On the reactivities of the tryptophan residues of human alpha-lactalbumin to 2-hydroxy-5-nitrobenzyl bromide.

The reaction of human alpha-lactalbumin with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide has been studied. This protein has 3 tryptophan residues (Trp-60, Trp-104 and Trp-118) all of which are accessible to the reagent at pH 2.7 or 7. Trp-60 of human alpha-lactalbumin is much more reactive than Trp-60 of bovine alpha-lactalbumin (Barman, T. E. (1972) Biochim. Biophys. Acta 257, 297-313). As with bovine alpha-lactalbumin, at pH 2.7, 2-hydroxy-5-nitrobenzyl bromide is specific for tryptophan but at pH 7 His-32 also reacts. When treated with the tryptophan reagent, both alpha-lactalbumins lose their specifier protein activities in the lactose synthase (UDPgalactose:D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) reaction.     

Phosphorylation of rabbit cardiac-muscle troponin I by phosphorylase kinase. The effect of adrenaline.

1. Incubation of rabbit cardiac-muscle troponin I with phosphorylase b kinase leads to the incorporation of .07-1.2 mol of Pi/mol. 2. The major site of phosphorylation is a serine residue at position 72. 3. Lesser amounts of phosphate are incorporated into threonine-138, threonine-162 and serine 20. 4. Serine-20 is the only site that contains a significant amount of phosphate before incubation with phosphorylase b kinase. 5. Unlike the situation with serine-20, the extent of phosphorylation of serine-72 and threonine-138 in the perfused rabbit heart does not change when the heart is exposed to adrenaline (4 microM).     

The binding of calmodulin to myelin basic protein and histone H2B.

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.     

Effects of chronic administration of antipsychotic drugs on GABA and other amino acids in the mesolimbic area of rat brain.

Albino rats were injected once daily with chlorpromazine (20 mg/kg) or haloperidol (3 mg/kg) for 100 days, and were sacrificed 24 hours or 35 days after the last injection. Brains were frozen rapidly in liquid nitrogen, after which the mesolimbic area (containing nucleus accumbens and olfactory tubercle) and striatum were dissected from each frozen brain. Amino acids were quantitated in the mesolimbic area, and GAD enzyme activity was measured in the striatum. Chronic administration of chlorpromazine and haloperidol did not alter brain GABA content or GAD activity in the treated rats, as compared to saline-injected controls, either 1 or 35 days after injections ended. However, a significant elevation of aspartate content and reduction of glycine content was found after chronic administration of haloperidol. These data suggest that alterations which may be found in the GABA system in autopsied brain from psychotic patients do not result from previous antipsychotic drug therapy.     

Affinity-chromatographic isolation and some properties of troponin C from different muscle types.

1. The formation of a complex between troponin I and troponin C that is stable in 6M-urea and dependent on Ca2+ was demonstrated in extracts of vertebrate striated and smooth muscles. 2. A method using troponin I coupled to Sepharose is described for the rapid isolation of troponin C from striated and smooth muscles of vertebrates. 3. Troponin C of rabbit cardiac muscle differs significantly in amino acid composition from troponin C of skeletal muscle. The primary structures of troponin C of red and white skeletal muscle are very similar. 4. The troponin C-like protein isolated from rabbit uterus muscle has a slightly different amino acid composition, but possess many similar properties to the forms of troponin C isolated from other muscle types. 5. The electrophoretic mobilities of the I-troponin C complexes formed from components isolated from different muscle types are determined by the troponin I component.     

BIOCHEMICAL EFFECTS IN MAN and RAT OF THREE DRUGS WHICH CAN INCREASE BRAIN GABA CONTENT

Abstract— Brain amino acids were measured in rats given aminooxyacetic acid (AOAA) by mouth, and in rats given sodium dipropylacetate (DPA) both orally and by intraperitoneal injection. Brain GABA content was significantly elevated by AOAA doses of 10mg/kg/day, but not by 5mg/kg/day. Approximately 4 times as much AOAA is required by mouth as by parenteral injection to raise brain GABA content in the rat. DPA (400mg/kg) increased brain GABA and lowered brain aspartate content significantly 1 h after a single injection. However, DPA given orally (350 mg/kg/day) produced no alterations of any amino acids in rat brain.
Amino acids were measured in plasma and urine from patients treated orally with isonicotinic acid hydrazide (INH) or DPA, and from a volunteer who took AOAA. INH (10–21 mg/kg/day) increased concentrations of β -alanine and ornithine in plasma, as well as urinary excretion of β -alanine. DPA had no such effect. AOAA in oral doses ranging from 1.25 to 5.0 mg/kg/day increased plasma concentrations of β -alanine, ornithine, β -aminoisobutyric acid, proline and hydroxyproline, and produced massive urinary excretion of β -alanine, β -aminoisobutyric acid, and taurine.
Both INH and AOAA, given in doses practical for human use, inhibit the transamination of β -alanine and ornithine in liver, and may also inhibit the transamination of GABA in brain. In addition, AOAA interferes with the catabolism of β -aminoisobutyric acid, proline, and hydroxyproline. AOAA, in the lowest dose employed, appeared more effective than INH as an inhibitor of GABA aminotransferase in man, and might therefore be useful in the treatment of neurological diseases in which brain GABA is deficient.     

Effect of growth substrate on thermal death of thermophilic bacteria.

The heat sensitivity of gram-negative, hydrocarbon-utilizing thermophilic bacteria was altered by a change in growth substrate. Thermophilic strains CC-6, BI-1, and LEH-1, grown with acetate or n-heptadecane as the carbon source, had a higher survival rate when incubated 5 degrees C above their maximum growth temperature than cells of the same organism after growth on glucose or glycerol. There was a correlation between the growth substrated, heat resistance, and the ratios of cellular n-hexadecanoic acid/branched hexadecanoic acid and n-heptadecanoic acid/branched heptadecanoic acid. The bacterial cells that were more heat resistant had ratios of straight-chain/branched-chain fatty acids above 1.0, whereas the heat-sensitive cells had ratios below 0.6.     

SEM analysis of amphibian mesodermal migration

Summary At the end of gastrulation, the lateral mesoderm of amphibian embryos migrates ventrally between the ectoderm and the endoderm. The present study is an examination of the morphology of the leading cells of the mesodermal sheet and of the substratum over which they move (the inner surface of the ectoderm). The cells of the leading edge of the mesoderm are generally round, with very short and narrow flattened projections in the forward direction. These projections do not have a ruffled morphology, regardless of whether fixation is carried out before or after the ectoderm and mesoderm are dissected away from the endoderm. The inner surface of the ectoderm is covered with fine (450–500A) filamentous extracellular material and the ectoderm cells sometimes extend cytoplasmic processes (approx. 0.1 wide) onto the leading surface of the mesoderm or onto adjacent ectoderm cells. These studies indicate that the morphology of cell migration in amphibians is closer to that seen inFundulus than to that characteristic of chick or mammalian cells.This paper is dedicated to the memory of Mac V. Edds, Jr., who warmly encouraged the developmental biologists of the Pioneer Valley     

Basic fibroblast growth factor upregulates steady-state levels of laminin B1 and B2 chain mRNA in cultured neuroepithelial cells

The growth of purified populations of murine neuroepithelial cells isolated from 10 day embryonic (E10) telencephalon and mesencephalon can be specifically enhanced by supplementing growth culture media with basic fibroblast growth factor (bFGF). One effect of bFGF on cultured neuroepithelial cells was to enhance the amount of laminin expressed at the protein level as detected by immunofluorescence. This was correlated with significant upregulation of steady-state levels of laminin B1 and B2 chain expression as analyzed at the mRNA level. When E12 neuroepithelial cells were split into precursor neuronal or glial subpopulations on the basis of differential expression of major histocompatibility class-1 antigens, only the glial progenitor fraction was found to be capable of detectable laminin synthesis. It is thus possible that a primary action of FGF is to increase the synthesis and release of extracellular matrix molecules from neural cells which act back in a paracrine manner to stimulate differentiation.