Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Heat shock proteins and aging in Drosophila melanogaster

Heat shock proteins (Hsps) are conserved molecular chaperones that are upregulated following exposure to environmental stress and during aging. The mechanisms underlying the aging process are only beginning to be understood. The beneficial effects of Hsps on aging revealed in mild stress and overexpression experiments suggest that these proteins are part of an important cell protection system rather than being unspecific molecular chaperones. Among the Hsps families, small Hsps have the greatest influence on aging and the modulation of their expression during aging in Drosophila suggest that they are involved in pathways of longevity determination.     

Acute angiotensin II increases plasma F2-isoprostanes in salt-replete human hypertensives

Angiotensin (Ang) II induces oxidative stress in vitro and in animal models of hypertension. We tested the hypothesis that Ang II increases oxidative stress in human hypertension, as assessed by plasma F2-isoprostane concentrations. Plasma F2-isoprostanes, hemodynamic and endocrine parameters were measured at baseline and following a 55 min infusion of 3 ng/kg/min Ang II in 13 normotensive and 13 hypertensive volunteers ingesting a high- (200 mmol/d) or low- (10 mmol/d) sodium diet. Mean arterial pressure (MAP) and body mass index were higher in hypertensive subjects. Ang II infusion increased MAP (p<.001) and plasma aldosterone concentrations (p<.001) and decreased plasma renin activity (p<.001) and renal plasma flow (p<.001) to a similar extent in both groups. Plasma F2-isoprostane concentrations were similar at baseline. There was no effect of Ang II on F2-isoprostane concentrations during low-salt intake in either group (normotensive 51.7 +/- 7.1 to 53.7 +/- 6.5 pg/ml and hypertensive 52.2 +/- 8.2 to 56.2 +/- 10.0 pg/ml; mean +/- SE). During high-salt intake, Ang II increased F2-isoprostane concentrations in the hypertensive group (52.3 +/- 7.2 to 63.2 +/- 10.4 pg/ml, p=0.010) but not in the normotensive group (54.2 +/- 4.4 to 58.9 +/- 6.6 pg/ml, p=0.83). Acute Ang II infusion increases oxidative stress in vivo in hypertensive humans. The renin-angiotensin system may contribute to oxidative stress in human cardiovascular disease.     

Misexpression of basic helix-loop-helix genes in the murine cerebral cortex affects cell fate choices and neuronal survival

To investigate the role(s) of basic helix-loop-helix genes (bHLH) genes in the developing murine cerebral cortex, Mash1, Math2, Math3, Neurogenin1 (Ngn1), Ngn2, NeuroD, NeuroD2 and Id1 were transduced in vivo into the embryonic and postnatal cerebral cortex using retrovirus vectors. The morphology and location of infected cells were analyzed at postnatal stages. The data indicate that a subset of bHLH genes are capable of regulating the choice of neuronal versus glial fate and that, when misexpressed, they can be deleterious to the survival of differentiating neurons, but not glia.     

Cytokine production in motor neurons by poliovirus replicon vector gene delivery

Poliovirus replicon vectors transiently express foreign proteins selectively in motor neurons of the anterior horn of the spinal cord. Here we intraspinally inoculated mice transgenic for the poliovirus receptor (PVR) with replicons encoding murine tumor necrosis factor alpha (mTNF-alpha). We detected high-level expression of mTNF-alpha in the spinal cords of these animals at 8-12 h post inoculation; this returned to background by 72 h. The mice exhibited ataxia and tail atony, whereas animals given a replicon encoding green fluorescent protein (GFP) exhibited no neurological symptoms. Histology of spinal cords from mice given the replicon encoding mTNF-alpha revealed neuronal chromatolysis, reactive astrogliosis, decreased expression of myelin basic protein, and demyelination. These animals recovered with only slight residual damage. This study shows that replicon vectors have potential for targeted delivery of therapeutic proteins to the central nervous system and provide a new approach for treatment of spinal cord trauma and neurological disease.     

Comparing ovulation synchronization protocols for artificial insemination in the scimitar-horned oryx (Oryx dammah)

Ovarian response and pregnancy success in scimitar-horned oryx (n=28) were compared, following treatment with two synchronization protocols and fixed-time artificial insemination (AI) with frozen-thawed semen. Each oryx received two injections of 500 microg of prostaglandin-F(2alpha) analogue (PGF(2alpha)-only) 11 days apart, and half received PGF(2alpha) in combination with an intravaginal progesterone-releasing device (CIDR11+PGF(2alpha)). Semen was collected by electroejaculation from anaesthetised adult oryx and cryopreserved. Anaesthetised females were transcervically inseminated 56.0+/-1.1 h (+/-S.E.M.) after PGF(2alpha) injection and/or device withdrawal using 28.0+/-1.5x10(6) motile thawed sperm. Ovarian endocrine response was monitored in 20 females by analysing faecal oestrogen and progesterone metabolites. Periovulatory oestrogen peaks were detected in 19/20 (95%) females after synchronization. There were no between-treatment differences in oestrogen concentrations or peak characteristics (P0.05). Luteal development after synchronization was delayed in half the progesterone treated (CIDR11+PGF(2alpha)) females, and faecal progestin excretion profiles indicated that the ovulatory follicle associated with synchronization either failed to ovulate or to fully lutenise. Pregnancy was diagnosed by ultrasonography and/or rectal palpation and was monitored by faecal progestin excretion. More (P=0. 013) pregnancies resulted from the PGF(2alpha)-only treatment (37.5%, 5/14) than from the CIDR11+PGF(2alpha) treatment (0/14), and four healthy scimitar-horned oryx calves were born, three after gestation intervals of 247 days and one after 249 days.     

The EGF receptor transmembrane domain: peptide-peptide interactions in fluid bilayer membranes

A peptide containing the transmembrane domain of the human EGF receptor was studied in fluid lipid bilayers for insight into receptor tyrosine kinase lateral associations in cell membranes. The peptide comprised the 23-amino acid hydrophobic segment thought to span the membrane (Ile(622) to Met(644) of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg(645) to Thr(654)). Probes for solid-state NMR spectroscopy were incorporated by deuteration of the methyl side chains of alanine at positions 623 and 637. (2)H-NMR spectra were recorded from 25 to 65 degrees C in membranes composed of 1-palmitoyl-2-oleoyl phosphatidylcholine, with and without 33% cholesterol, and relaxation times were measured. Peptide concentration ranged from 0. 5 to 10 mol %. The peptide behaved as predominant monomers undergoing rapid symmetric rotational diffusion; however, there was evidence of reversible side-to-side interaction among the hydrophobic transmembrane domains, particularly at physiological temperatures and in the presence of natural concentrations of cholesterol. The results of these experiments in fluid membranes are consistent with the existence of lipid-protein interactions that would predispose to receptor microdomain formation in membranes of higher animal cells.