Internalization of caveolin-1 scaffolding domain facilitated by Antennapedia homeodomain attenuates PAF-induced increase in microvessel permeability

We demonstrated previously that inhibition of endothelial nitric oxide synthase (NOS), using pharmacological inhibitors, attenuated the ionomycin- and ATP-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 272: H176-H185, 1997). Recently, the scaffolding domain of caveolin-1 (CAV) has been implicated as a negative regulator of endothelial NOS (eNOS). To examine the role of CAV-eNOS interaction in regulation of permeability in intact microvessels, the effect of internalized CAV on the platelet-activating factor (PAF)-induced permeability increase was investigated in rat mesenteric venular microvessels. Internalization of CAV was achieved by perfusion of individual vessels using a fusion peptide of CAV with Antennapedia homeodomain (AP-CAV) and visualized by fluorescence imaging and electron microscopy. Changes in microvessel permeability were evaluated by measuring hydraulic conductivity (Lp) in individually perfused microvessels. We found that the PAF (10 nM)-induced Lp increase was significantly attenuated from 6.0 +/- 0.9 (n = 7) to 2.0 +/- 0.3 (n = 5) times control after microvessels were perfused with 10 microM AP-CAV for 2 h. The magnitude of this reduction is comparable with that of the inhibitory effect of Nomega-monomethyl-l-arginine on the PAF-induced Lp increase. In contrast, perfusion with 10 microM AP alone for 2 h modified neither basal Lp nor the vessel response to PAF. These results indicate that CAV plays an important role in regulation of microvessel permeability. The inhibitory action of CAV on permeability increase might be attributed to its direct inactivation of eNOS. In addition, this study established a method for studying protein-protein interaction-induced functional changes in intact microvessels and demonstrated AP as an efficient vector for translocation of peptide across the cell membrane in vivo.     

Identification of Vitis vinifera (-)-alpha-terpineol synthase by in silico screening of full-length cDNA ESTs and functional characterization of recombinant terpene synthase

The flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes. Monoterpenes contribute to the final grape and wine aroma and flavour in form of free volatiles and as glycoside conjugates of monoterpene alcohols. Typical monoterpenol components of the cultivar Gewürztraminer and other aroma-rich grape varieties are linalool, geraniol, nerol, citronellol, and alpha-terpineol. In a functional genomics effort to identify genes for the formation of monoterpene alcohols in V. vinifera, a database of full-length cDNA sequences was screened in silico and yielded two clones for putative monoterpene synthases. The gene products were functionally characterized by expression in Escherichia coli, in vitro enzyme assay and gas chromatography-mass spectrometry (GC-MS) product identification as multi-product (-)-alpha-terpineol synthases.     

A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.     

CART peptide: central mediator of leptin-induced adipose tissue apoptosis?

Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague-Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 microl/day), leptin (15 microg/day), or CART55-102 at 2.4 microg/day (CART2.4) or 9.6 microg/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin (p<0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) (p<0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity (p<0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced (p<0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p<0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% (p<0.05) and increased adipose apoptosis by 48% (p<0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain.     

3-(7-Azaindolyl)-4-arylmaleimides as potent, selective inhibitors of glycogen synthase kinase-3

A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.     

Novel non-peptidic neuropeptide Y Y2 receptor antagonists

Through SAR studies of a piperidinylindoline cinnamide HTS lead, the first potent, non-peptide, low molecular weight selective Neuropeptide Y Y2 (NPY Y2) antagonists have been synthesized. The SAR studies around the piperidinyl, the indolinyl, and the cinnamyl moieties are discussed.     

Betaine improves growth, but does not induce whole body or hepatic palmitate oxidation in swine (Sus scrofa domestica)

Dietary betaine may reduce carcass fat in growing pigs. We explored the effects of betaine on short-term growth and in vivo and in vitro fatty acid oxidation. Pigs were housed in metabolism crates and fed diets containing either 0% (control), 0.125% or 0.5% betaine at 80% of ad libitum energy intake. Fatty acid oxidation was measured during intravenous infusions of 1-(13)C-palmitate and in hepatocytes incubated in the presence or absence of betaine and carnitine. CO2 and palmitate isotopic enrichments were determined by mass spectrometry. Pigs consuming 0.125% and 0.5% betaine for at least 9 days had growth rates that were 38% and 12% greater than controls, respectively. Feed efficiency was also improved with betaine. Fasting increased palmitate oxidation rates 7-8-fold (P < 0.01), but betaine had no effect in either the fed or fasted state (P > 0.1). For hepatocytes, carnitine but not betaine enhanced palmitate oxidation. This response suggests that previously observed reduction in adipose accretion must be via a mechanism other than oxidation. Betaine had no effect on plasma non-esterified fatty acids or urea nitrogen. Under the confinement conditions in this study, dietary betaine improved animal growth responses, but it had no apparent effect on either whole body or hepatic fatty acid oxidation.