The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.     

Relationship of visceral adiposity to cardiovascular disease risk factors in black and white teens

Objective: We tested the hypothesis that visceral adiposity, compared with general adiposity, would explain more of the variance in cardiovascular disease (CVD) risk factors. Research Method and Procedures: Subjects were 464 adolescents (238 black and 205 girls). Adiposity measures included visceral adipose tissue (VAT; magnetic resonance imaging), percent body fat (%BF; DXA), BMI, and waist girth (anthropometry). CVD risk factors were fasting insulin, fibrinogen, total to high‐density lipoprotein‐cholesterol ratio, triglycerides (TGs), systolic blood pressure, and left ventricular mass indexed to height^2.7. Results: After adjustment for age, race, and sex, all adiposity indices explained significant proportions of the variance in all of the CVD risk factors; %BF tended to explain more variance than VAT. Regression models that included both %BF and VAT found that both indices explained independent proportions of the variance only for total to high‐density lipoprotein‐cholesterol ratio. For TGs, the model that included both %BF and VAT found that only VAT was significant. For systolic blood pressure and left ventricular mass indexed to height^2.7, anthropometric measures explained more of the variance than VAT and %BF. Discussion: The hypothesis that visceral adiposity would explain more variance in CVD risk than general adiposity was not supported in this relatively large sample of black and white adolescents. Only for TGs did it seem that VAT was more influential than %BF. Perhaps the deleterious effect of visceral adiposity becomes greater later in life as it increases in proportion to general adiposity.     

Ten months of exercise improves general and visceral adiposity, bone, and fitness in black girls

Objective: The goal of this study was to evaluate the impact of a 10‐month after‐school physical activity (PA) program on body composition and cardiovascular (CV) fitness in young black girls. Research Methods and Procedures: Subjects were 8‐ to 12‐year‐olds recruited from elementary schools. Body composition was measured using anthropometrics {waist circumference and BMI, DXA [percentage body fat (%BF)] and bone mineral density (BMD)}, and magnetic resonance imaging [visceral adipose tissue (VAT)]. CV fitness was measured using a graded treadmill test. The intervention consisted of 30 minutes homework/healthy snack time and 80 minutes PA (i.e., 25 minutes skills instruction, 35 minutes aerobic PA, and 20 minutes strengthening/stretching). Analyses were adjusted for age, baseline value of the dependent variable, and sexual maturation (pediatrician observation). Results: Mean attendance was 54%. Compared with the control group, the intervention group had a relative decrease in %BF (p < 0.0001), BMI (p < 0.01), and VAT (p < 0.01) and a relative increase in BMD (p < 0.0001) and CV fitness (p < 0.05). Higher attendance was associated with greater increases in BMD (p < 0.05) and greater decreases in %BF (p < 0.01) and BMI (p < 0.05). Higher heart rate during PA was associated with greater increases in BMD (p < 0.05) and greater decreases in %BF (p < 0.005). Discussion: An after‐school PA program can lead to beneficial changes in body composition and CV fitness in young black girls. It is noteworthy that the control and intervention groups differed in change in VAT but not waist circumference. This suggests that changes in central adiposity can occur in response to PA, even in young children, but that waist circumference may not be a good indicator of central adiposity.     

Recent domoic acid closures of shellfish harvest areas in Washington State inland waterways

Several species of the toxigenic diatom Pseudo-nitzschia, together with low concentrations of domoic acid (DA) in shellfish have been observed in Puget Sound, Washington State, since 1991. However, for the first time in September 2003, high-density blooms of Pseudo-nitzschia forced the closure of recreational, commercial, and tribal subsistence shellfish harvesting in Puget Sound. Here we report on the environmental conditions associated with shellfish closures in two inland waterways of Washington State during the Fall 2005. In Sequim Bay, shellfish harvest losses occurred on September 12 following the measurement of elevated macronutrient levels on September 2, and a bloom of P. pseudodelicatissima (up to 13 million cells/L) on September 9. Ambient NH₄ concentrations >12 μM (measured on September 2) were likely due to anthropogenic sources, ostensibly from sewage inputs to Sequim Bay. The closure of a Penn Cove commercial shellfish farm on October 16 was caused by a bloom of P. australis that followed a period of sustained precipitation, elevated Skagit River flow, and persistent southeasterly winds. The relative importance of a number of environmental factors, including temperature, stratification caused by rivers, and nutrient inputs, whether natural or anthropogenic, must be carefully studied in order to better understand the recent appearance of massive blooms of toxigenic Pseudo-nitzschia in the inland waterways of Washington State.     

Platelet-derived growth factor BB mediated regulation of 12-lipoxygenase in porcine aortic smooth muscle cells

Platelet-derived growth factor BB (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). In the present study, we have examined the effects of PDGF on the 12-lipoxygenase (12-LO) pathway of arachidonate metabolism in porcine aortic VSMC (PVSMC). The rationale for this is previous studies showing that LO products have growth and chemotactic effects in VSMC and that another VSMC growth factor, angiotensin II, is a potent positive regulator of 12-LO activity and expression. We observed that PDGF causes a significant increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE) in PVSMC. In addition, PDGF also markedly increased leukocyte-type 12-LO messenger RNA and protein expression. PDGF-induced PVSMC migration was inhibited significantly by two LO blockers but not by a cyclooxygenase blocker. Furthermore, although the proliferative effects of PDGF on PVSMC were not altered by cell culture under hyperglycemic conditions (25 mM glucose, HG), the chemotactic effects of PDGF as well as those of 10% fetal calf serum were significantly greater in cells cultured in HG as compared to normal glucose conditions (5.5 mM), thus indicating a potential new mechanism for the accelerated cardiovascular disease usually observed in diabetes. These results indicate a novel mechanism for the biological effects of PDGF in leading to cardiovascular disease. © 1996 Wiley-Liss, Inc.     

Epigenetics underpins phenotypic plasticity of protandrous sex change in fish

Phenotypic plasticity is an important driver of species resilience. Often mediated by epigenetic changes, phenotypic plasticity enables individual genotypes to express variable phenotypes in response to environmental change. Barramundi (Lates calcarifer) are a protandrous (male‐first) sequential hermaphrodite that exhibits plasticity in length‐at‐sex change between geographic regions. This plasticity is likely to be mediated by changes in DNA methylation (DNAm), a well‐studied epigenetic modification. To investigate the relationships between length, sex, and DNAm in a sequential hermaphrodite, here, we compare DNAm in four conserved vertebrate sex‐determining genes in male and female barramundi of differing lengths from three geographic regions of northern Australia. Barramundi first mature as male and later sex change to female upon the attainment of a larger body size; however, a general pattern of increasing female‐specific DNAm markers with increasing length was not observed. Significant differences in DNAm between males and females of similar lengths suggest that female‐specific DNAm arises rapidly during sex change, rather than gradually with fish growth. The findings also reveal that region‐specific differences in length‐at‐sex change are accompanied by differences in DNAm and are consistent with variability in remotely sensed sea temperature and salinity. Together, these findings provide the first in situ evidence for epigenetically and environmentally mediated sex change in a protandrous hermaphrodite and offer significant insight into the molecular and ecological processes governing the marked and unique plasticity of sex in fish.     

Evaluation of an in vitro coculture model for the blood-brain barrier: Comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources

Summary Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.     

Fiber type composition of four hindlimb muscles of adult Fisher 344 rats

 The limb and trunk muscles of adult rats express four myosin heavy chain (MHC) isoforms, one slow (MHCI) and three fast (MHCIIa, MHCIId, and MHCIIb). The distribution of these isoforms correlates with fiber types delineated using myofibrillar actomyosin adenosine triphosphatase (mATPase) histochemistry. For example, type I fibers express MHCI and fiber types IIA, IID, and IIB express MHCIIa, MHCIId, and MHCIIb, respectively. Fibers containing only one MHC isoform have been termed ”pure” fibers. Recent evidence suggests that a population of ”hybrid” fibers exist in rat skeletal muscle which contain two MHC isoforms. The purpose of the present investigation was to document the entire range of histochemically defined ”pure” and ”hybrid” fiber types in untreated muscles of the young adult Fisher 344 rat hindlimb. The selected hindlimb muscles (soleus, tibialis anterior, extensor digitorum longus, and gastrocnemius muscles) were removed from 12 male rats and analyzed for muscle fiber type distribution, cross-sectional area, and MHC content. Care was taken to delineate eight fiber types (I, IC, IIC, IIA, IIAD, IID, IIDB, and IIB) using refined histochemical techniques. Hybrid fibers were found to make up a considerable portion of the muscles examined (a range of 8.8–17.8% of the total). The deep red portion of the gastrocnemius muscle contained the largest number of hybrid fibers, most of which were the fast types IIAD (8.5±2.8%) and IIDB (5.2±2.3%). In conclusion, hybrid fibers make up a considerable portion of normal rat limb musculature and are an important population that should not be ignored. Accepted: 15 October 1998     

Proapoptotic BH3-only Bcl-2 family member Bik/Blk/Nbk is expressed in hemopoietic and endothelial cells but is redundant for their programmed death

The BH3-only members of the Bcl-2 protein family are essential for initiation of programmed cell death and stress-induced apoptosis. We have determined the expression pattern in mice of the BH3-only protein Bik, also called Blk or Nbk, and examined its physiological function by gene targeting. We found that Bik is expressed widely in the hematopoietic compartment and in endothelial cells of the venous but not arterial lineages. Nevertheless, its loss did not increase the numbers of such cells in mice or protect hematopoietic cells in vitro from apoptosis induced by cytokine withdrawal or diverse other cytotoxic stimuli. Moreover, whereas loss of the BH3-only protein Bim rescued mice lacking the prosurvival protein Bcl-2 from fatal polycystic kidney disease and lymphopenia, loss of Bik did not. These results indicate that any function of Bik in programmed cell death and stress-induced apoptosis must overlap that of other BH3-only proteins.     

Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT)

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1alpha (HIF-1alpha) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between -165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.