Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O₂^·−) production upon desiccation in Castanea sativa seeds, mechanisms of O₂^·− production and the effect of exogenously supplied ROS. A transient increase in extracellular O₂^·− production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H₂O₂)-dependent O₂^·− production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of ∼50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O₂^·− production. H₂O₂, applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.     

Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an 'extended −10' motif and a core −10 element

σ³² controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ³² promoters is a long −10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of σ³² and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an 'extended −10' recognition motif that is recognized by K130, a residue universally conserved in β- and γ-proteobacteria. This residue is located in the α-helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α- and δ-/ε-proteobacteria and we found that σ³² from the α-proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ³² is the first Group 3 σ shown to use the 'extended −10' recognition motif.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Analysis of a cellodextrinase cloned from Ruminococcus flavefaciens FD-1

Abstract A cellulase gene from Ruminococcus flavefaciens FD-1 had previously been cloned in Escherichia coli . The product of this gene, CelA, was purified from E. coli and characterised. This 39 kDa cellulase is antigenically related, and of similar mass, to a protein in R. flavefaciens . The enzyme has cellodextrinase activity with predominantly exo-type action. CelA activity was optimal at pH 6.5 and 41°C, and was inhibited in the presence of divalent metal cations. The K _m and V _max were determined as 0.68 mM and 1.89 μmol min⁻¹ mg⁻¹ of CelA, respectively. Cellobiose was the major end product of cellodextrin hydrolysis, and our results suggest that celluboise is competitive inhibitor of CelA.     

Hemi-methylated oriC DNA binding activity found in non-specific acid phosphatase

The lacZ – hobH fusion clone, containing an Escherichia coli DNA segment located at 92 min on the chromosomal map, was screened as a producer of E. coli oriC hemi-methylated binding activity. We have purified the protein encoded by this locus to near homogeneity. The protein corresponds to the monomeric form of a non-specific acid phosphatase (NAP) whose gene has been designated aphA. oriC DNA footprinting experiments showed protection of hemi-methylated probe by partially purified NAP, but not by purified preparations. Yet, gel retardation experiments with an oriC oligonucleotide demonstrated DNA binding activity of purified NAP in the presence of Mg²⁺. This experiment also showed an increased affinity of the protein for the hemi-methylated probe compared with the fully or unmethylated form. Indirect immunofluorescence microscopy revealed the existence of discrete NAP foci at mid-cell in cells with two nucleoids, but at cell poles in those with one nucleoid.     

A calcium and calmodulin-dependent protein kinase present in differentiating Dictyostelium discoideum

Abstract A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca²⁺/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum . The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca²⁺/calmodulin-dependence upon standing at 4°C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [γ-³² P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca²⁺ and calmodulin but not Ca²⁺ alone. Ca²⁺/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.     

Methylotrophic growth of a mutant strain of the acetogenic bacterium Eubacterium limosum that uses acetate as co-substrate in the absence of CO2

Abstract Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col⁻, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col⁻ and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae and enhanced E. coli F-18Col⁻ colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX , which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two proteins (22 kDa and 26 kDa) encoded by genes immediately adjacent to leuX .     

Purification and characteristics of cytosolic chitinase from Piromyces communis OTS1

Abstract A chitinase was purified from the cytosolic fraction of the anaerobic rumen fungus Piromyces communis OTS1 by affinity chromatography using regenerated chitin, gel filtration and chromatofocusing. The chitinase was most active at pH 6.2 and at 60 °C in a 20-min assay. The molecular mass of the purified protein was estimated by SDS-PAGE to be 42 kDa and its pI was 4.9. The enzyme activity, which was of the 'endo' type, was inhibited by A⁺, Hg²⁺ and allosamidin. N -Acetyl- β -glucosaminidase and 'exo' type chitinase activity were absent from the purified preparation.     

Mutational analysis of Escherichia coli σ28 and its target promoters reveals recognition of a composite −10 region, comprised of an 'extended −10' motif and a core −10 element

σ²⁸ controls the expression of flagella-related genes and is the most widely distributed alternative σ factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of σ²⁸ promoters is a long −10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in σ²⁸ participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended −10 motif and is recognized by R91, a residue in Domain 3 of σ²⁸. The downstream CG is the upstream edge of −10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base-specific recognition of the promoter, R91, D81 and D84 are universally conserved in σ²⁸ orthologues. σ²⁸ is the second Group 3 σ shown to use an extended −10 region in promoter recognition, raising the possibility that other Group 3 σs will do so as well.     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p -nitrophenyl butyrate was 0·3 mmol l⁻¹ while on p -nitrophenyl caprylate, it was 0·04 mmol l⁻¹. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l⁻¹ phenylmethylsulphonyl fluoride, β-mercaptoethanol and N -ethylmaleimide, and was stimulated by Zn²⁺ at 1 mmol l⁻¹.