Structure and expression of a barley acidic β-glucanase gene

A barley acidic -1,3-glucanase gene was recovered from a barley genomic library by homology with a partial cDNA of barley basic -1,3-glucanase isoenzyme GII. The gene, Abg2, is homologous to the PR2 family of pathogenesis-related -1,3-glucanase genes. The ABG2 protein has 81% amino acid similarity to barley basic -1,3-glucanase GII. The ABG2 protein is encoded as a preprotein of 336 amino acids including a 28 amino acid signal peptide. A 299 bp intron occurs within codon 25. The mature ABG2 protein has a predicted mass of 32642 Da and a calculated isoelectric point of 4.9. The second exon of the Abg2 gene shows a strong preference for G+C in the third position of degenerate codons. The Abg2 gene was functionally expressed in Escherichia coli. Abg2 mRNA is constitutively expressed in barley root; leaf expression of Abg2 mRNA is induced by mercuric chloride and infection by Erysiphe graminis f. sp. hordei. Southern blot analysis indicates that Abg2 is a member of a small gene family.     

Genetic studies on the β-amylase isozymes of barley malt

From genetic studies on two -amylase forms (Sd1 and Sd2) observed in barley malts it is concluded that the Sd1 and Sd2 phenotypes are controlled by a pair of alleles acting without dominance. Gene dosage effects typical of characters determined by the endosperm were observed in F₁ hybrids, Preliminary studies on the distribution of Sd1 and Sd2 forms indicate that Sd1 is the rarer one; of thirty-one cultivars tested, twenty were Sd2, nine were typed as Sd1 and two cultivars had both Sd1 and Sd2 phenotypes. An analysis of joint segregations for Sd1, Sd2, the heat-stable -amylases and two esterase forms gave no evidence for linkage between these three loci.     

Gibberellic acid, β-1,3-glucanase and the cell walls of barley aleurone layers

Summary A glucanase from barley aleurone layers can be assayed using the algal polysaccharide laminarin as substrate. Gibberellic acid (GA₃) enhances the release of this enzyme from isolated aleurone layers but has no significant effect on its synthesis. Concentrations of GA₃ effective in stimulating this release are in the range of 3×10^-11-3×10^-7M. The time course of glucanase release was found to be significantly different from that of -amylase, glucanase release being completed before that of -amylase. Evidence based on using various histochemical stains suggests that barley aleurone cell walls contain a -1,3-linked polymer. Following treatment of aleurone layers with GA₃, digestion of these walls is seen to occur. These observations strongly suggest that the -1,3-glucanase produced by aleurone cells is resposible for the observed cell-wall digestion.Supported by National Science Foundation Grant GB-8332. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Influence of β-cyclodextrin on the Properties of Norfloxacin Form A

Cyclodextrins are able to form host–guest complexes with hydrophobic molecules to result in the formation of inclusion complexes. The complex formation between norfloxacin form A and β-cyclodextrin was studied by exploring its structure affinity relationship in an aqueous solution and in the solid state. Kneading, freeze-drying, and physical mixture methods were employed to prepare solid complexes of norfloxacin and β-cyclodextrin. The solubility of norfloxacin significantly increased upon complexation with β-cyclodextrin as demonstrated by a solubility isotherm of the A_L type along with the results of an intrinsic dissolution study. The complexes were also characterized in the solid stated by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier-transform infrared (FT-IR) spectroscopy, X-ray diffractometry, scanning electron microscopy (SEM), and solid-state nuclear magnetic resonance (ssNMR) spectrometry. The thermal analysis showed that the thermal stability of the drug is enhanced in the presence of β-cyclodextrin. Finally, the microbiological studies showed that the complexes have better potency when compared with pure drug.KEY WORDS: bioassay, complexation, intrinsic dissolution, norfloxacin, β-cyclodextrin     

Estimates of individual factors of the tryptophan requirement based on protein and tryptophan accretion responses to increasing tryptophan supply in broiler chickens 8 – 21 days of age

Ten diets (196?g CP and 13.0 MJ ME_N/kg) with graded levels of tryptophan (Trp) between 0.9 and 2.7?g/kg were offered ad libitum to Ross broilers from day 8?–?21 post-hatch. Each diet was allocated to three pens of ten birds each. In addition to growth and feed conversion, the accretions of protein, Trp, fat, and energy were determined by comparative whole body analyses. A sigmoidal function was fitted to the data. Responses to Trp supply were nonlinear and attained plateau values (y_max) within the studied range of Trp supply. The y_max values estimated for BW gain, whole body protein gain, and Trp gain during the 14 days under study were 615, 104, and 0.87?g/bird, respectively. Based on the response in whole body protein gain, the estimated requirement was 1.9?g Trp/kg of diet or 0.15?g Trp/MJ of ME_N. Estimates made from the other response criteria were similar to this value. While the protein concentration in gained BW was unaffected by Trp intake (169?g of protein per kg of gained BW), fat and energy concentrations of gained BW increased with increasing Trp supply, approaching plateau values of 146?g fat and 9.8 MJ energy per kg of gained BW. This is supposed to result from feed intake increasing with Trp supplementation. The Trp concentration in gained whole body protein (0.84?g Trp/16?g of gained N) was not significantly affected by Trp intake. As long as Trp intake was the limiting factor, 59% of supplemented Trp was transferred to gained whole body protein. During the 14 days of the study, broilers inevitably lost 87?mg Trp. This is equivalent to a daily loss of 30?mg/kg BW or a maintenance requirement of 52?mg/kg BW per day. Data determined for the individual factors may be used for future modelling of broilers' Trp requirement.     

Influence of novobiocin on the induction kinetics of β-galactosidase in Escherichiacoli

Novobiocin (0.05 μg/ml) reduced the growth rate of cultures of $\text{Escherichia}$$\text{coli}$ strain DK6 by about a factor of 2. The lag in appearance of β-galactosidase-forming capacity was extended from 50 sec to 85 sec by the drug. This appeared to be the result of a reduced rate of nascent mRNA elongation.     

Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases

Commercial β-galactosidase preparations from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae were evaluated as catalysts for the synthesis of lactulose. Among them, the enzyme from A. oryzae was selected for further studies. The effect of reaction conditions was then studied on product composition during the kinetically controlled synthesis of lactulose by transgalactosylation with A. oryzae β-galactosidase. Product composition was not affected by pH, temperature, total initial concentration of sugar (lactose plus fructose) and enzyme to substrate ratio within the ranges studied. However, lactose to fructose ratio strongly influenced product composition being then possible to control the lactulose to galacto-oligosaccharide ratio within ample margins. Maximum lactulose yield (0.282 g of lactulose per g initial lactose) was obtained using 1/8 lactose to fructose molar ratio, 50% (w/w) total initial sugars, 40 °C, pH 4.5 and enzyme to initial lactose ratio equivalent to 200 IU/g.     

Influence of C-H...O interactions on the structural stability of β-lactamases

β-Lactamases produced by pathogenic bacteria cleave β-lactam antibiotics and render them ineffective. Understanding the principles that govern the structural stability of β-lactamases requires elucidation of the nature of the interactions that are involved in stabilization. In the present study, we systematically analyze the influence of CH...O interactions on determining the specificity and stability of β-lactamases in relation to environmental preferences. It is interesting to note that all the residues located in the active site of β-lactamases are involved in CH...O interactions. A significant percentage of CH...O interactions have a higher conservation score and short-range interactions are the predominant type of interactions in β-lactamases. These results will be useful in understanding the stability patterns of β-lactamases.     

Influence of Ionic Surfactants on the Microstructure of Heat-Set β-Lactoglobulin-Stabilized Emulsion Gels

Using confocal microscopy, we studied the effect of heating (up to 85°C) on the microstructure of β-lactoglobulin-stabilized emulsions (20 vol% oil, pH 6.8) containing excess protein (total protein content 13.2%). Two different fluorescent dyes were used to separately visualize the oil droplets and the protein. In overlay micrographs, their location with respect to each other could then be determined. In the presence of a low salt concentration, flocculation of the emulsion without surfactant was inhibited, by a mechanism analogous to the “salting-in” of aqueous protein solutions. Addition of the anionic surfactant sodium dodecyl sulfate (SDS) caused weak flocculation, probably as a result of the formation of protein−SDS complexes. The final heat-set emulsion contained distinct pores for a surfactant/protein ratio of R = 1, but no pores for R = 2. Addition of the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) caused strong aggregation, as indicated by microscopic observation of the concentrated emulsion and light scattering of the diluted emulsion. For R = 1 with CTAB, there were aggregates consisting of oil droplets and excess protein. At R = 2, almost all the excess protein was aggregated into separate protein flakes. In the final emulsion gels containing CTAB, the protein was more spread out. Differing structural behavior with anionic and cationic surfactants has been interpreted in terms of different protein−surfactant interactions in aqueous solution and at the oil−water interface, both before and after protein denaturation.     

Mean surface temperature prediction models for broiler chickens—a study of sensible heat flow

Body surface temperature can be used to evaluate thermal equilibrium in animals. The bodies of broiler chickens, like those of all birds, are partially covered by feathers. Thus, the heat flow at the boundary layer between broilers’ bodies and the environment differs between feathered and featherless areas. The aim of this investigation was to use linear regression models incorporating environmental parameters and age to predict the surface temperatures of the feathered and featherless areas of broiler chickens. The trial was conducted in a climate chamber, and 576 broilers were distributed in two groups. In the first trial, 288 broilers were monitored after exposure to comfortable or stressful conditions during a 6-week rearing period. Another 288 broilers were measured under the same conditions to test the predictive power of the models. Sensible heat flow was calculated, and for the regions covered by feathers, sensible heat flow was predicted based on the estimated surface temperatures. The surface temperatures of the feathered and featherless areas can be predicted based on air, black globe or operative temperatures. According to the sensible heat flow model, the broilers’ ability to maintain thermal equilibrium by convection and radiation decreased during the rearing period. Sensible heat flow estimated based on estimated surface temperatures can be used to predict animal responses to comfortable and stressful conditions.     

Study of the effect of β-1,3-glucanase fromBasidiomycete QM 806 on yeast extract production

Summary Cell free supernatants, containing -1,3-glucanase fromBasidiomycete QM 806, dramatically augmented the effect of papain on yeast autolysis. This enables the process time to be significantly reduced and/or the yield of extract to be substantially increased.     

Influence of pH on the formation and location of β-glucosidase inAspergillus terreus grown on cellulose

Wild strainof Aspergillus terreus is very good producer of /gb-glucosidase. It produces 15 nkat/mL (0.9 IU/mL) of extracellular β-glucosidase at pH 5.0. The medium pH level strongly affects the production and binding of β-glucosidase on the cells and on residual cellulose. At pH 4.0 the rate of enzyme synthesis and the level of total activity is highest but 60—75 % of this activity is bound. At higher pH levels the enzyme is mainly released to the medium.     

Influence of proline and β-turn mimetics on the cyclization of penta- and hexapeptides

Summary Proline and Pro-derived peptidomimetics, such as meoxPro-Oic (4-methoxy-proline-octahydro indolic acid), and DBF (2-aminoethyl-6-dibenzofuran propionic acid) were introduced into thymopentin-derived penta-[SP5-] and hexa-[SP6-] peptides and penta-, hexa- and hepta-alanine. Surprisingly, we found that cyclomonomer formation in the investigated penta- and hexapeptides was drastically hindered by the presence of proline regardless of position.     

Influence of the human blood serum on contractility and β-adrenoreactivity of the isolated human myocardium

Influence of adrenaline (10⁻⁹ to 10⁻⁴ g/ml) on the contraction amplitude caused by electrostimuli (1Hz, 5 ms, 25–30 V) and inotropic and adrenomodulation activities of blood serum of nonpregnant women (at dilutions of 1 : 10 000, 1 : 1000, 1 : 500, 1 : 100, 1 : 50, 1 : 10, and 1 : 5) have been studied. The study has been carried out on isolated myocardium strips of the right atrial auricle that were taken from 43 patients with ischemic illness of the heart and 9 patients with valvular heart diseases of various etiologies upon venous cannula insertion during an aortocoronary bypass. Direct dependence of the contraction amplitude on the cardiac output according to Teicholz has been found. This meant that strips of the right atrial auricle reflected the contractility of the left ventricle myocardium. Adrenaline has been shown to dose-dependently increase the amplitude of evoked contractions in the concentration interval from 10⁻⁷ to 10⁻⁶ g/ml and had no influence from 10⁻⁹ to 10⁻⁸ g/ml (dissociation constant, 2 × 10⁻⁷ g/ml), which proved a decrease in the β-adrenoreceptor’s (β-AR) activation. Blood serum in a dilution range from 1 : 10 000 to 1 : 50 had no effect on the contraction amplitude, but an enhanced effect has been found in a dilution range from 1 : 10 and 1 : 5. The presence of the endogenous activator of myocytes contractility (EAMC) has explained this enhanced effect. The β-adrenomodulation activity of blood serum has been explained by the presence of the endogenous sensitizer of β-AR (ESBAR) and the endogenous blocker of β-AR (EBBAR). The ESBAR activity of blood serum (dilutions: 1 : 1000, 1 : 500, 1 : 100, and 1 : 50) has been found in experiments with a subthreshold adrenaline concentration (10⁻⁸ g/ml). ESBAR (dilutions: 1 : 50 and 1 : 10) and EBBAR (dilution 1 : 500) activities of blood serum have been found in experiments with the maximum effective concentration of adrenaline (10⁻⁶ g/ml). Therefore, blood serum endogenous modulators of β-adrenergic reactivity, ESBAR and EBBAR, can modulate the activation of β-AR of human cardiomyocytes. These prove the prospects of the ESBAR analogue application in cardiology.     

Rat small-intestinal β-galactosidases: Influence of pH on the hydrolysis of different substrates

1. The influence of pH and the kind of buffer on the hydrolysis of lactose and four hetero-β-galactosides (phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside) by homogenates of rat small-intestinal mucosa has been studied. 2. There are at least two β-galactosidases present in the homogenates, one with optimum pH3–4 and another with optimum pH5–6. 3. The enzyme with the lower pH optimum is mainly a heterogalactosidase. It hydrolyses lactose slowly. The other enzyme is mainly a disaccharidase, since it hydrolyses lactose much more rapidly than the heterogalactosides. 4. Under the conditions used, citrate had an inhibitory effect on the 6-bromo-2-naphthyl β-galactosidase activity at pH3–4, but did not influence the 6-bromo-2-naphthyl β-galactosidase activity at pH5–6 or the hydrolysis of the other substrates at any pH.