K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Ancient teeth and modern human origins: an expanded comparison of African Plio-Pleistocene and recent world dental samples

Previous research by the first author revealed that, relative to other modern peoples, sub-Saharan Africans exhibit the highest frequencies of ancestral (or plesiomorphic) dental traits and, thus, appear to be least derived dentally from an ancestral hominin state. This determination, in conjunction with various other lines of dental morphological evidence, was interpreted to be supportive of an African origin for modern humans. The present investigation expands upon this work by using: 1) direct observations of fossil hominin teeth, rather than data gleaned from published sources, 2) a single morphological scoring system (the Arizona State University Dental Anthropology System) with consistent trait breakpoints, and 3) data from larger and more varied modern human comparative samples. As before, a multivariate distance statistic, the mean measure of divergence, was used to assess diachronic phenetic affinities among the Plio-Pleistocene hominins and modern humans. The present study also employed principal components analysis on dental trait frequencies across samples. Both methods yielded similar results, which support the previous findings; that is, of all modern human samples, sub-Saharan Africans again exhibit the closest phenetic similarity to various African Plio-Pleistocene hominins-through their shared prevalence of morphologically complex crown and root traits. The fact that sub-Saharan Africans express these apparently plesiomorphic characters, along with additional information on their affinity to other modern populations, evident intra-population heterogeneity, and a world-wide dental cline emanating from the sub-continent, provides further evidence that is consistent with an African origin model.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Biomarkers of oxidative stress are significantly elevated in Down syndrome

There is convincing epidemiological and in vitro evidence of chronic oxidative stress in individuals with Down syndrome (DS). These individuals develop Alzheimer like changes in the brain in their 30s and 40s. The incidence of autoimmune diseases and cataracts is significantly increased, and the overall ageing process is accelerated. In vitro studies show that impaired viability of DS neurons may be amended by simple chemical antioxidants, such as vitamin E, BHT and propyl gallate, clearly indicative of oxyl radical involvement. However, because of the lack of in vivo experiments, the role of oxidative stress in DS remains controversial. We report here on the results of the chemical analyses of urine samples of 166 individuals, where DS subjects were matched by their siblings. The levels of 8-hydroxy-2′-deoxyguanosine (2.35 ± 1.69 in DS vs. 1.35 ± 1.04 in controls, P = 0.00011), a biomarker of oxidative damage to DNA, and malondialdehyde (0.255 ± 0.158 in DS vs. 0.204 ± 0.128 in controls, P = 0.033), a biomarker of lipid peroxidation, are significantly elevated in individuals with DS. Dietary influences failed to show any significant correlation with the oxidative stress biomarkers. These results provide direct evidence for increased oxidative stress in individuals with DS.     

Direct measurement of the adhesive strength of biofilms in pipes by micromanipulation

A novel micromanipulation technique has been developed to measure directly the adhesive strength of biofilms formed in pipe flows. A T-shaped probe was specially designed to pull Pseudomonas fluorescens biofilms away from the inner surface of a pipe to which they were attached. The adhesive strength between biofilms and the substratum was defined as the work required to remove the biofilms per unit area from the substratum. Results showed that the adhesive strength increased with the fluid velocity in which the biofilms were grown, with a typical value of 0.050.2 J/m2. © Rapid Science Ltd. 1998     

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well-defined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.     

Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy

Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques.     

The economics of producing sustainable aviation fuel: a regional case study in Queensland,Australia

The airline industry has a strong interest in developing sustainable aviation fuels, in order to reduce their exposure to increasing oil prices and cost liability for greenhouse gas emissions. The feasibility and cost of producing sustainable biomass‐based jet fuels at a sufficient scale to materially address these issues is an enormous challenge. This paper builds directly on the biophysical study by H.T. Murphy, D.A. O'Connell, R.J. Raison, A.C. Warden, T.H. Booth, A. Herr, A.L. Braid, D.F. Crawford, J.A. Hayward, T. Javonovic, J.G. McIvor, M.H. O'Connor, M.L. Poole, D. Prestwidge, N. Raisbeck‐Brown & L. Rye, In review, which examined a 25 year scale‐up strategy to produce 5% of projected jet fuel demand in Australia in 2020 (470 mL) in the Fitzroy region of Queensland, Australia. The strategy was based on the use of a mixed ligno‐cellulosic biomass feedstock and assumed, for the sake of exploring and quantifying the scenario, a simplified two‐step conversion process – conversion of biomass to crude bio‐oil within the region, and upgrade to jet fuel at a central Brisbane facility. This paper provides details on the costs of production in this scenario, focusing on two different strategies for biomass utilization, and two types of novel small–medium scale conversion technologies. The cost analyses have taken into account technology learning curves, different economies of scale and key cost sensitivities. The cost of biomass‐based jet fuels is estimated to be between 0.70 and 1.90 The airline industry has a strong interest in developing sustainable aviation fuels, in order to reduce their exposure to increasing oil prices and cost liability for greenhouse gas emissions. The feasibility and cost of producing sustainable biomass‐based jet fuels at a sufficient scale to materially address these issues is an enormous challenge. This paper builds directly on the biophysical study by H.T. Murphy, D.A. O'Connell, R.J. Raison, A.C. Warden, T.H. Booth, A. Herr, A.L. Braid, D.F. Crawford, J.A. Hayward, T. Javonovic, J.G. McIvor, M.H. O'Connor, M.L. Poole, D. Prestwidge, N. Raisbeck‐Brown & L. Rye, In review, which examined a 25 year scale‐up strategy to produce 5% of projected jet fuel demand in Australia in 2020 (470 mL) in the Fitzroy region of Queensland, Australia. The strategy was based on the use of a mixed ligno‐cellulosic biomass feedstock and assumed, for the sake of exploring and quantifying the scenario, a simplified two‐step conversion process – conversion of biomass to crude bio‐oil within the region, and upgrade to jet fuel at a central Brisbane facility. This paper provides details on the costs of production in this scenario, focusing on two different strategies for biomass utilization, and two types of novel small–medium scale conversion technologies. The cost analyses have taken into account technology learning curves, different economies of scale and key cost sensitivities. The cost of biomass‐based jet fuels is estimated to be between 0.70 and 1.90 $ L^?1 when the efficiency of conversion of biomass to biocrude and subsequently to aviation fuel is varied by ±10% of published values, with an average value of 1.10 $ L^?1. This is within the range of the projected 2035 conventional jet fuel price of 1.50 $ L^?1. Therefore, biomass‐based jet fuel has the potential to contribute to supply of Australia's jet fuel needs in the future.