Association between Membrane Phase Properties and Dehydration Injury in Soybean Axes

Axes of soybean seeds are tolerant to dehydration at 6 hours of imbibition, but susceptible to dehydration injury if dried at 36 hours of imbibition. Smooth microsomal membranes were isolated from axes imbibed for 6 hours (dehydration tolerant state) and 36 hours (dehydration susceptible state) before and after dehydration treatment. The phase properties and the lipid composition of the membrane fraction were investigated. Wide angle x-ray diffraction patterns of microsomal membranes from axes imbibed for 6 or 36 hours indicated a liquid-crystalline to gel phase transition at approximately 7°C. Membranes from axes dehydrated at 6 or 36 hours of imbibition and rehydrated for 2 hours exhibited a phase transition at 7°C and 47°C, respectively. Changes in fatty acid saturation did not account for the changes in phase properties. However, the increased phase transition temperature of the membranes from dehydration injured axes was associated with an increase in free fatty acid:phospholipid molar ratio and a decrease in phospholipid:sterol ratio. These results suggests that dehydration prompted a deesterification of the linkage between glycerol and fatty acid side chains of the phospholipid molecules in the membrane. The resultant increase in free fatty acid content in the membrane is thought to alter the fluidity and phase properties of the membrane and contribute to dehydration injury.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Constant helical pitch of the gramicidin channel in phospholipid bilayers.

X-ray diffraction has been applied in measuring the helical pitch of the gramicidin channel in oriented bilayers of dilauroylphosphatidylcholine (DLPC) and dimyristoylphosphatidylcholine (DMPC) at a polypeptide concentration of 9.1 mol %. The diffraction data show the helical pitch of gramicidin to be 4.7 +/- 0.2 A in both gel and liquid-crystalline phase bilayers, with and without monovalent cations. In addition, the width of the reflection due to the pitch of the helical gramicidin channel is consistent with a five turn helix.     

Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

The effect of water activity (a_w) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal a_w supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the a_w of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a_w when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower minimal a_w in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a_w levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the a_w levels studied.     

Highly efficient somatic-mutation identification using Escherichia coli mismatch-repair detection

The discovery of somatic mutations in cancer tissue is extremely laborious, time-consuming and costly. In an evaluation comparing mismatch repair detection (MRD) against Sanger sequencing for somatic-mutation detection, we found that MRD had a specificity of 96% and a sensitivity of 92%. Our results showed that MRD is a robust and cost-effective alternative to Sanger sequencing for identifying somatic mutations in human tumors.