Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.     

Accumulation of phosphatidic acid in microsomes from propranolol-treated retinas during short-term incubations

Abstract: The pool size and synthesis of phosphatidic acid derived from [2-³H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2-³H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation; labeling decreased rapidly thereafter. In drug-treated retinas,0.5 m M propranolol increased the endogenous content of phosphatidic acid and stimulated [2-³H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short-term incubations and was reversible. In pulse-chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol-induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short-term incubation with propranolol.     

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3′→5′ exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.     

Age-associated changes in central nervous system glycerolipid composition and metabolism

In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.     

Selective localization of phosphatidylcholine-derived signaling in detergent-resistant membranes from synaptic endings

Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats.     

Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice

Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less alpha2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of alpha-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express alpha-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-alpha2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-alpha1 and anti-alpha2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.     

Allozyme variation of oleaster populations (wild olive tree) (Olea europaea L.) in the Mediterranean Basin

As a result of the early domestication and extensive cultivation of the olive tree throughout the Mediterranean Basin, the wild-looking forms of olive (oleasters) presently observed constitute a complex, potentially ranging from wild to feral forms. Allozyme variation was analysed at 10 loci in 31 large and 44 small oleaster populations distributed in various habitats of the Mediterranean Basin and in two populations of the wild subspecies Olea europaea subsp (ssp) guanchica, endemic to the Canary islands and closely related to oleasters. At eight polymorphic loci, 25 alleles were identified. Genetic evidence that nondomesticated oleasters still survive locally was provided by the occurrence of four and one alleles shared exclusively by the eight western and two eastern oleaster populations, respectively, which were collected in forests potentially containing genuinely wild forms according to environmental, historical and demographic criteria. As reported previously from cytoplasmic and RAPDs analysis, substantial genetic differentiation was observed between the eastern oleaster populations genetically close to most olive clones cultivated in the Mediterranean Basin, and the western populations that are related to the wild Canarian populations. In addition, the occurrence of significantly lower heterozygosity in cultivated olive than in oleasters, whatever their origin, suggests that intensive selection involving inbreeding has taken place under cultivation to obtain particular characteristics in the olive cultivars.     

Age-associated changes of insulin action on the hydrolysis of diacylglycerol generated from phosphatidic acid

Age-related changes in insulin action on diacylglycerol (DAG) degradation was studied in rat cerebral cortex synaptosomes. The generation of monoacylglycerol (MAG) and water soluble products (WSP, glycerol plus glycerol-3-phosphate) from DAG was studied in cerebral cortex (CC) synaptosomes from adult (4-month-old) and aged (28-month-old) rats. Additionally, the effect of porcine insulin and tyrosine phosphorylation was evaluated in the same group of animals. In this study we demonstrate that the age-related increase in WSP generation was accompanied by unmodified MAG levels. In the presence of diacylglycerol lipase (DAG lipase) inhibitor, RHC-80267, a lower inhibitory effect on MAG production was observed in CC synaptosomes from aged rats with respect to that in adult membranes. Under these experimental conditions, WSP formation was only diminished in aged membranes. Insulin stimulated MAG and WSP formation at long incubation times (30 min) in adult animals, while it had an inhibitory effect in aged animals. Insulin plus vanadate (as tyrosine-phosphatase inhibitor) inhibited MAG production at short incubation times whereas the same effect was observed in aged animals at long times of incubation. WSP formation was stimulated by insulin plus vanadate both in adult and aged animals at 30 min of incubation. Our results show that insulin differentially modulates MAG and WSP production from exogenous PA in CC synaptosomes from aged rats compared with adult rats.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

A general role for surface membrane proteins in attachment to chitin particles and copepods of environmental and clinical vibrios

AIMS: To investigate the role of surface membrane proteins (MP) to promote attachment to chitin particles and copepods of different environmental and clinical vibrios. METHOD AND RESULTS: The role of surface MP to promote attachment to chitin particles and the copepod Tigriopus fulvus was investigated in several environmental and clinical Vibrio strains by inhibition test methods. Attachment to both substrates was significantly inhibited by homologous MP treatment in all strains and percentages of inhibition were comparable with the ones observed with N-acetyl glucosamine (GlcNAc). Sarkosyl-insoluble MP extracted from tested strains were added to chitin particles either in the presence or in the absence of GlcNAc and the fraction bound to chitin in both conditions was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitin-binding proteins (CBP) defined as Sarkosyl-insoluble MP that bound chitin in the absence of GlcNAc but did not in the presence of the sugar were isolated in all strains. CONCLUSION: CBP are common in both environmental and clinical Vibrio strains and they have an important general role in mediating cell interactions with chitin-containing surfaces. SIGNIFICANT AND IMPACT OF THE STUDY: The role of CBP should be taken into account when investigating environmental persistence of aquatic vibrios.