Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Bovine lens aldose reductase: identification of two enzyme forms

Two structurally different forms of bovine lens aldose reductase have been identified. Freshly prepared lens extracts contain an unactivated "b form" (ARb) which is sensitive to inhibition by Sorbinil. Upon incubation of the extracts with oxygen radical generating systems, ARb is converted to a more active "a form" (ARa), which is not inhibited by Sorbinil. ARa and ARb were purified to electrophoretic homogeneity.     

The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil.

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

In vitro detachment of bacteria from ruminal digesta by buffered sodium oleate solutions

The effectiveness of a buffered sodium oleate solution was evaluated for detaching bacteria from ruminal digesta samples. A response surface derived from an octagonal design was used to determine the pH and concentration combination for maximum detachment of total and cellulolytic bacteria. The total number of bacteria detached increased up to 81% over control with treatment of a pH 8.8 and 1.5% sodium oleate solution. The recovery of cellulolytic bacteria was decreased to 35% of control with treatment of a pH 9.0 and 0.1% sodium oleate solution. Attempts to improve the recovery of viable bacteria exposed to sodium oleate solutions were unsuccessful. This response surface design identified an optimal pH and concentration that were consistent with existing information regarding detachment of total bacteria, and suggested that sodium oleate, at the concentrations tested, was toxic to the cellulolytic population of the rumen.     

Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.     

Ca- and Mg-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca²⁺- and Mg²⁺-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca²⁺. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca²⁺-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 μg/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Organic Constituents and Complexation of Nickel(II), Iron(III), Cadmium(II), and plutonium(IV) in Soybean Xylem Exudates

The xylem exudates of soybean (Glycine max cv Williams), provided with fixed N, were characterized as to their organic constituents and in vivo and in vitro complexation of plutonium, iron, cadmium, and nickel. Ion exchange fractionation of whole exudates into their compound classes (organic acid, neutral, amino acid, and polyphosphate), followed by thinlayer electrophoresis, permitted evaluation of the types of ligands which stabilize each element. The polyvalent elements plutonium(IV) and iron(III) are found primarily as organic acid complexes, while the divalent elements nickel(II) and cadmium(II) are associated primarily with components of the amino acid/peptide fraction. For plutonium and cadmium, it was not possible to fully duplicate complexes formed in vivo by back reaction with whole exudates or individual class fractions, indicating the possible importance of plant induction processes, reaction kinetics, and/or the formation of mixed ligand complexes. The number and distribution of specific iron- and nickel-containing complexes varies with plant age and appears to be related to the relative concentration of organic acids and amino acids/peptides being produced and transported in the xylem as the plant matures.     

The MgADP-induced decrease of the SH1-SH2 fluorescence resonance energy transfer distance of myosin subfragment 1 occurs in two kinetic steps

The fluorescence resonance energy transfer distance between 5-[2-[iodoacetyl)amino)ethyl]aminoaphthalene-1-sulfonic acid covalently attached to the SH1 thiol of myosin subfragment 1 as the energy donor and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide attached to SH2 as the energy acceptor has been found to decrease by about 7 A in the presence of MgADP (Dalby, R. E., Weiel, J., and Yount, R. G. (1983) Biochemistry 22, 4696-4706; Cheung, H. C., Gonsoulin, F., and Garland, F. (1985) Biochim. Biophys. Acta 832, 52-62). Fluorescence stopped-flow experiments on the same system have yielded biphasic traces which are resolvable into a fast and slow component, k1 and k2, respectively. Results of experiments in which k1 and k2 were measured as a function of excess ADP concentration showed: 1) a nonlinear dependence of the apparent rate constants on [ADP]; 2) k1 is a factor of 10 faster than k2. These results are consistent with the 3-step mechanism previously proposed for nucleotide binding to myosin S1 (Garland, F., and Cheung, H. C. (1979) Biochemistry 18, 5281-5289). Kinetic experiments in which the anisotropy of the donor was monitored show this quantity to be unchanged over the course of the reaction. The biphasic decrease of donor intensity is assigned to an increase in energy transfer efficiency which, from the above results, is due to a decrease in donor-acceptor distance, occurring in two steps. The fast step is associated with a 4-5-A decrease of the donor-acceptor separation, while the slow step is associated with a further decrease of approximately 2 A.     

Altricial and precocial mammals: A model of neural and muscular development

This model of growth offers a quantitative definition for altricial and precocial newborns, makes muscular strength a benchmark for locomotor independence, and discriminates related genera as well as genera across major taxonomic divides. The model contrasts four theoretical conditions of the neonate (I, small brain, weak musculature; II, small brain, strong musculature; III, large brain, weak musculature; IV, large brain, strong musculature) with species from three orders of placental mammal. Each species exhibits a distinct mother-infant strategy from the altricial red panda cub (condition I) and the golden lion tamarin (condition III) to the precocial wildebeest calf (condition IV). The model proposes that early growth rates of brain and muscle correlate with nutrition, maternal effort during gestation and lactation, and parental care, whereas postnatal muscular growth correlates directly with adult body size and locomotor repertoire. An example of condition II (small brain, strong musculature) has not been found. This suggests that muscle does not grow in advance of the brain and that the brain acts as a pacemaker of growth. In order to increase our understanding of exotic species, noninvasive measures (body weight and length) and observations (opening of the eyes and ears, hair density, weaning, and the abilities to ther-moregulate and to move) should be supplemented with analysis of the differential tissue and organ growth. In both theoretical and practical ways analysis of deceased individuals contributes to the understanding of all species.