Carbohydrate digestion in the adult moth Heliothis zea

Extracts from the ventriculus and the salivary glands of the adult corn earworm, Heliothis zea, were tested for carbohydrase activity. The hydrolysis that occurred among the 12 carbohydrate substrates tested and the evidence from thin-layer chromatography indicated only one carbohydrase, a β-fructosidase, from the salivary glands and two, a β-fructosidase fnd an α-glucosidase, from the ventriculus. Optimum pH was 6·5 for the β-fructosidase from both the ventriculus and the salivary glands and 5·5 for the α-glucosidase. The K_m for the β-fructosidase from the salivary glands was 112 mM.     

Biochemical characterisation of digestive proteases and carbohydrases of the pistachio fruit hull borer,Arimania komaroffi Ragonot (Lepidoptera: Pyralidae)

Pistachio fruit hull borer, Arimania komaroffi Ragonot (Lep.: Pyralidae), is one the most important pests of pistachio in Iran. The larvae spin web as well as bore into young fruits, and the infested fruits fall off the trees. The second-generation adult moths appear in August and September, and their offspring feed on the fruit hull. Results indicated the presence of α-amylase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and some proteases in the digestive tract of the pest. Highest activities of α-amylase, α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase were at pH 10, 7, 7, 6 and pH 6, respectively. Highest activities of trypsin, chymotrypsin and elastase of larval midgut were at pH 11. Zymogram analysis of α-amylase, α-glucosidase, β-glucosidase, tryptic, chymotryptic and elastase using native-PAGE revealed 1, 1, 2, 3, 3 and 2 bands of activity respectively, in A. komaroffi. One band was disappeared in the presence of the inhibitor TLCK, but no further inhibition by the inhibitors TPCK was observed. The results can be of help for designing new strategies for controlling the pistachio fruit hull borer based on natural proteases and carbohydrase inhibitors.     

Lytic bodies from cereals hydrolysing maltose and starch

Protein bodies and spherosomes from sorghum contained carbohydrase activity against maltose, starch and p-nitrophenyl-α-d-glucoside. Maltase activities in sorghum and also in maize lytic bodies were very high; carbohydrase activities of lytic bodies from whole wheat, whole barley, sorghum aleurone, sorghum embryo and maize embryo were considerably lower. The pH response of sorghum lytic bodies was bimodal with an optimum in the range of 3·4–4·2 and a minimum or a shoulder near pH 3·8. Protein bodies from sorghum, maize, wheat and barley reduced the iodine-colouring capacity of soluble starch to give a purple colour typical of a β-limit dextrin. With spherosomes colour reduction was usually more rapid, eventually taking the breakdown of starch beyond the achroic point. The lytic bodies produce both maltose and glucose from starch, except in the case of maize when only glucose was found. The data suggest that protein bodies contain a linked β-amylase-maltase system and that spherosomes contain a linked α-amylase-maltase system.     

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

The Relationship of Some Glycosidases to the Endogenous and IAA-induced Growth of Light-grown Cucumber Hypocotyls

Some glycosidases in light-grown cucumber (Cucumis sativus L. cv. Aonaga-jibae) hypocotyl sections were examined with respect to their localization and relation to endogenous and IAA-induced growth. Frozen-thawed sections were used directly for measurement of enzyme activities, and β-glucosidase, α- and β-galactosidases and β-xylosidase were assayed by using p- or o-nitro-phenylglycopyranosides as substrates. The order of the activity of these enzymes were β -glucosidase > β -galactosidase =α-galactosidase > β-xylosidase. No activity of α-glucosidase was detected. High glycosidase activities were found in the youngest region of the hypocotyl, where the endogenous growth rate was highest. However, there was no significant difference in the activities of this region between seedlings at different growth stages. Among the enzymes tested, β -glucosidase showed a high correlation with the endogenous growth rate. β-glucosidase was found to be mostly associated with the cell wall fraction, while β-galactosidase was rather found in the soluble fraction of the cell. Separation of the epidermis from the section showed that a very high activity of β-glucosidase was associated with the epidermis. In both whole sections and isolated cell wall fractions, IAA was shown to have no effect on the activities of β-glucosidase and β-galactosidase.     

Expression from two Drosophila promoters in embryos of the migratory locust

We have accomplished gene transfer into embryos of Locusta migratoria, the African migratory locust. Freshly oviposited eggs were injected with circular or linear plasmids containing the Drosophila hsp70 promoter and the choramphenicol acetyltransferase (CAT) reporter gene (hsp-cat), or with circular plasmid containing the Drosophila copia promoter fused to CAT (copia-cat). Southern blot analysis showed that the hsp-cat plasmid persisted extrachromosomally for at least 8 days after injection. There was no evidence for plasmid replication. Transient expression from the introduced promoters was determined by monitoring CAT enzyme activity. After injection of hsp-cat, activity was detected at varying levels in 6–8% of day 3 and day 9 embryos. Embryos injected with copia-cat, assayed on day 3, had a greater frequency but no higher level of expression. The described gene transfer system is promising for analysis of other promoters, including those of Locusta.     

Wall-bound enzymes in callus of Convolvulus arvensis

Isolated cell walls of Convolvulus callus contain α- and β-galactosidase, α- and β-glucosidase, α- and β-mannosidase, acid invertase and acid phosphatase activities. No neutral invertase or alkaline phosphatase activities could be detected. Acid invertase activity per mg cell wall increased considerably during incubation of callus fragments in nutrient solution, as opposed to the activities of the other enzymes mentioned.     

Comparison of α- and β-mannosidase activity in the three cereal pests,Haplothrips tritici Kurdjumov (Thysanoptera: Phlaeothripidae), Rhopalosiphum padi L. (Homoptera: Aphididae) and Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

The Sunn pest, Eurygaster integriceps, the bird cherry-oat aphid, Rhopalosiphum padi, and wheat thrips, Haplothrips tritici are the major pests of wheat and other cereals in a wide area of the world. All these three insect species could produce damage to the wheat to some extent. Therefore, the purpose of the present study was to determine α- and β-mannosidase of the three mentioned insect pests. These insects were collected from the wheat farm and their guts (the Sunn pest and the aphid) and salivary glands of Sunn pest were removed. However, regarding tiny body of thrips, the whole body used in order to extract the enzymes. The enzymes, including α- and β-mannosidase activity, were measured by the hydrolysis of p-nitrophenyl-α-d-mannopyranoside (pNPαGal) and p-nitrophenyl-β-d-mannopyranoside (pNPβGal), respectively, using phosphate citrate buffer (pH 5.0). Mannosidases were not active in all three tested insect species, and also there were significant differences in activities of the two enzymes in three species. The greatest activity of α-mannosidases was observed in the Sunn pest salivary glands, E. integriceps, and the least activity was found in Sunn pest midgut with no activity. However, the activity of β-mannosidase was established in Sunn pest midgut, but there was no activity in the aphid midgut, R. padi. Activities of these two enzymes were modest in the thrips, H. tritici. The greatest amount of β-mannosidases in the Sunn pest midgut makes sense, since the Sunn pest is the main pest in the wheat farm that can feed on wheat grains. In the wheat grains, the highest amount of glycoproteins and glycolipids are present. Thus, it has been known that these enzymes (α- and β-mannosidases) are active on digestion of carbohydrates.     

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.     

Use of specific glycosidases to probe cellular interactions in the sea urchin embryo

We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used α- and ß-amylase and α- and ß-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of α-glucosidase and ß-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ß-glucosidase and α-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the α- and ß-glycosidases.     

Digestive glucosidases in the midgut of Apodiphus amygdali Germar (Hemiptera: Pentatomidae)

Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30?°C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K⁺ and Ca²⁺ significantly increased enzymatic activity, Na⁺ had no effect, and Cu²⁺, Fe²⁺ and Mg²⁺ had the significant negative effects on the enzyme activity. Ca²⁺ and Fe²⁺ increased β-glucosidase activity in the midgut of A. amygdali, Na⁺ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N?,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests.     

Nkx6.1 and nkx6.2 regulate α- and β-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells

In mice, the Nkx6 genes are crucial to α- and β-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in β-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of α-cells but has no effect on β-, δ-, or ε-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of β-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both β- and α-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for α-cell than for β-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control α- and β-cell differentiation.     

Chromatographic methods for the determination of alpha- and beta-5-phospho-D-ribose-alpha-1-pyrophosphate pools in bacteria.

Different methods for the determination of 5-phospho-d-ribose-α-1-pyrophosphate (PRPP) are described. PRPP from ³²P-labeled microorganisms is determined directly either alone or together with the ribo- or deoxyribonucleosidetriphosphates in thin-layer chromatographic systems, using chemicals of high purity. As little as 0.1 pmol of both the α- and β-anomeric forms can be detected. α-PRPP is also determined by a new enzymatical radioactive microassay; the lower limit of detection is 1 pmol. An anomerization from α- to β-PRPP was found to take place during the application of the PRPP onto polyethyleneimine cellulose plates if formic acid or cell extract or both is added to α-PRPP. If the α-PRPP is purified from the cell extract before application, no anomerization will take place. It is also found that PRPP is stable in dilute organic acids. During accumulation of PRPP in purine-starved S. typhimurium, 10% of the PRPP was found as β-PRPP in vivo.     

Sucrose-dependent increase in oligosaccharide production and associated glycosidase activities in the potato aphid Macrosiphum euphorbiae (Thomas)

Osmotic stress due to high sucrose concentration was imposed on the potato aphid, Macrosiphum euphorbiae (Thomas), in an artificial diet situation. Thin layer chromatography resolved melezitose and six additional honeydew oligosaccharides. Significantly higher levels of these oligosaccharides were recovered from the honeydew of this species in response to feeding on increased dietary sucrose concentrations, pointing to an osmoregulatory response. In addition, carbohydrase activities from the potato aphid were described. A degree of specificity toward hydrolysis of the sucrose substrate over other α-glucosyl sugars was demonstrated. Also, sucrose was optimal for the formation of the oligosaccharides. A strong α-galactosidase activity was found and transgalactosylation ability was indicated. These major trends in carbohydrase activity were also found in the foxglove aphid, Acyrthosiphon solani (Kaltenbach), and the oleander aphid, Aphis nerii Fonscolombe.     

Artemia hemoglobins: Increase in net synthesis of the β-polypeptide (relative to the α-polypeptide) in hypoxia

Previous studies have shown that in the brine shrimp there are three dimeric hemoglobins with polypeptide composition α₂, αβ, β₂. Concentrations of the α- and β-polypeptides increase in hypoxia. We now report a two-dimensional electrophoretic method for assay of radiolabelled polypeptidesin each hemoglobin. Net synthesis (synthesis minus degradation) of the β-chain, relative to that of the α-chain, increases more than 3-fold (in male and female adults) within 3 days following a downshift in oxygen concentration from 0.2 to 0.1 mM in the culture medium. 3 days after downshift (2 days after in vivo incorporation of radiolabelled leucine), the β-homodimer contained 10–20% of the radiolabel in the three hemoglobins although β₂ was usually not detectable in the protein stain of an overloaded gel. The amount of radioactive leucine incorporated per unit amount of protein was more than 300-times greater in the β₂ homodimer than in the β-subunit of the heterodimer, suggesting that β₂ does not dissociate rapidly during electrophoresis on the first dimension non-denaturing gel. This evidence for stable association of the two β-monomers and the 5–8 heme-binding domains within each monomer (in vivo and during electrophoresis on non-denaturing gels) allows us to exclude one of two alternative interpretations of genetic data published previously. We present an independent line of evidence for the dimer model of the native hemoglobins (which states that each polypeptide has many heme-binding domains).     

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas

Cell lineage analysis is critical in understanding the relationship between progenitors and differentiated cells as well as the mechanism underlying the process of differentiation. In order to study the zebrafish endocrine pancreas cell lineage, transgenic expression of diphtheria toxin gene A chain (DTA) under two cell type-specific promoters derived from the insulin (ins) and somatostatin2 (sst2) genes was used to ablate the two types of endocrine cells: insulin-producing β-cells and somatostatin-producing δ-cells, respectively. We found that ablation of β-cells resulted in a reduction of not only β-cells but also glucagon-producing α-cells; in contrast, δ-cells were largely unaffected. Ablation of δ-cells led to reduction of all three types of endocrine cells: α-, β-, and δ. Interestingly, α-cells were more profoundly affected in both β- and δ-cell ablations and were frequently reduced together with β- and δ-cells. By taking advantage of Tg(ins:gfp) and Tg(sst2:gfp) lines, we also monitored the changes of different types of endocrine cells in vivo after ablation and found that both β- and δ-cell populations significantly recovered by 3 dpf after their ablation and it seemed that δ-cells had a better capability of recovery than β-cells. Thus, our current observations indicated differential interdependence of these three cell lineages. The development of zebrafish α-cells, but not δ-cells, is dependent on β-cells, while the development of both α- and β-cells is dependent on δ-cells. In contrast, the development of δ-cells was independent of β-cells.     

Terpenoids of Perezia hebeclada

Perezone (I), hydroxyperezone (IIa), hydroxyperezone monoisovalerate (IIb), α-(IIIa) and β-pipitzols (IVa) and the new compounds α- (Va), β- (VIa) and γ-perezols (Ve) were found in the roots of P. hebeclada. The structures and stereochemistry of the perezols were established by chemical evidence and comparison of their spectral properties with those of the known α- (IIIa) and β-pipitzols (IVa).     

Biochemical characterization of digestive α-amylase, α-glucosidase and β-glucosidase in pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

The pistachio green stink bug, Brachynema germari, has 3–5 generations per year and causes severe damages to pistachio crops in Iran. Physiological digestive processes, such as digestive carbohydrases, can be used to design new strategies in IPM programs for controlling this pest. The enzyme α-amylase digests starch during the initial stage of digestion. Complete breakdown of carbohydrates takes place in the midgut where α- and β-glucosidic activities are highest. Alpha-amylase and α- and β-glucosidase activities were found in the midgut and salivary glands of pistachio green stink bug adults. Overall enzyme activities were significantly higher in the midgut than in salivary glands. The highest α-amylase and α- and β-glucosidase activities were in section v3, whereas the lowest activities were in section v4. V_max was higher and K_m was lower in the midgut than in the salivary glands for these enzymes. In the pistachio green stink bug, the optimal pH was pH 5–6.5 and the optimal temperature was 30 °C to 35 °C for these enzymes. Alpha-amylase activity in the midgut and salivary glands decreased as the concentrations of MgCl₂, EDTA and SDS increased. Enzyme activities in both midgut and salivary glands increased in the presence of NaCl, CaCl₂, and KCl. NaCl had a negative effect on alpha-amylase extracted from salivary glands.