Comparative gas phase and pulsed liquid phase sequencing on a modified Applied Biosystems 477A sequencer

A simple and inexpensive modification of the Applied Biosystems 477A sequencer, to run in the pulsed liquid-phase and in the gas-phase mode of the Edman chemistry, is described. This modification is especially useful for sequencing samples on polyvinylidene difluoride (PVDF) membranes. Additional carriers are required if a sample on a PVDF membrane is sequenced with the pulsed liquid-phase degradation program of the 477A. In the gas-phase mode no such carriers are needed. This eliminates time-consuming preconditioning sequencer cycles and reduces the sequencer background. In addition, initial coupling yields in the gas-phase mode exceeded those in the pulsed liquid-phase mode, whereas the average repetitive yields were similar. Samples spotted onto glass fiber filters pretreated with polybrene and samples spotted or electroblotted onto PVDF membranes were examined. A number of advantages of the gas-phase mode are presented.     

Modifications of ganglioside composition in peripheral nerve of myelin deficient trembler mutant mouse

Ganglioside distribution was studied in peripheral nerves of normal controls and those of Trembler mutant mouse with defect in Schwann cell differentiation and myelination. Neuraminic acid content was considerably decreased in the mutant. Ganglioside distribution as evaluated by densitometry of resorcinol positive spots on thin-layer chromatography revealed a major peak for GDl_a in normal controls. In the mutant, the relative proportion was modified with qualitative modifications in the GDl_a area and a tremendous increase in GM₃ content. The relation with the intense Schwann cell proliferation observed in the mutant is discussed.     

Cell surface T antigen in cells infected with simian virus 40 or an adenovirus-simian virus 40 hybrid, Ad2+D2.

Cell surface T antigen, detected by a radioimmune assay that uses 125I-labeled Staphylococcus aureus protein A and antibodies against either authentic T antigen or D2 hybrid T antigen, was found in simian virus 40-transformed and -infected cells and in cells infected with an adenovirus-simian virus 40 hybrid, Ad2+D2. In simian virus 40 lytic infection, the surface T antigen appeared at the same time as the nuclear T antigen.     

Study of relationship among species ofVibrio,Photobacterium, and terrestrial enterobacteria by an immunological comparison of glutamine synthetase and superoxide dismutase

The amino acid sequence divergence of glutamine synthetase (GS) from species ofVibrio, Photobacterium, Aeromonas, Escherichia, Salmonella, Citrobacter, Enterobacter, Serratia, Proteus, Erwinia, Xenorhabdus, andPlesiomonas was determined by quantitative microcomplement fixation, using antisera to GS fromVibrio alginolyticus andEscherichia coli. A similar study was performed with superoxide dismutase (SOD), using antiserum to the enzyme fromV. alginolyticus. A comparison of the results for GS and SOD, relative to the enzymes fromV. alginolyticus, as well as a comparison of these data with the results of previous ribosomal RNA (rRNA)/DNA homology studies indicated a high degree of congruence (correlation coefficients≥0.9). The results with both enzymes suggested four major groupings among these genera: (i)Vibrio, (ii)Photobacterium, (iii)Aeromonas, (iv) a large and heterogeneous group which included the peritrichously flagellated terrestrial enterobacteria.     

A rapid assay of acyl-coenzyme A:lysolecithin acyltransferase activity

A simple and rapid procedure for the assay of acyl-coenzyme A:1-acyl-sn-glycero-3-phosphocholine acyltransferase (lysolecithin acyltransferase, LLAT [EC 2.3.1.23]) activity in crude enzyme preparations is described. The incubation system utilizes lysolecithin and [1-14C]-oleoyl-coenzyme A as substrates. Labeled fatty acid released due to accompanying acyl-coenzyme A hydrolase [EC 3.1.2.2]activity is first removed by di-isopropyl ether extraction. The labeled lecithin produced due to LLAT action is then quantitatively recovered by partition of the incubation medium with di-isopropyl ether-n-butanol 60:40 (v/v). Selective extraction of the labeled lecithin formed and avoidance of customary thin-layer chromatographic isolation procedures permits assay of LLAT activity with excellent accuracy at a substantial saving of time. The entire assay can be completed in less than 30 min as compared to 2-3 hrs when following conventional procedures.     

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).