Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.     

Impacts of climate warming on lake fish community structure and potential effects on ecosystem function

Fish play a key role in the trophic dynamics of lakes, not least in shallow systems. With climate warming, complex changes in fish community structure may be expected owing to the direct and indirect effects of temperature, and indirect effects of eutrophication, water-level changes and salinisation on fish metabolism, biotic interactions and geographical distribution. We review published and new data supporting the hypotheses that, with a warming climate, there will be changes in: fish community structure (e.g. higher or lower richness depending on local conditions); life history traits (e.g. smaller body size, shorter life span, earlier and less synchronised reproduction); feeding mode (i.e. increased omnivory and herbivory); behaviour (i.e. stronger association with littoral areas and a greater proportion of benthivores); and winter survival. All these changes imply higher predation on zooplankton and macroinvertebrates with increasing temperatures, suggesting that the changes in the fish communities partly resemble, and may intensify, the effects triggered by eutrophication. Modulating factors identified in cold and temperate systems, such as the presence of submerged plants and winter ice cover, seem to be weaker or non-existent in warm(ing) lakes. Consequently, in the future lower nutrient thresholds may be needed to obtain clear-water conditions and good ecological status in the future in currently cold or temperate lakes. Although examples are still scarce and more research is needed, we foresee biomanipulation to be a less successful restoration tool in warm(ing) lakes without a strong reduction of the nutrient load.     

Understanding the Anodic Mechanism of a Seafloor Fuel Cell: Interactions Between Geochemistry and Microbial Activity

Seafloor fuel cells made with graphite electrodes generate electricity by promoting electron transfer in response to a natural voltage difference (−0.7 to −0.8 V) between anoxic sediments and overlying oxic seawater. Geochemical impacts of a seafloor fuel cell on sediment solids and porewaters were examined to identify the anodic mechanisms and substrates available for current production. In an estuarine environment with little dissolved sulfide, solid-phase acid volatile sulfide and Cr²⁺-reducible sulfur minerals decreased significantly toward the anode after 7 months of nearly continuous energy harvesting. Porewater iron and sulfate increased by millimolar amounts. Scanning electron microscope images showed a biofilm overcoating the anode, and electron microprobe analyses revealed accumulations of sulfur, iron, silicon and phosphorus at the electrode surface. Sulfur deposition was also observed on a laboratory fuel cell anode used to generate electricity with only dissolved sulfide as an electron donor. Moreover, current densities and voltages displayed by these purely chemical cells were similar to the values measured with field devices. These results indicate that electron transfer to seafloor fuel cells can readily result in the oxidation of dissolved and solid-phase forms of reduced sulfur producing mainly S⁰ which deposits at the electrode surface. This oxidation product is consistent with the observed enrichment of bacteria most closely related to Desulfobulbus/Desulfocapsa genera within the anode biofilm, and its presence is proposed to promote a localized biogeochemical cycle whereby biofilm bacteria regenerate sulfate and sulfide. This electron-shuttling mechanism may co-occur while these or other bacteria use the anode directly as a terminal electron acceptor.     

Influence of cholecystokinin on central monoaminergic pathways

Dopamine (DA) and cholecystokinin octapeptide carboxy-terminal (CCK-8) have been found to coexist in some mesolimbic neurons. The present investigation was undertaken in order to study the biochemical and behavioral interactions between CCK-8 and some central monoaminergic pathways. The action of the sulfated form of CCK-8 (10 micrograms/10 microliter intracerebroventricularly) on DA turnover in nucleus accumbens, olfactory tubercles and corpus striatum of the rat was determined after DA synthesis inhibition with alpha-methyl-p-tyrosine (250 mg/kg i.p.). Also, CCK-8 action (1-30 micrograms intracisternally) on DA synthesis was assessed by measuring accumulation of dihydroxyphenylalanine (DOPA) after DOPA-decarboxylase inhibition with NSD-1015 (m-hydroxybenzylhydrazine, 100 mg/kg i.p.). The contents of DA and its main metabolites, dihydroxyphenylacetic acid and homovanillic acid, together with serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in different brain areas after direct injection of CCK-8 into the ventral tegmental area (A10) or nucleus accumbens. Further, the effect of CCK-8 on amphetamine-induced locomotion and apomorphine-induced stereotypies was studied along with changes in spontaneous locomotion and rearing after CCK-8 injection into the ventral tegmental area and nucleus accumbens. No consistent statistically significant effects of CCK-8 on biochemical or behavioral assessments on measures of DA function were observed. However, injection of high doses of CCK-8 into the ventral tegmental area significantly decreased levels of 5-HIAA in the nucleus accumbens, olfactory tubercles and striatum.     

Some like it hot: body and weapon size affect thermoregulation in horned beetles

Body size and shape affect thermoregulatory properties of organisms, and in turn are believed to have shaped macroevolutionary patterns of morphological diversity across many taxa. However, it is less clear whether thermoregulation plays a role in shaping intraspecific morphological diversity such as sexual dimorphisms or the conditional expression of exaggerated secondary sexual traits. Here, we investigate individual thermoregulatory properties in two species of horned beetles that share similar ecologies and body size ranges, but differ substantially in degree of sexual and male dimorphism. We find that intraspecific variation in body size had an unexpectedly large effect on thermal preference behavior and the ability to passively regulate body temperature. Furthermore, we find that the presence or absence of exaggerated secondary sexual traits dramatically altered thermal preference behavior, consistent with a thermoregulatory cost of horn possession. Lastly, we show that the increase in surface area associated with the expression of enlarged horns is, by itself, insufficient to account for the radically altered thermoregulatory behavior observed in horn-bearing males, and discuss possible alternative, physiological explanations. These findings are among the first to link intra-and interspecific variation in body- and weapon size to thermal preferences within and between insect species.     

Administration of chlordiazepoxide affects [3H][3-MeHis2]thyrotropin-releasing hormone binding in rat brain.

The effects of acute and subchronic administration of chlordiazepoxide (CDZ) on [3H][3-methyl-histidyl2]thyrotropin-releasing hormone binding to thyrotropin-releasing hormone (TRH) receptors in membrane preparations from various regions of rat brain were examined. Acute administration of CDZ (50 mg/kg x 3 within 24 h) did not alter either the equilibrium dissociation constant (Kd) or the maximum number of binding sites (Bmax) in cerebellum (CB), olfactory bulbs (OB), frontal cortex (Cx), hypothalamus (HT) or corpus striatum (ST). However, the Kds of the pyriform cortex/amygdala (PC/A) (Kd = 3.6 +/- 0.1 nM compared to 1.9 +/- 0.1 nM in the control group; p less than 0.01) and the hippocampus (HP) (Kd = 7.8 +/- 0.7 nM compared to 2.1 +/- 0.1 nM in the control group; p less than 0.01) were increased. There were no changes in Bmax. Subchronic administration of CDZ (50 mg/kg/day for 7 days) increased the Kd of the PC/A complex (p less than 0.05), the OB (p less than 0.05) and the HP (p less than 0.01) without altering in Bmax. These results, showing regional differences in the response of TRH receptors to acute and subchronic CDZ administration, suggest that reduced affinity of TRH receptors in the PC/A complex, OB and HP may be related to some of the neurobiological actions of CDZ and/or its metabolites.     

The Arabidopsis STICHEL gene is a regulator of trichome branch number and encodes a novel protein

Here, we analyze the STICHEL (STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis. We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles. The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III gamma-subunits. Because endoreduplication was found to be normal in sti mutants the molecular function of STI in cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway. Northern-blot analysis shows that STI is expressed in all organs suggesting that STI function is not trichome specific. The analysis of sti alleles and transgenic lines overexpressing STI suggests that STI regulates branching in a dosage-dependent manner.     

Chaperones involved in hepatitis B virus morphogenesis

Little is known about host cell factors necessary for hepatitis B virus (HBV) assembly which involves envelopment of cytosolic nucleocapsids by the S, M and L transmembrane viral envelope proteins and subsequent budding into intraluminal cisternae. Central to virogenesis is the L protein that mediates hepatocyte receptor binding and envelopment of capsids. To serve these topologically conflicting roles, L protein exhibits an unusual dual membrane topology, disposing its N-terminal preS domain inside and outside of the virion lipid envelope. The mixed topology is achieved by posttranslational preS translocation of about half of the L protein molecules across a post-endoplasmic reticulum membrane. Here we identify and characterize a preS-specific sequence that confers the suppression of cotranslational translocation even of a model reporter. This cytosolic anchorage sequence specifically binds the cognate heat shock protein Hsc70, thus indicating chaperone participitation in HBV morphogenesis. Conversely, the M envelope protein needs the assistance of the chaperone calnexin for proper folding and trafficking. Calnexin selectively binds to the N-glycan, specific for M, rather than to the N-glycan, common to all three envelope proteins. As inhibition of the calnexin-M interaction blocks the secretion of viral envelopes, we propose an essential role for calnexin, as well as for Hsc70, in chaperoning HBV assembly.