Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Spatial and temporal variation in distribution of Gelidium canariensis (Rhodophyta) from natural populations of the Canary Islands

This study was designed to investigate spatial and temporal variation in Gelidium canariensis populations at two shores in northern Gran Canaria during two years. Spatial scales ranged from some hundred meters (distance between shores), 10 to 30 m (distance between plots) to less than 3 m (distance between quadrats). Gelidium individuals were defined as distinct Gelidium clumps. The results show a significant difference in size of clumps between shores, but not on the smaller spatial scales. No significant temporal variation was found. There was no significant temporal or spatial variation in standing crop or density (counts made in quadrats where Gelidium was present, rather than counts for the total shore). Sporophytic and gametophytic clumps were also distinguished by identifying reproductive structures in the field. The total proportion of sporophytes was larger than the proportion of gametophytes, but at a smaller scale there could be a shift in dominance. The survival rate of clumps was similar between shores with a mean survival rate of 85%, but there was a significant difference in recruitment between shores. The results indicate a stable population structure. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis.

The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2 mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.     

An improved negative staining technique using a thin quartz membrane as sample support

A negative staining technique is presented based on the use of 40-60 nm quartz membrane supported by a silicon grid. The quartz membrane is fabricated by thermal growth of silicon dioxide on a silicon substrate followed by an anisotropic silicon etching step giving rectangular holes in the silicon substrate. The hydrophilic membrane is shown to be ideally suited for negative staining due to its spreading characteristics, homogeneity, heat resistance and mechanical stability. Micrographs of phage lambda are presented showing the detailed structure of the tail. A simple method of calculating the number of adsorbed particles based on diffusion limited association is also presented.     

Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Formation and characterisitcs of hepatic dexamethasone-receptor complexes of different molecular weight

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 Å and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [³H]dexamethasone in vivo or in vitro (reconstitution experiments with [³H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30–36 Å and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 × g homogenate supernatant to low ionic strenght during preparation of cytosol resulted in conversion of the 61 Å to a 36 Å complex very similar to the intranuclear form of dexamethasone receptor. 61 → 36 Å complex-verting activity was present in both the 100 × g ?10 000 × g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 Å dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 Å. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 Å and 36 Å dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 Å complex revealed that this form had been converted to the 30–36 Å complex.Further digestion of teh 61 and 36 Å [³H]dexamethasone-receptor complexes with hypotonic extract of the 1000 × g ?10 000 × g sediment of liver homogenate or with trypsin resulted in formation of a third complex with a Stokes radius of 19 Å and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 Å dexamethasone-receptor complexes were calculated as 102 000, 46 00 and 19 000, respectively, and the frictional ratios of the molecules as 1. 84, 1. 38 amd 1.00, respectively.It is concluded that the nuclear 30–36 Å dexamethasone-receptor complex is formed from the cytosol 61 Å complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.     

Cell wall polypeptides of Polyphysa (Acetabularia) cliftonii: amino acid composition of stalk and cap cell wall polypeptides

The amino acid composition of stalk and cap cell wall polypeptides of the unicellular alga $\text{Polyphysa (A.) cliftonii}$ has been investigated. In spite of chemical and physical differences between stalk and can cell wall polysaccharides, the amino acid composition of the cell wall polypeptides appeared qualitatively similar in both structures. however, quantitative differences have been observed. The results are discussed on the basis of a possible role of the polypeptides in the growth of the cell wall.