Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5'-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N(6)-(1-methylbutyl)-ATP and genetically engineered CaMKKα mutant, CaMKKα (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKKα (Phe(230)Gly), but not with wild-type kinase, in the presence of N(6)-(1-methylbutyl)-ATP and Ca(2+)/CaM, induced significant threonine phosphorylation of a 50kDa protein as well as CaMKI phosphorylation at Thr(177). The 50kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKKα and β isoforms at Thr(355)in vitro. Phosphorylation of HA-Syndapin I at Thr(355) in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca(2+)-signaling pathways that are independent of downstream protein kinases.

Previously, we demonstrated increased Angiotensin II type I receptor expression in leukocytes from patients with untreated, but not in treated, essential hypertension (essential hypertension). We hypothesized that the Angiotensin II AT1 receptor is also increased in leukocytes from patients with chronic kidney disease, however and can still be corrected with combined anti-hypertensive treatment with renin-angiotensin system (RAS) blockers and statins. Blood pressure, cholesterol, renal function oxidative stress parameters, inflammation, and leukocyte Angiotensin II AT1 receptor mRNA expression were measured both on and (6 weeks) off treatment. Data were compared to data of 10 healthy control subjects. Untreated chronic kidney disease patients (n=20) had higher blood pressure, cholesterol and leukocyte Angiotensin II AT1 receptor mRNA expression, but no different ox-LDL, thiobarbituric acid reactive substances, paraoxonase activity or hs-CRP. OxLDL and Lipoprotein(a) were increased in untreated chronic kidney disease. Angiotensin II AT1 receptor expression inversely correlated with renal function (R(2)=0.15, P<0.03) and Lipoprotein(a) but not with the other parameters. Treatment with RAS blockers and statins normalized blood pressure and cholesterol, however it did not correct enhanced leukocyte Angiotensin II AT1 receptor expression. Leukocyte Angiotensin II AT1 receptor expression is inappropriately high in chronic kidney disease, correlates inversely with renal function and does not depend on antihypertensive and lipid-lowering treatment. The uremic environment seems to dominate over previously reported actions of high blood pressure and cholesterol to enhance leukocyte Angiotensin II AT1 receptor expression.

Children with large optic nerve head cups often pose diagnostic difficulty due to concern over possible glaucoma. This study's purpose was to evaluate optical coherence tomography (OCT) measurement of the peripapillary retinal nerve fiber layer (RNFL) and macular thickness in healthy eyes of black and white children, comparing values for eyes with large cup-to-disc ratios against those with small cup-to-disc ratios (normal controls). Using Stratus OCT (OCT 3) (Carl Zeiss Meditec, Dublin, CA) a fast macular thickness and fast RNFL 3.4 protocol were performed on normal eyes of normal children. Included were children (aged 5-17 years) with normal ophthalmic examinations, drawn from data generated during an ongoing study of OCT in children. Excluded were eyes with corrected vision below 20/20, spherical equivalent >5 diopters, intraocular pressure >21 mm Hg, abnormal optic nerve rim, prematurity, family history of glaucoma. Clinical examination identified children with large cup-disc-ratios (0.5-0.8), and each was matched for age and race to a "normal control" with a small cup-to-disc ratio (<0.5). OCT-3 analyses were compared between eyes with optic nerve heads showing large cup-to-disc ratios (≥0.5) vs. those showing small cup-to-disc ratios (<0.5) for different sections of the macula and the peripapillary RNFL thickness. Analyses included OCT values and optic cup grading for 162 eyes of 162 children. White children with large cup-to-disc ratios had thinner mean RNFL thickness and macular volume compared to those with small cup-to-disc ratios (average RNFL = 100 ± 9 vs. 110 ± 8 μm, respectively, p = 0.0001, macular volume = 6.84 ± 0.41 vs. 7.03 ± 0.33 mm(3), respectively, p = 0.0186). On the other hand, black children with large cup-to-disc ratios had an equivalent mean RNFL thickness and macular volume compared to those with small cup-to-disc ratios average RNFL = 107 ± 10 vs. 107 ± 8 for both groups, macular volume = 6.81 ± 0.34 vs. 6.85 ± 0.28 respectively. Positive equivalence testing assumed a clinically significant difference of 10 μm for RNFL, and 0.2 mm(3) for macular volume. We report that white children with large cup-to-disc ratios have evidence of reduced/thinner RNFL and macular volume versus white children with smaller cup-to-disc ratios, as assessed by OCT. By contrast, this distinction was not found for black children in the present study.

High myopia, which is extremely prevalent in the Chinese population, is one of the leading causes of blindness in the world. Genetic factors play a critical role in the development of the condition. To identify the genetic variants associated with high myopia in the Han Chinese, we conducted a genome-wide association study (GWAS) of 493,947 SNPs in 1088 individuals (419 cases and 669 controls) from a Han Chinese cohort and followed up on signals that were associated with p < 1.0 × 10(-4) in three independent cohorts (combined, 2803 cases and 5642 controls). We identified a significant association between high myopia and a variant at 13q12.12 (rs9318086, combined p = 1.91 × 10(-16), heterozygous odds ratio = 1.32, and homozygous odds ratio = 1.64). Furthermore, five additional SNPs (rs9510902, rs3794338, rs1886970, rs7325450, and rs7331047) in the same linkage disequilibrium (LD) block with rs9318086 also proved to be significantly associated with high myopia in the Han Chinese population; p values ranged from 5.46 × 10(-11) to 6.16 × 10(-16). This associated locus contains three genes-MIPEP, C1QTNF9B-AS1, and C1QTNF9B. MIPEP and C1QTNF9B were found to be expressed in the retina and retinal pigment epithelium (RPE) and are more likely than C1QTNF9B-AS1 to be associated with high myopia given the evidence of retinal signaling that controls eye growth. Our results suggest that the variants at 13q12.12 are associated with high myopia.

Disodium 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (8) has been synthesized using cholesterol (1) as starting material. Sulfation was performed using trimethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. The acetylcholinesterase inhibitory activity of compound 8 was evaluated and compared to that of disodium 2β,3α-dihydroxy-5α-cholestane disulfate (10) and diols 7 and 9. Compounds 8 and 10 were active with IC(50) values of 14.59 and 59.65 μM, respectively. Diols 7 and 9 showed no inhibitory activity (IC(50)>500 μM).

Studies have revealed abnormalities in resting-state functional connectivity in those with major depressive disorder specifically in areas such as the dorsal anterior cingulate, thalamus, amygdala, the pallidostriatum and subgenual cingulate. However, the effect of antidepressant medications on human brain function is less clear and the effect of these drugs on resting-state functional connectivity is unknown. Forty volunteers matched for age and gender with no previous psychiatric history received either citalopram (SSRI; selective serotonergic reuptake inhibitor), reboxetine (SNRI; selective noradrenergic reuptake inhibitor) or placebo for 7 days in a double-blind design. Using resting-state functional magnetic resonance imaging and seed based connectivity analysis we selected the right nucleus accumbens, the right amygdala, the subgenual cingulate and the dorsal medial prefrontal cortex as seed regions. Mood and subjective experience were also measured before and after drug administration using self-report scales. Despite no differences in mood across the three groups, we found reduced connectivity between the amygdala and the ventral medial prefrontal cortex in the citalopram group and the amygdala and the orbitofrontal cortex for the reboxetine group. We also found reduced striatal-orbitofrontal cortex connectivity in the reboxetine group. These data suggest that antidepressant medications can decrease resting-state functional connectivity independent of mood change and in areas known to mediate reward and emotional processing in the brain. We conclude that hypothesis-driven seed based analysis of resting-state fMRI supports the proposition that antidepressant medications might work by normalising the elevated resting-state functional connectivity seen in depressed patients.

Neuropathological events featuring early stages of Alzheimer's disease (AD) appear in the entorhinal cortex (EC), subiculum (SB) and cornu ammonis 1 (CA1) of hippocampus, which may account for associative memory deficits in non-demented people with mild cognitive impairment (MCI). To test this hypothesis in vivo, we investigated whether volume changes in these regions are related to failures in associative memory in MCI as compared to cognitively normal (CN) elderly subjects. Volume changes in EC and hippocampal subfields were determined by using deformation-based morphometry techniques applied to probabilistic cytoarchitectonic maps derived from post mortem human brains. CN subjects were distinguished from MCI patients by firstly identifying local volume differences in EC and hippocampus, and then evaluating the way in which these anatomical changes correlated with performance in a non-intentional face-location association task. MCI patients not only performed worse than CN elders in building new associations, but they were further unable to benefit from semantic encoding to improve episodic binding. According to our initial hypothesis, local volume reductions in both EC and hippocampal CA accounted for group differences in associative memory whereas atrophy in CA, but not in EC, accounted for semantic encoding of associations. Two main conclusions can be drawn from the present study: i) access to semantic information during encoding does not reduce the episodic deficit in MCI; and ii) EC and hippocampal CA, two regions early affected by AD neuropathology, are responsible, at least partially, for associative memory deficits observed in MCI patients.

Our goal was to evaluate the expression patterns for voltage gated sodium channels in odontoblasts of developing and mature rat teeth.

High-resolution in vivo imaging of higher vertebrate embryos over short or long time periods under constant physiological conditions is a technically challenging task for researchers working on cardiovascular development. In chick embryos, for example, various studies have shown that without appropriate maintenance of temperature, as one of the main environmental factors, the embryonic heart rate drops rapidly and often results in an increase in regurgitant flow. Hemodynamic parameters are critical stimuli for cardiovascular development that, for a correct evaluation of their developmental significance, should be documented under physiological conditions. However, previous studies were mostly carried out outside of an incubator or under suboptimal environmental conditions. Here we present, to the best of our knowledge, the first detailed description of an optical coherence tomography (OCT) system integrated into an examination incubator to facilitate real-time in vivo imaging of cardiovascular development under physiological environmental conditions. We demonstrate the suitability of this OCT examination incubator unit for use in cardiovascular development studies by examples of proof of principle experiments. We, furthermore, point out the need for use of examination incubators for physiological OCT examinations by documenting the effects of room climate (22°C) on the performance of the cardiovascular system of chick embryos (HH-stages 16/17). Upon exposure to room climate, chick embryos showed a fast drop in the heart rate and striking changes in the cardiac contraction behaviour and the blood flow through the vitelline circulation. We have documented these changes for the first time by M-mode OCT and Doppler M-mode OCT.

Rex1 (Zfp42), GeneID 132625, is a gene whose expression is closely associated with pluripotency/multipotency in both mouse and human embryonic stem cells. To study the function of the murine Rex1 gene in vivo, we have used cre/lox technology to create Rex1(floxed) mice and mice deficient in Rex1 gene function. Rex1(-/-)males are characterized by an age-associated decrease in sperm counts, abnormal sperm morphology, and mild testicular atrophy. We characterized global patterns of gene expression in primary germ cells by microarray and identified the growth hormone responsive gene, GRTP1, as a transcript present at a 4.5 fold higher level in wild type (WT) compared to Rex1(-/-) mice. We analyzed immature germ cell (Dazl), proliferating (PCNA), and Sertoli cell populations, and quantitated levels of apoptosis in Rex1(-/-) as compared to WT testes. We evaluated the expression of proteins previously reported to correlate with Rex1 expression, such as STAT3, phospho-STAT3, p38, and phospho-p38 in the testis. We report a distinct cellular localization of total STAT3 protein in Rex1(-/-) affected testes. Our data suggest that loss of Rex1 leads to impaired testicular function.

Circadian rhythms are regulated by a synchronized system of central and peripheral clocks. Here, we show that a clock in the Drosophila fat body drives rhythmic expression of genes involved in metabolism, detoxification, the immune response, and steroid hormone regulation. Some of these genes cycle even when the fat body clock is disrupted, indicating that they are regulated by exogenous factors. Food is an important stimulus, as limiting food availability to a 6 hr interval each day drives rhythmic expression of genes in the fat body. Restricting food to a time of day when consumption is typically low desynchronizes internal rhythms because it alters the phase of rhythmic gene expression in the fat body without affecting the brain clock. Flies maintained on this paradigm produce fewer eggs than those restricted to food at the normal time. These data suggest that desynchrony of endogenous rhythms, caused by aberrant feeding patterns, affects reproductive fitness.

Mitochondria are organelles centrally important for bioenergetics as well as regulation of apoptotic death in eukaryotic cells. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromatin-associated protein which maintains nuclear homeostasis, is also a critical regulator of mitochondrial function and morphology. We show that heat shock protein beta-1 (HSPB1 or HSP27) is the downstream mediator of this effect. Disruption of the HSPB1 gene in embryonic fibroblasts with wild-type HMGB1 recapitulates the mitochondrial fragmentation, deficits in mitochondrial respiration, and adenosine triphosphate (ATP) synthesis observed with targeted deletion of HMGB1. Forced expression of HSPB1 reverses this phenotype in HMGB1 knockout cells. Mitochondrial effects mediated by HMGB1 regulation of HSPB1 expression serve as a defense against mitochondrial abnormality, enabling clearance and autophagy in the setting of cellular stress. Our findings reveal an essential role for HMGB1 in autophagic surveillance with important effects on mitochondrial quality control.

Human chorionic gonadotropin (hCG) is one of the earliest signals secreted by the implanting embryo. In addition to its well-known luteotropic function in early pregnancy, hCG also acts directly on decidualizing endometrium. Recently, we demonstrated that recombinant hCG (rhCG) prevented apoptosis in decidualizing human endometrial stromal cells (HESCs) exposed to oxidative stress. Two hCG preparations are widely used clinically: rhCG, produced by recombinant DNA technology, and urinay hCG (uhCG), extracted from urine of post-menopausal women. However, an analysis of the direct effects of rhCG and uhCG on the decidual phenotype of HESCs has not yet been done. In this study, we investigated the effects of uhCG and rhCG on the morphological and functional profiles of decidualizing HESCs. We demonstrate that neither rhCG nor uhCG alter the morphological appearance of the decidual HESC cultures, although rhCG but not uhCG attenuated prolactin expression, a major decidual marker protein. Moreover, rhCG, but not uhCG, protected decidualizing HESCs from oxidative cell death, mediated at least in part by two major mechanisms. First, rhCG, but not uhCG, enhances the expression of manganese superoxide dismutase, a cardinal enzyme in the cellular defense against oxidative damage. Second, rhCG signaling selectively limits activation of the apoptotic machinery in decidualizing HESCs by enhancing Bcl-2 expression whereas uhCG induces the expression of Fas ligand. Our results suggest that rhCG might be a preferable agent to protect the maternal decidua against oxidative damage in pregnancy, especially at the time of implantation and beyond.

Dileptid and tracheliid ciliates have been traditionally classified within the subclass Haptoria of the class Litostomatea. However, their phylogenetic position among haptorians has been controversial and indicated that they may play a key role in understanding litostomatean evolution. In order to reconstruct the evolutionary history of dileptids and tracheliids, and to unravel their affinity to other haptorians, we have used a cladistic approach based on morphological evidence and a phylogenetic approach based on 18S rRNA gene sequences, including eight new ones. The molecular trees demonstrate that dileptids and tracheliids represent a separate subclass, Rhynchostomatia, that is sister to the subclasses Haptoria and Trichostomatia. The Rhynchostomatia are characterized by a ventrally located oral opening at the base of a proboscis that carries a complex oral ciliature. We have recognized two orders within Rhynchostomatia. The new order Tracheliida is monotypic, while the order Dileptida comprises two families: the new, typically bimacronucleate family Dimacrocaryonidae and the multimacronucleate family Dileptidae. The Haptoria evolved from the last common ancestor of the Litostomatea by polarization of the body, the oral opening locating more or less apically and the oral ciliature simplifying. The Trichostomatia originated from a microaerophylic haptorian by further simplification of the oral ciliature, possibly due to an endosymbiotic lifestyle.

It was previously demonstrated that diet potently modulates the toxic effects of an acute lethal dose of the nerve agent soman. The current investigation was undertaken to examine the influence of diet on the cumulative toxicity of repeated soman administration. Rats were fed one of four distinct diets (standard, choline-enriched, glucose-enriched, or ketogenic) for four weeks prior to and throughout a repeated soman dosing and recovery regimen. Each diet group included animals exposed to an equivalent volume of saline that served as negative controls. In exposure Week 1, animals received three consecutive daily doses of 0.4 LD(50) soman. In exposure Week 2, animals received four consecutive daily doses of 0.5 LD(50) soman. In exposure Week 3, animals received five consecutive daily doses of 0.5 LD(50) soman. Week 4 constituted a post-exposure recovery evaluation. Throughout the experiment, behavioral function was assessed by a discriminated avoidance test that required intact sensory and motor function. Survival and body weight changes were recorded daily. Differences in toxicity as a function of diet composition became apparent during the first week. Specifically, rats fed the glucose-enriched diet showed pronounced intoxication during Week 1, resulting in imperfect survival, weight loss, and deteriorated avoidance performance relative to all other groups. All rats fed the glucose-enriched diet died by the end of exposure Week 2. In contrast, only 10% of animals fed the standard diet died by the end of Week 2. Also in Week 2, weight loss and disrupted avoidance performance were apparent for all groups except for those fed the ketogenic diet. This differential effect of diet composition became even more striking in Week 3 when survival in the standard and choline diet groups approximated 50%, whereas survival equaled 90% in the ketogenic diet group. Avoidance performance and weight loss measures corroborated the differential toxicity observed across diet groups. Upon cessation of soman exposure during the final week, recovery of weight and avoidance performance in survivors was comparable across diet groups. These results systematically replicate previous findings demonstrating that diet composition exacerbates or attenuates toxicity in rodents exposed acutely to organophosphorus compounds.

Studies of human neonates, and in animal experiments, suggest that birth asphyxia results in functional compromise of the hippocampus, even when structural damage is not observable or resolves in early postnatal life. The aim of this study was to determine if changes in hippocampal function occur in a model of birth asphyxia in the precocial spiny mouse where it is reported there is no major lesion or infarct. Further, to assess if, as in human infants, this functional deficit has a sex-dependent component. At 37 days gestation (term=39 days) spiny mice fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5min of in utero asphyxia causing systemic acidosis and hypoxia. At 5 days of age hippocampal function was assessed ex vivo in brain slices, or brains were collected for examination of structure or protein expression. This model of birth asphyxia did not cause infarct or cystic lesion in the postnatal day 5 (P5) hippocampus, and the number of proliferating or pyknotic cells in the hippocampus was unchanged, although neuronal density in the CA1 and CA3 was increased. Protein expression of synaptophysin, brain-derived neurotrophic factor (BDNF), and the inositol trisphosphate receptor 1 (IP(3)R1) were all significantly increased after birth asphyxia, while long-term potentiation (LTP), paired pulse facilitation (PPF), and post-tetanic potentiation (PTP) were all reduced at P5 by birth asphyxia. In control P5 pups, PPF and synaptic fatigue were greater in female compared to male pups, and after birth asphyxia PPF and synaptic fatigue were reduced to a greater extent in female vs. male pups. In contrast, the asphyxia-induced increase in synaptophysin expression and neuronal density were greater in male pups. Thus, birth asphyxia in this precocial species causes functional deficits without major structural damage, and there is a sex-dependent effect on the hippocampus. This may be a clinically relevant model for assessing treatments delivered either before or after birth to protect this vulnerable region of the developing brain.

Disrupted in schizophrenia 1 (DISC1) is known to play a major role during brain development and is a candidate gene for schizophrenia. Cortical thickness is highly heritable and several MRI studies have shown widespread reductions of cortical thickness in patients with schizophrenia. Here, we investigated the effects of variation in DISC1 on cortical thickness. In a subsequent analysis we tested whether the identified DISC1 risk variant is also associated with neural activity during working memory functioning.

Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca(2+)) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca(2+) channel inhibitors, 2-ABP, and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca(2+) signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca(2+) in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.

Chagas disease has a variable clinical course with different manifestations and heterogenous geographical distribution. Some studies suggest that this clinical variability could be influenced by the genetic variability of T. cruzi. Here we present the differential protein expression among trypomastigotes and amastigotes of T. cruzi group I isolates from patients with acute and chronic form of Chagas disease from Santander, Colombia. A total of 29 proteins were identified by MALDI-TOF and LC-MS/MS; twenty in trypomastigote and nine in amastigote stage. The 29 proteins identified were grouped in 7 functional categories: 1) metabolism 31%, 2) assembly of cytoskeleton 13.7%, 3) protein destination 13.7%, 4) defenses antioxidants 20.6%, 5) protein synthesis and cellular cycle 13.7%, 6) catabolism 6.8%, and 7) adhesion 3.4%. Tryparedoxin peroxidase, lipoamide dehydrogenase, tyrosine amino transferase and HSP70 were overexpressed in the acute Chagas isolate. Tryparedoxin peroxidase overexpression in the acute isolate was confirmed by Western blot analysis. Most of these proteins are associated with resistance to oxidative stress facilitating their survival within host cells. Therefore, these proteins may represent virulence factors associated with the development of the acute form of the disease and could be used as biomarkers of the clinical course of disease and as drug targets.

A recent Monographs Working Group of the International Agency for Research on Cancer (IARC) concluded that there is sufficient evidence for a causal association between exposure to asbestos and ovarian cancer. We performed a meta-analysis to quantitatively evaluate this association.