Transient and long-term differential modulations of branched-chain α-keto acid decarboxylase activity in hypophysectomized rats

Hypophysectomy caused a marked but transient increase in branched-chain α-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain α-keto acids. The activities then fall to a new steady state in three weeks with α-ketoisovalerate decarboxylase activity within the normal range, α-keto-β-methylvalerate decarboxylase activity at twice normal, and α-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.     

Secretory IgA as a measure of immunocompetence

The field of psychoimmunology has rapidly expanded in recent years and various parameters of the immune system have been examined in relation to psychological factors. The secretory immune system is one of the more interesting aspects of the entire immune system because it protects mucosal membranes from invading organisms. Stress-produced changes in secretory immunoglobulin A (s-IgA) as measured by radial immunodiffusion assays have been reported in several studies. We present three reasons why total s-IgA protein, the measure derived from radial immunodiffusion assays, may not be a reasonable measure of immune system functioning, and we suggest an alternative method for examining secretory IgA that focuses on s-IgA antibody response to a novel antigen.     

Inhibition of DNA methylation in Friend erythroleukemia cells by actinomycin D

Actinomycin D caused the production of hypomethylated DNA in cultured Friend erythroleukemia cells at cell culture concentrations of 1-4 ng per ml. Inhibition of DNA methyltransferase in cell-free assays was kinetically complex, with mixed-type inhibition. Cornish-Bowden graphical analysis was used to derive a Ki of about 35 nmol Act D per mg DNA. Although nuclei from drug-treated cells were found to contain hypomethylated DNA and DNA methyltransferase could be extracted from the nuclei, the methyl-accepting ability of DNA in whole nuclei themselves was not elevated. We conclude that the low level of Act D bound to DNA in the nuclei is sufficient to prevent the remethylation of hypomethylated sites.     

The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.     

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron

Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a non-heme iron, tetrahydropterin-dependent enzyme which catalyzes the rate-limiting step in the biosynthesis of catecholamines. The highly purified bovine adrenal enzyme contains an unusual blue-green chromophore with lambda max at around 700 nm (epsilon = 1.3 (mM subunit enzyme)-1 cm-1). On excitation at 605.2 nm, resonance-enhanced Raman vibrations are observed at 454, 494, 527, 604, 635, 835, 1130, 1271, 1320, 1426, and 1476 cm-1. The excitation profiles of the modes of 1276 and 1476 cm-1 (from 488 to 620 nm) follow the contour of the 700 nm absorption band. The vibrations observed strongly indicate the presence of a bidentate catecholamine-Fe(III) complex in the enzyme as isolated which gives rise to the characteristic charge-transfer transitions. This is further supported by the release of 0.11 +/- 0.04 mol of noradrenaline and 0.25 +/- 0.06 mol of adrenaline per mol of enzyme subunit on denaturation of the enzyme. The energies of the catecholate to Fe(III) charge-transfer transitions indicate a mixture of histidines and carboxylate(s) coordinated to the iron center in tyrosine hydroxylase. At neutral pH, the enzymatic activity was inhibited more than 50% by 10 microM dopamine, noradrenaline, and adrenaline. The high affinity of the catecholamines to the nonphosphorylated form of tyrosine hydroxylase may have significance in vivo since catecholamines are potent feedback inhibitors of the enzyme.     

Evidence for enzyme subunit complementation in somatic cell heterokaryons

Somatic cell heterokaryons derived from normal human fibroblasts which had different glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants, types A and B, were examined for their G6PD pattern. A hybrid band of activity with intermediate migration, in addition to the A and B bands, was observed in such heterokaryons. These results directly demonstrate that enzyme subunit complementation can take place in somatic cell heterokaryons, and suggest that this technique may be important for elucidating the molecular basis of the genetic heterogeneity seen with many human single enzyme defects.