Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.     

Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli

The interaction between some polyhexamethylene biguanides and the cell envelope of Escherichia coli has been investigated. An amine-ended dimer, (AED, n = 2), a polydisperse mixture (ICI plc) available as the active ingredient of Vantocil IB, (PHMB, n = 5.5), and a high molecular weight fraction, (HMW, n = greater than or equal to 10) of PHMB were used. The sensitivity of batch cultures depleted of magnesium (M-dep), phosphorus (P-dep) or glycerol (C-dep) towards the biocides was assessed by monitoring the rate and extent of potassium ion leakage. P-dep suspensions were particularly resistant to all these agents and possessed less than half the quantity of phospholipid of other cell types. This was compensated for by a proportionate increase in fatty acid and neutral lipid content of the cells. The reduction in phospholipid content was accounted for by decreases in phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) and phosphatidylserine (PS) content of the cultures remained unaffected by the depleting nutrient. Fourier-transform n.m.r. spectroscopy was used to study proton nuclei during the interaction of HMW, AED and PHMB with various phospholipid-vesicle preparations. The results strongly suggest that the biocides acted preferentially on the acidic phospholipids PG and DPG, rather than towards PE or PS. Resistance of P-dep cultures therefore reflected reductions in PG content. A molecular basis for the interaction of these compounds and membranes is proposed.     

Islet cell and 64K autoantibodies are associated with plasma IgG in newly diagnosed insulin-dependent diabetic children

There is a high prevalence of islet cell antibodies (ICA) and autoantibodies detected against an islet cell protein of Mr 64,000 at the time of clinical diagnosis of insulin-dependent diabetes (IDDM). In view of the biphasic immune response after antigen presentation, the purpose of this study was to determine the presence of ICA and antibodies against the 64,000 islet antigen after separation of IgM from IgG to prevent interference between the two antibody classes. Plasma samples from 10 newly diagnosed IDDM children and 10 healthy controls were precipitated with polyethylene glycol (PEG), and the crude Ig was subjected to Sephacryl S-300 chromatography to separate IgM and IgG. ICA determined by indirect immunofluorescence on frozen sections of human pancreas showed reduced background immunofluorescence intensity in the purified fractions compared with crude plasma. The number of ICA-positive samples among the IDDM patients increased from 7/10 in plasma to 9/10 in the IgG fraction. There was an increase in the ICA titer in 6/9 of the positive samples. All purified IgM samples were ICA negative. Immunoprecipitation experiments by using Nonidet P-40 detergent lysates of [35S]methionine-labeled neonatal rat islets demonstrated that the 64,000 autoantibodies were in the IgG fraction. We found 7/10 IDDM samples to be positive, whereas all controls were negative. The background in the autoradiographic analysis was markedly reduced in the IgG fractions compared with immunoprecipitates with crude or PEG-purified plasma and the IgM fraction. ICA titers did not correlate to the ability of the IgG fraction to precipitate the 64,000 autoantigen. It is concluded that both the ICA and 64,000 autoantibodies are primarily of the IgG class at the time of clinical onset of IDDM, and that purification of IgG from human IDDM plasma facilitates the detection of the rat islet cell 64,000 antigen.     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

Glycosylation of catalase inhibitor necessary for activity

A protein isolated from maize scutella which inhibits catalase in vitro has been shown to contain 12% carbohydrate in the form of galactose. This corresponds to four galactose molecules per inhibitor subunit. Removal of the carbohydrate with β-galactosidase or blockage with a galactose-specific lectin abolished activity of the inhibitor.     

Mutagenicity testing of dichloromethane in short-term mammalian test systems

Several short-term mammalian test systems were used for mutagenicity testing of the organic solvent dichloromethane. The compound was negative in the forward mutation test on the HGPRT locus in Chinese hamster cells and the unscheduled DNA synthesis test in both human and hamster cells. In the test on DNA synthesis inhibition, dichloromethane caused an aspecific inhibition in both human and hamster cells, but in this test the effect did not indicate a DNA-damaging action. A weak positive effect was found in the test on sister-chromatid exchanges in hamster cells.