Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Triphosphoinositide breakdown and dense body release as the earliest events in thrombin-induced activation of human platelets

The activation by thrombin of human platelets prelabelled with 32P induced a 30-40% decrease in 32P-triphosphoinositides (TPI) in the first 10 sec; the decrease in the other 32P-labelled phosphoinositides occurred by 20-30 sec. At 10 sec., the intensity of these effects was maximum with 0.2-0.4 U/ml thrombin. Under these conditions, 53, 20 and 15% of the dense granule, alpha-granule and lysosome constituents, respectively were released and thromboxane B2 synthesis reached only 10% of its maximum. Together with experiments carried out with chlorpromazine - or PGE1 - treated platelets, our results suggest the existence of a close relationship between TPI-breakdown and dense body release which appear to be the earliest events resulting from the activation of human platelets by thrombin.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Changes in membrane phospholipid distribution during platelet activation

(1) Exposure of phospholipids at the outer surface of activated and control platelets was studied by incubation with a mixture of phospholipase A₂ from Naja naja and bee venom, solely or in combination with sphingomyelinase from Staphylococcus aureus, using conditions under which cell lysis remained below 10%. (2) Incubation with phospholipase A₂ alone revealed a markedly increased susceptibility of the phospholipids in platelets activated by a mixture of collagen plus thrombin, by the SH-oxydizing compound diamide, or by calcium ionophore A23187, as compared to control platelets or platelets activated separately by collagen or thrombin. (3) Collagen plus thrombin, diamide, and ionophore treated platelets revealed an increased exposure of phosphatidylserine at the outer surface accompanied by a decreased exposure of sphingomyelin, as could be concluded from incubations with a combination of phospholipase A₂ and sphingomyelinase. These alterations were much less apparent in platelets activated either by thrombin or by collagen alone. (4) The increased exposure of phosphatidylserine in activated platelets is accompanied by an increased ability of the platelets to enhance the conversion of prothrombin to thrombin by coagulation factor Xa, in the presence of factor Va and calcium. (5) It is concluded that the altered orientation of the phospholipids in the plasma membrane of platelets activated by collagen plus thrombin, by diamide, or by calcium ionophore, is the result of a transbilayer movement. Moreover, the increased exposure of phosphatidylserine in platelets stimulated by the combined action of collagen and thrombin might be of considerable importance for the hemostatic process.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

Accurate assay of dopa decarboxylase by preventing nonenzymatic decarboxylation of dopa

The nonenzymatic decarboxylation of dopa was completely blocked by both 2-mercaptoethanol and EDTA together over the wide range of pH. This finding made it possible to measure the activity of dopa decarboxylase precisely even at an alkaline pH value. The pH optimum of dopa decarboxylase was found to be pH 7.0 and the Km value for dopa was determined to be 4 X 10(-5) M.     

Effects of Light on Dopamine Metabolism in the Chick Retina

The effect of prolonged exposure to light on the activity of dopaminergic neurons and dopamine (DA) metabolism of chick retinae was investigated. alpha-Fluoromethyldopa, a potent and specific irreversible inactivator of aromatic amino acid decarboxylase, was used to assess DA turnover after inhibition of synthesis, and also to assess in vivo tyrosine hydroxylase activity by dihydroxyphenylalanine accumulation. After 48 h of light exposure, retinal DNA in 12-day-old chicks was about 30% higher (p less than 0.005) whereas dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated two to three times (p less than 0.005) the level of controls kept in the dark for the same period. DA turnover was about twofold faster in the light (t 1/2 = 31 min) than in the dark (t 1/2 = 65 min). Tyrosine hydroxylase, assayed in vitro with saturating levels of cofactor and substrate, increased by about 50% after light exposure. The apparent tyrosine hydroxylase activity in vivo was approximately sixfold higher in the light than the dark. These results are interpreted and discussed in terms of the regulation of DA synthesis, and the use of DOPAC and HVA as indices of DA function in the retina.     

The steroids and fatty acids of the basidiomycete Scleroderma polyrhizum

The fruit bodies of the Basidiomycete Scleroderma polyrhizum have been shown to contain the steroids ergosta-4,6,8(14) 22-tetraen-3-one and 5α,8α-epidoxyergosta-6,22-dien-3β-ol and also palmitic and oleic acids.     

Role of aminocyclopropane-l-carboxylic acid in ethylene release by distal tissues following localized application of ethephon in Cucurbita pepo

The application of ethephon to a single leaf of Cucurbita pepo L. cv. Trailing Marrow plants caused a huge increase in ethylene production from the treated organ and an increased rate of ethylene production from other parts of the plant. These increases were particularly marked in the shoot apex and expanding leaf. Prior treatment with aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor, blocked the increased production of ethylene at sites distant from the point of ethephon application. This strongly suggests that the increased ethylene production at these distant sites is due to ethylene biosynthesis and not a result of the translocation of ethylene released by the breakdown of ethephon at the site of application. Assays of 1-aminocyclopropane-l-carboxylic acid (ACC), an ethylene precursor, showed that it increased substantially after ethephon application but was at undetectable levels in the presence of AVG. It is proposed that the application of ethephon stimulates ethylene biosynthesis, but that transport through the plants is effected by ACC which is then converted to ethylene at the shoot apex and leaves.     

1H nmr studies of complexes of ferric leghemoglobin with substituted pyridines and nicotinic acids

The ¹H nuclear magnetic resonance (nmr) spectra of complexes of soybean ferric leghemoglobin with 3-substituted pyridines and 5-substituted nicotinic acids have been recorded in order to determine the influence of axial ligands on heme electronic structure. The hyperfine shifted resonances of the heme group were assigned by analogy to previous assignments for the pyridine and nicotinic acid complexes of leghemoglobin. The spectra are characteristic of predominantly low-spin ferric heme complexes. For the pyridine complexes, the rate of ligand exchange was found to increase with decreasing ligand pK_A. For many of the complexes, optical and nmr spectra reveal the presence of an equilibrium mixture of high- and low-spin states of the iron atom. The percentage of high-spin component increases with decreasing ligand pK_A Smaller hyperfine shifts are noted for leghemoglobin complexes with ligands capable of weak ligand → metal π bonding. The pattern of hyperfine shifted resonances is similar for all complexes studied and indicates that the overall heme electronic structure is dominated by the bonding to the proximal histidine.