A rapid and sensitive method for detection of proteins in polyacrylamide SDS gels: Staining with ethidium bromide

We describe here a fluorometric method of detection of proteins fractionated by electrophoresis in polyacrylamide-SDS gels. This method, using ethidium bromide as fluorescent dye, is performed within 40 minutes after the end of the electrophoretic run. It does not require treatment of proteins prior to electrophoresis, and entails neither fixation of proteins in the gel, nor destaining. It is sufficiently sensitive to detect 0.5–1.0 g of protein per band. Furthermore, the simultaneous electrophoretic resolution and detection of protein and RNA on a single SDS-polyacrylamide gradient gel is reported.     

Solubilization and separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid-delta4-delta5-isomerase from bovine adrenal cortex microsomes.

A physical separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid delta4-delta5-isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3beta-ol-17-one, pregnen-3beta-ol-20-one and pregn-5-en-(3beta,17alpha)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.     

Allergic delayed skin reactions from lipid fractions of trichophytin.

Crude trichophytin was fractionated to find out if its lipid fraction could cause inflammatory delayed allergic skin reactions in dermatophyte-sensitized guinea pigs. The following fractions were obtained: polysaccharide-peptide, total lipids, total lipids without free fatty acids and free fatty acids. The crude trichophytin and polysaccharide-peptide fraction gave rise to strong and equal allergic delayed skin reactions after 24 h. The total lipids gave statistically significant weaker, but clearly positive reactions, and of the same degree as the free fatty acid fraction. The total lipids without free fatty acids did not produce reactions in the sensitized animals, indicating that free fatty acids are responsible for the allergic skin reactions. In some cases the free fatty acids showed comparatively intense reactions. It can be concluded that free fatty acids are antigenic substances that are, sometimes, involved in the allergic delayed skin reactions in dermatophytosis.     

Cholinesterases (acetyl and pseudocholinesterase) in human amniotic fluid]

Study on 41 cases. The cholinesterasic activity of human amniotic fluid, free from blood, is very weak, in average 0,10 microkatal, approximatively 300 times weaker than mother's serum. In this "total cholinesterase", we find a large amount (about 40%) of acetylcholinesterase. Amniotic cholinesterases have an prominent placentary origin, with, in addition, a likely activity proceeding from cellular elements.     

Studies on autoimmune encephalomyelitis in the guinea pig. II. An in vitro investigation on the nature, properties, and specificity of the serum-demyelinating factor.

Complement-dependent demyelinating activity of whole brain homogenate (WBH)-induced experimental allergic encephalomyelitis (EAE) sera was tested on long term tissue cultures of in vitro myelinated fetal guinea pig cerebellum. Complement-fixing (CF) auto-antibodies were shown to be the responsible agents, as demonstrated in experiments where all reagents belonged to the same species: guinea pigs of outbred (Hartley) and even of inbred (S2 or S13) strains. These antibodies were of the IgG2 class as shown by Sephadex G-200 and DEAE cellulose fractionation experiments. The corresponding auto-antigen was present in the homogenate and myelin of the central nervous system (CNS) tissue. It was different from the encephalitogenic basic protein of CNS myelin (BP), as shown in experiments where the demyelinating auto-antibodies were induced, detected, and absorbed by WBH or by CNS myelin but not by BP. They were neither induced by nor cross-reacting with cerebroside and peripheral nervous system (PNS) tissue.     

Amino acid sequence at the phosphorylated site of rat liver pyruvate kinase

One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(³²P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(³²P)SerP-Leu.     

Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12.

Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes. Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav. = 1.983. Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.     

A factor analysis of morphogenetic fields in the human dentition

The traditional morphogenetic fields of the human dentition were evaluated by means of factor analysis of dental dimensions taken from a series of human crania. When crown length, crown width and crown index were considered separately, factors emerged which could be identified with the tooth group fields. But a combined crown length-crown width analysis generated factors which extended beyond the regional tooth groups. Crown width itself was revealed to be an important axis of morphologic intergration. It was concluded that univariate methods are not adequate for identifying morphogenetic fields; the teeth must be treated as multidimensional units where the correlation among dimensions is accounted for.     

Plasma normetanephrine: A biochemical marker of sympathetic nerve function in man

The concentration of the catecholamines in plasma are regarded by many investigators as biochemical markers of sympathoadrenal activity in man. This study was designed to: 1) assess the relationship of normetanephrine (NMN), the metabolite of norepinephrine (NE) most likely to represent degration of newly synthesized and released “active” norepinephrine, to that of norepinephrine in plasma of normal volunteers and 2) to determine regional variations in plasma normetanephrine concentrations at venous and arterial sites of patients undergoing cardiac catherization. The findings suggest that 1) plasma normetanephrine reflects sympathoadrenal activity and neurotransmitter production at both peripheral neuronal and adrenal medullary sites and 2) there is net removal of normetanephrine by liver and kidney. Plasma normetanephrine should provide assistance in the biochemical assessment of sympathoadrenal tone in man and in patients with various autonomic disorders.