Electrophysiological evidence for the autoregulation of β-cell secretion by insulin

Electrophysiological studies of cultured rat pancreatic β-cells using intracellular microelectrodes show that exogenous insulin over the range of 0.1–10.0 μg/ml inhibits the electrical activity due to 27.8 mM glucose in a dose-related manner. This inhibitory effect is manifested by a mean increase of the membrane potential from about ?20 to ?30 mV and inhibition of the manner of cells impaled showing spike activity from 60 to less than 10%. The inhibitory influence of insulin is rapid occuring within 5 min for the highest level used. The results provide evidence for a negative feedback role of insulin in regulating its own release.     

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F1F0-ATPase disrupt ATP-driven proton translocation

Three missense mutants in subunit a of the Escherichia coli F1F0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp119----His, Ser152----Phe, or Gly197----Arg substitutions in subunit a. Function was not completely abolished by any of the mutations. The F0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F1F0-ATPase. The H+ translocation capacity of F0 was reduced in all three mutants. ATP-driven H+-translocation was also reduced, with the response in the Gly197----Arg mutant being almost nil and that in the Asp119----His and Ser152----Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly197----Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp119----His and Ser152----Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp231----stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB+ plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp231----stop) background strain was shown to bind F1-ATPase nearly normally despite lacking subunit a in its membrane.     

Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.     

The Presence and Role of Transmembrane Transforming Growth Factor-α in Cultures of Rat Liver Epithelial Cells

We assessed the presence and the role of membrane TGF-α in two rat liver epithelial cell lines, either parental or transfected with c-fos proto-oncogene. c-fos overexpressing cells had more TGF-α-like activity in their membranes. When TGF-α was removed by elastase or neutralized, the growth rates of both cell lines were markedly reduced, but to a higher extent for parental cells. If membrane TGF-α seemed to play a key contribution in normal cell growth, both cell lines were unable to react to the addition of soluble TGF-α, showing that these two forms of growth factors are not equivalent.     

Changes in the Microvasculature and Interstitium of Aged Human Mandibles and Maxillae1

The present study describes the age changes to the microvasculature and connective tissue interstitium of the osteons and periosteums of aged human mandibles and maxillae. The mandibles and maxillae obtained from 14 and 19 year old males, respectively, were also studied. In the nutrient canals of the aged osteons, the walls of the arterioles and venules stained intensely PAS positive, and alcian blue negative. The walls of the blood capillaries were thick and strongly PAS positive. There was a deposition of PAS positive material in the connective tissue stroma of the nutrient canals which progressed to the obliteration of the canal space. Many of the nutrient canals exhibited diffuse calcification within the connective tissue interstitium localized around the blood vessels. The lacunae and canaliculi of those osteons in which the nutrient canals were partially or completely obliterated were filled with PAS material. None of these histochemical changes were seen in the osteons of young individuals. The microvasculature of the aged periosteum showed similar changes. The periosteal tissue consisted of thick collagenous bundles and few osteogenic cells. There was a thin darkly stained amorphous calcified layer forming the bone surface.     

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity

Cell capacity for cytosolic NADPH regeneration by NADP‐dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O₃) treatment (120 ppb for 17 days). Two genotypes with differential O₃ sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O₃ flux (POD₀), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O₃ flux, CO₂ assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP‐dehydrogenases above their constitutive level, i.e. NADP‐G6PDH (EC 1.1.1.49), NADP‐ME (malic enzyme) (EC 1.1.1.40) and NADP‐ICDH (NADP‐isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non‐phosphorylating NADP‐GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP‐G6PDH and NADP‐ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O₃‐treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.     

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.