Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 μM DA or a dopaminergic D₁ agonist, fenoldopam, but not with the dopaminergic D₂ agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase α1 and β1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-δ, or isozyme-specific inhibitor peptides for PKC-δ or PKC-ε inhibited the DA-mediated increase in Na,K-ATPase. PKC-δ and PKC-ε, but not PKC-α or -β, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-δ– and PKC-ε–specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-δ and PKC-ε.     

Chemistry,distribution, and metabolism of tomato carotenoids and their impact on human health

Recent epidemiological studies have suggested that the consumption of tomatoes and tomato-based food products reduce the risk of prostate cancer in humans. This protective effect has been attributed to carotenoids, which are one of the major classes of phytochemicals in this fruit. The most abundant carotenoid in tomato is lycopene, followed by phytoene, phytofluene, zeta-carotene, gamma-carotene, beta-carotene, neurosporene, and lutein. The distribution of lycopene and related carotenoids in tomatoes and tomato-based food products has been determined by extraction and high-performance liquid chromatography-UV/Visible photodiode array detection. Detailed qualitative and quantitative analysis of human serum, milk, and organs, particularly prostate, have revealed the presence of all the aforementioned carotenoids in biologically significant concentrations. Two oxidative metabolites of lycopene, 2,6-cyclolycopene-1,5-diols A and B, which are only present in tomatoes in extremely low concentrations, have been isolated and identified in human serum, milk, organs (liver, lung, breast, liver, prostate, colon) and skin. Carotenoids may also play an important role in the prevention of age-related macular degeneration, cataracts, and other blinding disorders. Among 25 dietary carotenoids and nine metabolites routinely found in human serum, mainly (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, lycopene, and their metabolites were detected in ocular tissues. In this review we identified and quantified the complete spectrum of carotenoids from pooled human retinal pigment epithelium, ciliary body, iris, lens, and in the uveal tract and in other tissues of the human eye to gain a better insight into the metabolic pathways of ocular carotenoids. Although (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their metabolites constitute the major carotenoids in human ocular tissues, lycopene and a wide range of dietary carotenoids have been detected in high concentrations in ciliary body and retinal pigment epithelium. The possible role of lycopene and other dietary carotenoids in the prevention of age-related macular degeneration and other eye diseases is discussed.     

Metabolic and therapeutic lessons from genetic manipulation of GLUT4

This review focuses on the effects of varying levels of GLUT4, the insulin-sensitive glucose transporter, on insulin sensitivity and whole body glucose homeostasis. Three mouse models are discussed including MLC-GLUT4 mice which overexpress GLUT4 specifically in skeletal muscle, GLUT4 null mice which express no GLUT4, and the MLC-GLUT4 null mice which express GLUT4 only in skeletal muscle. Overexpressing GLUT4 specifically in the skeletal muscle results in increased insulin sensitivity in the MLC-GLUT4 mice. In contrast, the GLUT4 null mice exhibit insulin intolerance accompanied by abnormalities in glucose and lipid metabolism. Restoring GLUT4 expression in skeletal muscle in the MLC-GLUT4 null mice results in normal glucose metabolism but continued abnormal lipid metabolism. The results of experiments using these mouse models demonstrates that modifying the expression of GLUT4 profoundly affects whole body insulin action and consequently glucose and lipid metabolism.     

Influence of 50 Hz electromagnetic fields on recurrent embryogenesis and germination of cork oak somatic embryos

Plant tissue culture techniques are carried out under environmentally controlled conditions in phytotrons. However, electric components of phytotrons generate electromagnetic fields that may act as a environmental factor influencing plant growth and morphogenesis. Isolated somatic embryos of Quercus suber, picked from embryogenic lines, were chronically exposed to a 50 Hz and 15 μT electromagnetic field generated in a Helmholtz-coil system for 8 weeks, in order to examine if the extremely low frequency (ELF) magnetic field (MF) affected the morphogenic behaviour of embryogenic cultures during recurrent embryogenesis. Germination of somatic embryos from genotype G7.1 was carried out under the same electromagnetic field, and also under conditions in which the local geomagnetic field was suppressed. The ELF MF did not influence the growth of embryogenic clumps of the assayed genotypes, but reduced the number of detachable embryos produced by genotype G3.27. The ELF MF did not modify the percentages of germination or plant formation of somatic embryos. However, somatic embryos had better germination when cultured under the suppressed geomagnetic field condition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex.

In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.     

Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.     

Genetic organization of the duplicated vap region of the Dichelobacter nodosus genome.

The recombinant plasmid pJIR318 contains a fragment of the Dichelobacter nodosus genome which is associated with virulence. Sequence analysis of the pJIR318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the Escherichia coli F plasmid and the Neisseria gonorrhoeae cryptic plasmid (M. E. Katz, R. A. Strugnell, and J. I. Rood, Infect. and Immun. 60:4586-4592, 1992). The plasmid pJIR318 hybridizes to three regions of the D. nodosus genome, each of which has now been isolated. Regions 1 and 3 were found to be adjacent in the genome of D. nodosus A198, and the order of the vap genes in vap regions 1 and 2 were shown to be identical. Partial sequence analysis and Southern blot analysis of the vap regions showed that the three regions probably arose by a duplication event(s) followed by insertions and/or deletions. A recombinant plasmid, pJIR749, was isolated from a library of a benign D. nodosus strain, 305. This plasmid contained sequences from both ends of vap region 2. Analysis of pJIR749 showed that the sequences on either side of vap region 2 were separated by 324 bp in the genome of benign strain 305 and that the orientations of the sequences were different. It is clear that a simple insertion or deletion event did not generate the benign and virulent strains studied. A model which describes the evolution of the duplicated vap regions in D. nodosus A198 is presented.     

Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease virus in chickens

Abstract Dimethyl diotadecyl ammonium bromide (DDA), a lipophilic quaternary amine, was evaluated in adult chickens for potentiation of immunological responses to subcutaneously administered inactivated Newcastle disease virus (NDV) vaccines. DDA enhanced humoral and cell-mediated immune (CMI) responses to levels which were significantly higher than those induced by the vaccine alone The haemagglutination inhibition antibody titers induced by DDA were slightly lower than those induced by mineral oil although neutralizing antibody titers seemed to be higher. DDA induced strong CMI (DTH and lymphocyte proliferation) responses, more than those induced by Freund's complete adjuvant and mineral oil. Both DDA and mineral oil induced comparable high levels of protection to challenge doses of 200 000 LD₅₀ per chicken. No toxic effects or local tissue damage were observed in any of the inoculated chickens.