met diagnostic criteria for any previously described clinical syndrome. A candidate-gene approach was undertaken and culminated in the identification of a novel Jagged 1 (JAG1) missense mutation (C234Y) in the first cysteine of the first epidermal-growth-factor-like repeat domain of the protein. JAG1 is a cell-surface ligand in the Notch signaling pathway. Mutations in JAG1 have been identified in patients with Alagille syndrome. Our findings revealed a unique phenotype with highly penetrant deafness, posterior embryotoxon, and congenital heart defects but with variable expressivity in a large kindred, which demonstrates that mutation in JAG1 can cause hearing loss.     

Differential response to low-fat diet between low and normal HDL-cholesterol subjects

Heart attacks frequently occur in normolipidemic subjects with low concentration of high density lipoproteins (35 mg/dL). We hypothesized that as subjects with low HDL-C already have low HDL concentrations, the major decrease of HDL-C will occur in subjects with normal HDL-C when a low-fat diet is consumed. Normolipidemic male subjects consumed three diets differing in total fat and saturated fat composition (AAD: 37%, Step-1: 28%, Step-2: 24% total fat) for 6 weeks in a three-period double-blind randomized crossover design. Plasma lipids and apolipoproteins were determined and changes in distribution of HDL subpopulations were evaluated. As a result of a low-fat diet, low HDL-C individuals slightly decreased their HDL-C, but substantially decreased their LDL-C resulting in a significant improvement in the LDL-C/HDL-C ratio. However, subjects with normal HDL-C levels decreased both their LDL-C and HDL-C resulting in an unchanged LDL-C/HDL-C ratio. We also observed significant differences in response to low-fat diets in HDL-C and alpha(1) concentrations between low and normal HDL-C subjects. In the normal HDL-C group, consumption of a low-fat diet also resulted in redistribution of apoA-I-containing HDL subpopulations, indicated by a decrease in the large apoA-I-only alpha(1) subpopulation. These data demonstrate that male subjects with low HDL-C respond to a low-fat diet differently than individuals with normal HDL-C.     

Experimental study of umbilical cord length as a marker of fetal alcohol syndrome

Umbilical cord length has long been investigated as a potential marker of intrauterine events that may place the neonate at risk for future adverse developmental sequelae. Experimentally, significantly shortened cords have been reported in association with prenatal exposure to common drugs of abuse. This study in rats reports the time course of effects on umbilical cord length of a daily maternal ethanol gavage (3,200 mg/kg) from gestational day 6 through termination of pregnancy at either day 17, 18, 19, or 20. A total of 786 fetuses derived from 60 litters were examined. Control fetuses demonstrated a linear increase in umbilical cord length and body weight gain during late gestation, findings that support previous studies. The body weights of the ethanol-exposed fetuses were reduced significantly on all gestational days examined, indicating intrauterine growth retardation, a characteristic of fetal alcohol syndrome. Similarly, acute fetal akinesia as well as long-term sequelae stemming from impaired neurological development would result from the elevated blood ethanol levels achieved in this study. The umbilical cords of ethanol-exposed fetuses were significantly shorter on gestational days 19 and 20 in comparison to their controls, while cord lengths on days 17 and 18 were not shortened significantly. A stretch hypothesis has been proposed suggesting that the degree of fetal activity is the main determinant of umbilical cord length. In rats, there is a physiologic diminution of the volume of amniotic fluid (oligohydramnios) in late gestation (day 19 to term), which restricts fetal movements but does not appear to alter the linear relationships between gestational age and cord length in controls, thus arguing against the stretch hypothesis. However, cord lengths in the ethanol-exposed fetuses plateaued in late gestation, suggesting possible adherence to a stretch hypothesis. This dichotomy is discussed emphasizing fetal growth and activity as well as intrauterine space.     

Sequence correlations between Cro recognition helices and cognate O(R) consensus half-sites suggest conserved rules of protein-DNA recognition

The O(R) regions from several lambdoid bacteriophages contain the three regulatory sites O(R)1, O(R)2 and O(R)3, to which the Cro and CI proteins can bind. These sites show imperfect dyad symmetry, have similar sequences, and generally lie on the same face of the DNA double helix. We have developed a computational method, which analyzes the O(R) regions of additional phages and predicts the location of these three sites. After tuning the method to predict known O(R) sites accurately, we used it to predict unknown sites, and ultimately compiled a database of 32 known and predicted O(R) binding site sets. We then identified sequences of the recognition helices (RH) for the cognate Cro proteins through manual inspection of multiple sequence alignments. Comparison of Cro RH and consensus O(R) half-site sequences revealed strong one-to-one correlations between two amino acids at each of three RH positions and two bases at each of three half-site positions (H1-->2, H3-->5 and H6-->6). In each of these three cases, one of the two amino acid/base-pairings corresponds to a contact observed in the crystal structure of a lambda Cro/consensus operator complex. The alternate amino acid/base combinations were rationalized using structural models. We suggest that the pairs of amino acid residues act as binary switches that efficiently modulate specificity for different consensus half-site variants during evolution. The observation of structurally reasonable amino acid-to-base correlations suggests that Cro proteins share some common rules of recognition despite their functional and structural diversity.     

Effects of polyunsaturated fatty acids and clofibrate on chicken stearoyl-coA desaturase 1 gene expression

In chicken, adiposity is influenced by hepatic stearoyl-CoA desaturase (SCD) 1. This gene is up-regulated by low-fat high-carbohydrate diet and down-regulated by addition of polyunsaturated fatty acids (PUFA). In this study, we present evidence for an inhibition of chicken SCD1 expression by PUFA using reporter gene constructs in transient transfection assays. This inhibition does not involve the peroxisome proliferator-activated receptor pathway, in contrast with what has been observed in rodents. We were able to localise a PUFA as well as an insulin response element within the -372/+125 bp region of the promoter. Sequence analyses of this region allowed identification of several cis-regulatory elements: A sterol regulatory element (SRE) and a juxtaposed NF-Y element which have been shown to be involved in the regulation of mouse SCD genes by PUFA. In addition, we identified an overlapping Sp1/USF motif, which was described to play a role in insulin/glucose and PUFA regulation of fatty synthase, ATP-citrate-lyase, and leptin genes. These data provide the first characterisation of the chicken SCD1 promoter and putative cis-sequences involved in the regulation of this gene by PUFA and insulin.     

Physical Fitness and Physical Activity in Obese and Nonobese Flemish Youth

Objective: To assess different aspects of physical fitness and physical activity in obese and nonobese Flemish youth. Research Methods and Procedures: A random sample of 3214 Flemish schoolchildren was selected and divided into an “obese” and “nonobese” group based on body mass index and sum of skinfolds. Physical fitness was assessed by the European physical fitness test battery. Physical activity was estimated by a modified version of the Baecke Questionnaire. Results: Obese subjects had inferior performances on all tests requiring propulsion or lifting of the body mass (standing‐broad jump, sit‐ups, bent‐arm hang, speed shuttle run, and endurance shuttle run) compared with their nonobese counterparts (p < 0.001). In contrast, the obese subjects showed greater strength on handgrip (p < 0.001). Both groups had similar levels of leisure‐time physical activity; however, nonobese boys had a higher sport index than their obese counterparts (p < 0.05). Discussion: Results of this study show that obese subjects had poorer performances on weight‐bearing tasks, but did not have lower scores on all fitness components. To encourage adherence to physical activity in obese youth, it is important that activities are tailored to their capabilities. Results suggest that weight‐bearing activities should be limited at the start of an intervention with obese participants and alternative activities that rely more on static strength used.