Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Airborne <Emphasis Type="Italic">Legionella</Emphasis> bacteria from pulp waste treatment plant: aerosol particles characterized as aggregates and their potential hazard

In response to reported outbreaks of Legionnaires’ disease near a biological waste treatment plant, Legionella-containing aerosols were characterized at this location. Culturable bacteria-containing particles were assessed with slit-to-agar samplers and liquid impingement. Results showed that the air at this location contained a higher level of airborne Legionella particles compared to that of a control clean location 90 km away. On average, about 1 of 50 culturable particles contained Legionella (2%). The median particle size was estimated to 3.5 μm permitting an estimation of a maximum about 147 Legionella cells per aggregate. This implies that in <11 h, a human under such conditions (worse-case), may inhale an estimated lethal dose of individual Legionella cells (1,000 cells, 100% alveolar retained deposition level). Additionally, 44 taxonomically different airborne bacterial genera were measured at the treatment plant using denaturing gradient-gel electrophoresis (DGGE) and 16S rDNA analysis of air samples. Of these, 64% of the genera were not detected at the clean location. Our findings suggest that the treatment facility may emit respirable particles, as complex aggregates, containing a mixture of several Legionella cells and other bacterial cells. These findings can potentially have an impact on assessing environmental risk exposure and health hazard prediction modeling.     

Uptake of Glycine into Synaptic Vesicles Isolated from Rat Spinal Cord

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.     

Development of Benzodiazepine and Picrotoxin (t-Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.     

Effects of ketamine anaesthesia, stress and repeated bleeding on the haematology of vervet monkeys

Haematology values are presented for the vervet monkey (Cercopithecus aethiops), and the relative effects of high dose ketamine anaesthesia, stress of capture and repeated bleedings assessed. Anaesthesia resulted in decreased WBC and RBC values, attributed to depression of cardiovascular function. These effects were the reverse of those of alarm and strenuous exercise (leukocytosis and polycythaemia) during capture. Stress resulted in relatively high white and low red blood cell counts. Opposing effects of stress and anaesthesia led to comparable haematological values for trained, non-anaesthetized vervets and stressed, anaesthetized vervets. Effects of repeated bleedings were opposite in anaesthetized and non-anaesthetized animals. These effects, however, along with those of ketamine anaesthesia and stress, were relatively insignificant compared with the wide variation in haematological values found among individuals. The biological importance of these effects thus appeared to be slight. The concept of 'normal values' is discussed.     

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.     

Clozapine and Some Other Antipsychotic Drugs May Preferentially Block the Same Subset of GABAA Receptors

Selective blockade of a subset of GABA_A receptors may be involved in the antipsychotic effects of Clozapine and several other antipsychotic drugs. Seven antipsychotic drugs, and 11 drugs classified as antidepressants that only partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, do not yield additive reversal when tested pairwise with Clozapine, which also only partially reverses the inhibitory effect of GABA. This suggests that all of these antipsychotic/antidepressant drugs may block a common subset of GABA_A receptors. DMCM and Ro 5-4864 are also partial reversers of GABA's inhibitory effect, but they yield additive reversals when tested pairwise with the antipsychotic/antidepressant drugs, and also with each other, suggesting that DMCM, Ro 5-4864, and the antipsychotic drugs define three heterogeneous subsets of GABA_A receptors, with variable overlap, depending on the drug. Several potent ligands for benzodiazepine binding sites can block the GABA inhibitory effects of DMCM and Ro 5-4864, but with different patterns: the ligands generally blocked DMCM less potently, but more completely than Ro 5-4864, Ro 5-4864 was not blocked by Flumazenil or CGS-8216, ligands that potently blocked DMCM. Nine additional antipsychotic/antidepressant drugs, as well as Clozapine, and 7 classical GABA_A receptor blockers, all of which reversed GABA nearly completely, when tested at lower concentrations that only reverse 20–35%, yielded almost complete additivity when tested pairwise with DMCM or Ro 5-4864. Another convulsant benzodiazepine, KW-1937, a positional isomer of Brotizolam, fully reverses the inhibitory effect of 1 M GABA. At a lower concentration yielding about 50% reversal, KW-1937 is completely additive with DMCM, but entirely nonadditive with Ro 5-4864. The 50% reversal obtained with KW-1937 was potently blocked by Triazolam, but with a plateau similar to that obtained with Ro 5-4864. The results with KW-1937 suggest that its 50% reversal largely corresponds to the reversal obtained with Ro 5-4864, and that virtually all of the [³⁵S]TBPS binding sites inhibited by 1 M GABA are coupled to benzodiazepine binding sites. The fraction of GABA_A receptors preferentially blocked by all the antipsychotic/antidepressant drugs, roughly 25% of the [³⁵S]TBPS binding sites inhibited with 1 M GABA, are sensitive to KW-1937, but not to DMCM or to Ro 5-4864.     

Seasonal variation in light- and temperature-dependent growth of marine planktonic diatoms in in situ dialysis cultures in the Trondheimsfjord,norway (63°N)

Three species of diatoms, Skeletonema costatum (Grev.) Cleve, Thalassiosira gravida Cleve, and T. pseudonana (Hustedt) Hasle et Heimdal, were grown in in situ dialysis culture in the Trondheimsfjord at depths of 0.5 and 4 m. The rates of growth and the chemical composition of exponentially growing cells were monitored and related to seasonal changes in illumination and temperature. Functions correlating growth rate with temperature were deduced. Growth took place from February to November. During this period temperature ranged from ?1 to 16°C, the average photon flux density (ifI) (per 24 h) from 9 to 570 μE · m^?2 · s^?1 (0.5 m depth), and the length of the days (I > 1 μE · m^?2 · s^?1) from 6 to 24 h. Light-limited growth was evident when the product of the average daily light and the chlorophyll/N ratio was < 10; this occurred mostly in early spring and late autumn. Peak densities (> 800 for the Thalassiosira spp. and > 1300–1400 μE · m^?2 · s^?1 for Skeletonema) seem to inhibit growth. The highest rates recorded were ≈1.6 doubl. · day^?1 (July, 15–16°C).The three species exhibit different ecological behaviour. Skeletonema is eurythermal (Q₁₀ = 1.8), whereas Thalassiosira pseudonana favours high temperatures, and T. gravida temperatures < 10°C. Moreover, Skeletonema has generally less chlorophyll and more phosphorus and ATP (≈ 1.4 ×) than the other two species. In Skeletonema, the ATP level seems related to the light-governed growth rate, and independent of temperature. In Thalassiosira no such correlation was found.     

Influences of separation and adjacent sequences on the use of alternative 5' splice sites.

Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.