L-Fucose Is a Potent Inhibitor of myo-Inositol Transport and Metabolism in Cultured Neuroblastoma Cells

It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.     

Coordinate Regulation of the Cyclic AMP System with Firing Rate and Expression of Tyrosine Hydroxylase in the Rat Locus Coeruleus: Effects of Chronic Stress and Drug Treatments

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amygdala Kindling Alters Protein Kinase C Activity in Dentate Gyrus

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.     

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue,cell, and model membrane preparations

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Behavioral responses of female oriental fruit flies to the odor of papayas at three ripeness stages in a laboratory flight tunnel (Diptera: Tephritidae)

Behavioral responses of adult female oriental fruit flies, Dacus dorsalisHendel, to the odor of papayas from three ripeness classes were studied using a threechoice flight tunnel bioassay. Laboratoryreared flies were allowed to respond freely to any of three papaya odors (mature green, colorbreak to one-fourth ripe, and one-half to full ripe) emanating from identical (spherical) fruit models. Five behaviors were measured in assessing the fly's relative attraction to the odors (number of landings), arrestment (total fly seconds on sphere), fly-fly interactions on the fruit model (maximum and modal fly density), and acceptance for oviposition (total eggs laid). Females showed no significant difference in total fly landings based on all age classes combined. Significant differences were noted among age classes. Females spent more total time on the sphere and showed a higher maximum density and modal fly density to ripe fruit than to green fruit odors. Ovipositional acceptance of fruit models based on the total number of eggs laid in a sphere was greater in response to the ripefruit odor than to the other two odor classes. Olfactorystimulated behavioral responses of females to the odor of ripe papayas were significantly different from the other ripeness classes for all behaviors at 8 days postemergence and then declined in 11-day-old flies. Behavioral responses were greater during the afternoon than in the morning. Observations of wild oriental fruit flies to papayas in the field indicated a preference for residing on riper fruit. The results of this study are discussed with regard to the role of olfactory inputs generated by the odor of ripening fruit on female attraction and oviposition behavior resulting in infestation of papayas by oriental fruit fly.     

Benzoyl cellulose beads in the pure polymeric form as a new powerful sorbent for the chromatographic resolution of racemates

Cellulose-based stationary phases are known to be very efficient and versatile chiral sorbents for the chromatographic resolution of racemates. Except for microcrystalline cellulose triacetate (CTA I), basically all other cellulose-based phases have been prepared by coating of ca. 20% weight polymer on a wide pore silica gel used as a carrier. In this work we describe the preparation of benzoylcellulose (TBC) beads in the pure polymeric form (without inorganic carrier) from an emulsion of the organic polymer. The new material has been fully characterized and used as a chiral stationary phase for the resolution of various classes of racemic compounds such as benzylic alcohols or acetate derivatives of aliphatic alcohols and diols. The structural variety of the separated solutes as well as the irrational influence of the aromatic substituent in different classes of aryl compounds suggest that multiple interaction sites are involved in the complexation, making a prediction of the separation difficult. The benzoyl cellulose beads exhibit a very high loading capacity, which is particularly useful for preparative purposes as demonstrated for selected examples.