The cell survival kinase SGK1 and its targets FOXO3a and NDRG1 in aged human brain.

AIMS: Serum- and glucocorticoid-inducible kinase 1 (SGK1) protects neuronal cells from injury stimuli in vitro, and exerts anti-apoptotic effects via downstream targets including the forkhead-like transcription factor FOXO3a. SGK1 is a homolog of Akt, a related survival kinase that is up-regulated in Alzheimer's disease (AD). Here we aimed to examine the expression pattern of SGK1 and FOXO3a in aged human cerebral cortex. METHODS: Cortical tissue from aged donors without brain disease (aged controls, AC, n = 19) and from severe AD patients (Braak stage V-VI; n = 14) were examined by immunohistochemistry and immunoblot analysis. RESULTS: SGK1 was present in all samples (detected by immunohistochemistry and immunoblotting). Large cortical neuronal cells were strongly positive for SGK1, with predominantly nuclear labelling. Some astrocytes and oligodendrocytes were also labelled. SGK1 was not seen in nerve tracts (axons or myelin) and rarely seen in CD68-positive cells (microglia, perivascular macrophages) or vascular cells (myocytes or endothelia). The fraction of large cortical neurones with nuclear FOXO3a was lower in AD cases relative to AC (54%, 70%, respectively, P < 0.001). In immunoblots no difference in SGK1 abundance was detected between AC and AD tissues. Phosphorylation of NDRG1 (an SGK1-specific target) was greater in AD, relative to AC cases (approximately twofold, P = 0.023). CONCLUSIONS: Neuronal expression of SGK1 in aged human brain and its nuclear compartmentalization suggest a possible neuroprotective role. FOXO3a and NDRG1 data suggest augmented SGK1 activity (as reported for Akt) in severe AD. Increased intracellular SGK1 may complement enhanced Akt, with a distinct subcellular localization.

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr346/356/366 phosphorylation.

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na(+) conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 muM) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na(+) absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

Alcohol and backward masking of visual information.

Alcohol increased the time necessary to transfer information from the initial sensory information storage system into the short-term memory system.

Decrease of iconic memory after alcohol.

Alcohol did not alter the rate of information loss from iconic memory. However, there was a dose-related decrease in the total amount of information reported which was independent of the rate of information loss.

mVps34 is activated by an acute bout of resistance exercise.

Resistance-exercise training results in a progressive increase in muscle mass and force production. Following an acute bout of resistance exercise, the rate of protein synthesis increases proportionally with the increase in protein degradation, correlating at 3 h in the starved state. Amino acids taken immediately before or immediately after exercise increase the post-exercise rate of protein synthesis. Therefore a protein that controls protein degradation and amino acid-sensitivity would be a potential candidate for controlling the activation of protein synthesis following resistance exercise. One such candidate is the class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting mutant 34). Vps34 controls both autophagy and amino acid signalling to mTOR (mammalian target of rapamycin) and its downstream target p70 S6K1 (S6 kinase 1). We have identified a significant increase in mVps34 (mammalian Vps34) activity 3 h after resistance exercise, continuing for at least 6 h, and propose a mechanism whereby mVps34 could act as an internal amino acid sensor to mTOR after resistance exercise.

The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection.

The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein was isolated from [35S]methionine- and [33P]orthophosphate-labelled RSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role of tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580, an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependent tyrosine kinase activity and that this modification influences its cellular distribution.

Mechanism of phosphatidylinositol 3-kinase-dependent increases in BAC1.2F5 macrophage-like cell density in response to M-CSF: phosphatidylinositol 3-kinase inhibitors increase the rate of apoptosis rather than inhibit DNA synthesis.

OBJECTIVE AND DESIGN: To determine the role of phosphatidylinositol 3-kinase (PI 3-kinase) in macrophagecolony stimulating factor (M-CSF)-induced macrophage proliferation. MATERIALS: The M-CSF-dependent BAC1.2F5 murine macrophage cell line was used. METHODS: PI 3-kinase activity, Protein kinase B activation, increased cell numbers, induction of DNA synthesis and apoptosis were measured in response to serum, M-CSF and PI 3-kinase inhibitors. RESULTS: Wortmannin or LY294002 inhibited M-CSF-stimulated increases in BAC1.2F5 cell density. Further analysis showed that inhibition of PI 3-kinase had an insignificant effect on DNA synthesis, but significantly induced apoptosis. Other co-factors in serum mediated cell survival and prevented programmed cell death, in a PI 3-kinase-dependent manner. Stimulation of BAC1.2F5 macrophages with M-CSF induced phosphorylation of PKB/Akt as detected by activation-specific antibodies. Activation of PKB/Akt correlated with PI 3-kinase activation, suggesting that the protection from apoptosis in these cells is mediated by PKB/Akt. CONCLUSIONS: These results indicate that the lack of increase in cell numbers when cells are stimulated with M-CSF in the presence of PI 3-kinase inhibitors is due to a preferential PI 3-kinase requirement for protection against apoptosis, rather than a requirement for PI 3-kinase activation during the proliferation signal.

Division by 3 of optical frequencies by use of difference-frequency generation in noncritically phase-matched RbTiOAsO(4).

A new scheme for coherently connecting optical frequencies in a 3:1 ratio has been demonstrated. To phase lock a Nd:YAG laser at 1064 nm with a CO overtone laser at 3192 nm, we generated their difference frequency in RbTiOAsO(4) (RTA) and beat it against the second harmonic of 3192 nm that was generated in AgGaSe(2).

Predictors of aspiration pneumonia: how important is dysphagia?

Aspiration pneumonia is a major cause of morbidity and mortality among the elderly who are hospitalized or in nursing homes. Multiple risk factors for pneumonia have been identified, but no study has effectively compared the relative risk of factors in several different categories, including dysphagia. In this prospective outcomes study, 189 elderly subjects were recruited from the outpatient clinics, inpatient acute care wards, and the nursing home care center at the VA Medical Center in Ann Arbor, Michigan. They were given a variety of assessments to determine oropharyngeal and esophageal swallowing and feeding status, functional status, medical status, and oral/dental status. The subjects were followed for up to 4 years for an outcome of verified aspiration pneumonia. Bivariate analyses identified several factors as significantly associated with pneumonia. Logistic regression analyses then identified the significant predictors of aspiration pneumonia. The best predictors, in one or more groups of subjects, were dependent for feeding, dependent for oral care, number of decayed teeth, tube feeding, more than one medical diagnosis, number of medications, and smoking. The role that each of the significant predictors might play was described in relation to the pathogenesis of aspiration pneumonia. Dysphagia was concluded to be an important risk for aspiration pneumonia, but generally not sufficient to cause pneumonia unless other risk factors are present as well. A dependency upon others for feeding emerged as the dominant risk factor, with an odds ratio of 19.98 in a logistic regression model that excluded tube-fed patients.

Specific requirement for the p85-p110alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells.

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.