Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

Cadmium-2-acetylaminofluorene interaction in isolated rat hepatocytes

Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 M) of ¹⁰⁹Cd. This was followed by a 10 hr exposure to 1 M [³H]AAF. Cellular distribution of Cd and ³H was determined. Sephadex G-75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of ³H was found in the low molecular weight components. Further analysis of IMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some ³H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of ³H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.Abbreviations AAF 2-acetylaminofluorene - Cd cadmium - DMSO dimethyl sulfoxide - HBSS Hank's balanced salt solution - LDH lactate dehydrogenase - MT metallothionein - UDS unscheduled DNA synthesis     

Sustained primary culture of lobster (Panulirus argus) olfactory receptor neurons

Appropriate conditions were developed for primary sustained culture of olfactory neurons of the spiny lobster Panulirus argus. Neurons were cultured in a modified Liebowitz L15 media supplemented with Panulirus salts, basic minimal essential (BME) vitamins, L-glutamine, low dextrose, and either fetal calf serum (FCS) or lobster haemolymph. Neurite outgrowth and cell viability was strongly affected by choice of adherent substratum, presence of serum, and length of animal captivity. Neither nerve growth factor 7s (NGF-7s), HEPES, nor preconditioned media from the target organ, the olfactory lobe, had any gross effect on either longevity or neurite outgrowth. Five morphologically distinct neuronal cell types (8-16 mum soma diameter) could be defined based on their number and type of processes. All of these cells were electrically excitable (N = 50), and many (56%) produced either inward or outward currents in response to stimulation with single odors. The proportion of cells responding to odors increased (80%) when 10 cells were sequentially presented with a series of 3-5 odors. The finding that cultured cells maintain responsiveness to odors yet are morphologically more compact than their counterparts in situ, argues for the prospect of using these dissociated cultured olfactory receptor neurons to study signal transduction in olfaction.     

Novel DNA structures resulting from dTam3 excision in tobacco

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated thattrans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing thetrans-activation of a dTam3 element, resulting in aberrant excision.     

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.     

The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot.

Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot. Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB. The position of terB and the flanking sequences had little effect on deletion hot spot activity. About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site. We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.     

Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation

Summary Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G₅₀) and 36,000 (G₃₆), as determined on SDS-gels. G₃₆ was ADP-ribosylated by pertussis toxin. Thus, G₅₀ could represent a G_sα subunit, whereas G₃₆ could be G_iα or G_oα. G₅₀ was phosphorylated by cAMP dependent protein kinase and protein kinase C. G₃₆ was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G₃₆ by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities withras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.     

Seasonal change in photoperiodic response of the larvae of the lacewing Nineta pallida

Larvae of the lacewing Nineta pallida (Schneider), collected in the field during two seasons, from September to July, were reared in the laboratory under short- or long-day light conditions at 21°C. In autumn and winter, artificial short days delayed the first ecdysis. The influence on the duration of the first instar was maximal (3.4 times longer) when the short days began at hatching time, and later regularly diminished. In spring, the second and third instars showed a reversed response so that the long days now increased the duration of development, although development took no more than 1.4 time as long as in short days. A similar effect appeared in field-collected third instars on and after mid June, reaching its maximum (1.8 time until the cocoon spinning) in July. This sort of photoperiodic effect on the larval development is new to the seasonal adaptation of the life cycle in insects.

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Résumé Des formes préimaginales (oeufs, puis larves) de N. pallida sont récoltées sur des conifères de montagne (Pyrénées), chaque mois depuis septembre jusqu'en juillet en deux saisons (1983/84 et 1985/86). Elles sont ensuite élevées au laboratoire à 21°C, soit en jours longs (JL=L16:D8), soit en jours courts (JC=L8:D16).Le développement embryonnaire est légèrement plus long s'il se fait en JC. Pour les larves de premier stade récoltées en automne et en hiver, les JC retardent considérablement la première mue et prolongent aussi le deuxième stade qui en provient. L'influence retardatrice est maximale (3,4 fois) lorsque les JC agissent dès l'éclosion. Elle diminue ensuite progressivement et devient insignificante pour les larves récoltées à artir de février.Au printemps, les larves récoltées au deuxième stade ainsi que les troisièmes stades qui en découlent présentent une réaction inverse: ce sont alors les JL qui augmentent la durée du dévelopement, toutefois, pas plus de 1,4 fois par rapport aux JC. Un effet de même ordre se manifeste sur les larves de troisième stade récoltées à partir de juin, atteignant son maximum (1,8 fois) dans le lot de larves de juillet, c'est-à-dire peu avant la fin de la croissance pondérale larvaire et le coconnage.Un tel retardement du développement larvaire hivernal, prolongé au printemps et au début de l'été par une inversion de la réponse à la photopériode, est nouveau comme élément d'adaptation saisonnière du cycle naturel chez les insectes.