Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.     

Expression of a pathogen-induced gene can be mimicked by auxin insensitivity

Following perception of a pathogenic attack, plants are able to develop a strong response with the corresponding activation of a plethora of defense-related genes. In this study we have characterized the mode of expression of the CEVI-1 gene from tomato plants, which encodes an anionic peroxidase. CEVI-1 expression is induced during the course of compatible viral and subviral infections, like many other defense-related genes, but is induced neither in incompatible interactions nor by signal molecules such as salicylic acid, ethylene, or methyl jasmonate. Additionally, CEVI-1 is induced in detached leaf tissues following a pathway distinct from that related to the classical wound response. We also describe the characterization of the structural CEVI-1 gene and compare the mode of expression in different transgenic plant species harboring a CEVI-1::GUS construct. Furthermore, we have isolated mutants in Arabidopsis, called dth mutants, that are deregulated in the control of expression of this gene. From the initial analysis of some of these mutants it seems that activation of CEVI-1 gene expression correlates with a defect in the perception of auxins by the plant. All these results may suggest that, during systemic infections with viruses, auxin homeostasis is one of the components participating in the regulation of the overall defense response.     

Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Acetylcholinesterase activation and enhanced lipid peroxidation after long-term exposure to low levels of aluminum on different mouse brain regions

Aluminum (Al), oxidative stress and impaired cholinergic functions have all been related to Alzheimer's disease (AD). The present study evaluates the effect of aluminum on acetylcholinesterase (AChE) and lipid peroxidation in the mouse brain. Mice were loaded by gavage with Al 0.1 mmol/kg/day 5 days per week during 12 weeks. The mice were divided into four groups: (1) control; (2) 10 mg/mL of citrate solution; (3) 0.1 mmol/kg of Al solution; (4) 0.1 mmol/kg of Al plus 10 mg/mL of citrate solution. AChE activity was determined in the hippocampus, striatum, cortex, hypothalamus and cerebellum and lipid peroxidation was determined in the hippocampus, striatum and cortex. An increase of AChE activity was observed in the fourth group (Al + Ci) in the hippocampus (36%), striatum (54%), cortex (44%) and hypothalamus (22%) (p<0.01). The third group (Al) presented a decrease of AChE activity in the hypothalamus (20%) and an enhancement in the striatum (27%). Lipid peroxidation, measured by TBARS (thiobarbituric acid reactive substances), was elevated in the hippocampus and cerebral cortex when compared with the control (p < 0.01). The effect of aluminum on AChE activity may be due to a direct neurotoxic effect of the metal or perhaps a disarrangement of the plasmatic membrane caused by increased lipid peroxidation.     

Morphology and metamorphosis of Eupsophus calcaratus tadpoles (Anura: Leptodactylidae)

Eupsophus calcaratus, a leptodactyloid frog from the austral Andean forests of Argentina and Chile, has endotrophic, nidicolous tadpoles. We studied a metamorphic series from Stages 31 to 46 of Gosner's developmental table (1960). Other than the scarce pigmentation, proportionately large eyes, and massive developing hindlimbs, the remaining external characters are similar to those of generalized, exotrophic larvae. At the same time, internal morphology does not reveal any character state attributable to the endotrophic-nidicolous way of life; conversely, structures such as the hyobranchial skeleton and the mandibular cartilages are similar to those of exotrophic-macrophagous tadpoles. The metamorphic process is characterized by the delayed development of diverse structures (e.g., ethmoid region, palatoquadrate, and hyobranchial apparatus), and the retention of some larval characters (e.g., parietal fenestrae, overall absence of ossification) with the absence of development of some "juvenile" characters (e.g., adult otic process, several bones) in metamorphosed individuals. These heterochronic processes and truncation of larval development are related to a shorter larval life (when compared to other species of the austral Andean region) and to the small size at metamorphosis.     

Mechanisms of blood pressure variability-induced cardiac hypertrophy and dysfunction in mice with impaired baroreflex

Enhanced blood pressure variability contributes to left ventricular hypertrophy and end-organ damage, even in the absence of hypertension. We hypothesized that the greater number of high-blood pressure episodes associated with enhanced blood pressure variability causes cardiac hypertrophy and dysfunction by activation of mechanosensitive and autocrine pathways. Normotensive mice were subjected to sinoaortic baroreceptor denervation (SAD) or sham surgery. Twelve weeks later, blood pressure variability was doubled in SAD compared with sham-operated mice. Blood pressure did not differ. Cardiac hypertrophy was reflected in greater heart/body weight ratios, larger myocyte cross-sectional areas, and greater left ventricular collagen deposition. Furthermore, left ventricular atrial and brain natriuretic peptide mRNA expression was greater in SAD than in sham-operated mice. SAD had higher left ventricular end-diastolic pressures and lower myocardial contractility indexes, indicating cardiac dysfunction. Cardiac protein content of phosphorylated p125 focal adhesion kinase (p125 FAK) and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was greater in SAD than in sham-operated mice, indicating activation of mechanosensitive pathways of cardiac hypertrophy. Furthermore, enhanced cardiac renin and transforming growth factor-beta1 (TGFbeta1) protein content indicates activation of autocrine pathways of cardiac hypertrophy. Adrenal tyrosine hydroxylase protein content and the number of renin-positive glomeruli were not different, suggesting that sympathetic activation and the systemic renin-angiotensin system did not contribute to cardiac hypertrophy. In conclusion, more frequent blood pressure rises in subjects with high blood pressure variability activate mechanosensitive and autocrine pathways leading to cardiac hypertrophy and dysfunction even in the absence of hypertension.     

First cases in the Czech Republic of the Hallervorden-Spatz disease resulting from mutation in the pantothenate kinase 2 gene

Hallervorden-Spatz disease (HSD) was and is known as a rare disorder primarily characterized by progressive extrapyramidal dysfunction and dementia alongside optic nerve atrophy or retinal degeneration and pyramidal signs. The rate of occurence of HSD is thus far unknown. Progress in DNA diagnostics stirred up a nomenclature and from HSD, or, perhaps better put, the Hallervorden-Spatz syndrome, crystallized the pantothenate kinase-associated neurodegeneration (PKAN) as a clearly defined entity on the level of DNA. In this paper, we present our first results and experience in the diagnosis of PKAN in the Czech Republic and discuss questions related to differential diagnosis.     

Dual Effect of Pyruvate in Isolated Nerve Terminals: Generation of Reactive Oxygen Species and Protection of Aconitase

Generation of reactive oxygen species (ROS) in synaptosomes was investigated in the presence of different substrates. When pyruvate was used as a substrate an increased rate of hydrogen peroxide formation was detected by the Amplex Red fluorescent assay, but aconitase, which is known to be a highly sensitive enzyme to ROS was not inhibited. In contrast, pyruvate exerted a partial protection on aconitase against a time-dependent inactivation that occurred when synaptosomes were incubated in the absence of substrates. Disruption of synaptosomal membranes with Triton X-100 prevented the protective effect of pyruvate. It is suggested that citrate and/or isocitrate formed in the metabolism of pyruvate could be responsible for a partial protection of aconitase. Therefore while pyruvate could have a prooxidant effect it could also exert a protective effect on the aconitase. Special issue dedicated to Dr. Bernd Hamprecht.     

Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium.