Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR.

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.     

Basal activation of p70S6K results in adipose-specific insulin resistance in protein-tyrosine phosphatase 1B -/- mice

Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance.     

Identification of six putative human transporters with structural similarity to the drug transporter SLC22 family

The solute carrier family 22 (SLC22) is a large family of organic cation and anion transporters. These are transmembrane proteins expressed predominantly in kidneys and liver and mediate the uptake and excretion of environmental toxins, endogenous substances, and drugs from the body. Through a comprehensive database search we identified six human proteins not yet cloned or annotated in the reference sequence databases. Five of these belong to the SLC22 family, SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, and the sixth gene, SVOPL, is a paralog to the synaptic vesicle protein SVOP. We identified the orthologs for these genes in mouse and rat and additional homologous proteins and performed the first phylogenetic analysis on the entire SLC22 family in human, mouse, and rat. In addition, we performed a phylogenetic analysis which showed that SVOP and SV2A-C are, in a comparison with all vertebrate proteins, most similar to the SLC22 family. Finally, we performed a tissue localization study on 15 genes on a panel of 30 rat tissues using quantitative real-time polymerase chain reaction.     

SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.     

Models of sequestration and receptor cross-talk for explaining multiple mutants in plant stem cell regulation

Background  

Stem cells reside in a plant's shoot meristem throughout its life and are main regulators of above-ground plant development. The stem cell maintenance depends on a feedback network between the CLAVATA and WUSCHEL genes. The CLAVATA3 peptide binds to the CLAVATA1 receptor leading to WUSCHEL inhibition. WUSCHEL, on the other hand, activates CLAVATA3 expression. Recent experiments suggest a second pathway where CLAVATA3 inhibits WUSCHEL via the CORYNE receptor pathway. An interesting question, central for understanding the receptor signaling, is why the clavata1-11 null mutant has a weaker phenotype compared with the clavata1-1 non-null mutant. It has been suggested that this relies on interference from the mutated CLAVATA1 acting on the CORYNE pathway.     

Antisecretory factor modulates GABAergic transmission in the rat hippocampus

Antisecretory Factor (AF) is a protein that has been implicated in the suppression of intestinal hypersecretion and inflammation. Intestinal secretion and inflammation are partly under local and central neural control raising the possibility that AF might exert its action by modulating neural signaling. In the present study we have investigated whether AF can modulate central synaptic transmission. Evoked glutamatergic and GABAergic synaptic transmissions were investigated using extracellular recordings in the CA1 region of hippocampal slices from adult rats. AF (0.5 microg/ml) suppressed GABA(A)-mediated synaptic transmission by about 40% while having no effect on glutamatergic transmission. Per oral administration of cholera toxin as well as feeding of rats with a diet containing hydrothermally processed cereals, known to upregulate endogenous AF plasma activity, mimicked the effect of exogenously administered AF on hippocampal GABAergic transmission. Our results identify AF as a neuromodulator and further raise the possibility that the hippocampus and AF are involved in a gut-brain loop controlling intestinal secretion and inflammation.     

The verprolin family of proteins: regulators of cell morphogenesis and endocytosis

The verprolin family of proteins, WIP, CR16 and WIRE/WICH, has emerged as critical regulators of cytoskeletal organisation in vertebrate cells. The founding father of the family, verprolin, was originally identified in budding yeast and later shown to be needed for actin polymerisation during polarised growth and during endocytosis. The vertebrate verprolins regulate actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. Interestingly, also the vertebrate verprolins have been implicated in endocytosis, demonstrating that most of the functional modules in this fascinating group of proteins have been conserved from yeast to man.