Characterization of the catalytic domains of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endoglucanase I,II, and III,expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.     

Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10^–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10^–5 m), kinetin (Kn) (5×10^–6 m), 1-indolebutyric acid (IBA) (2×10^–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l^–1), or MS with 2,4-D (1×10^–5 m), 6-benzylaminopurine (BAP) (1×10^–5 m), and soluble PVP (250 mg l^–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10^–6 m), Kn (5×10^–6 m) and soluble PVP (250 mg l^–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10^–6 m) and IBA (2.5×10^–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced. Received: 16 April 1998 / Revision received: 25 September 1998 / Accepted: 10 October 1998     

Age and composition of carbonate shoreface sediments, Kailua Bay, Oahu, Hawaii

The origin, age, and dynamics of carbonate sediments in Kailua Bay on Oahu, Hawaii, are described. The shoreface (from shoreline to 4 km offshore) consists of a broad (5 km²) fringing coral reef ecosystem bisected by a sinuous, shore-normal, sand-filled paleostream channel 200–300 m wide. The median grain diameter of surface sands is finest on the beach face (<0.3 mm) and increases offshore along the channel axis. Kailua sands are >90% biogenic carbonate, dominated by skeletal fragments of coralline algae (e.g. Porolithon, up to 50%) followed by the calcareous green alga Halimeda (up to 32%), coral fragments (1–24%), mollusc fragments (6–21%), and benthic foraminifera (1–10%). Sand composition and age across the shoreface are correlated to carbonate production. Corals and coralline algae, principal builders of the reef framework, are younger and more abundant in sands along the channel axis and in offshore reefal areas, while Halimeda, molluscs, and foraminifera are younger and more dominant in nearshore waters shoreward of the main region of framework building. Shoreface sediments are relatively old. Of 20 calibrated radiocarbon dates on skeletal constituents of sand, only three are younger than 500 years b.p.; six are 500–1000 years b.p.; six are 1000–2000 years b.p.; and five are 2000–5000 years b.p. Dated fine sands are older than medium to coarse sands and hence may constitute a reservoir of fossil carbonate that is distributed over the entire shoreface. Dominance of fossiliferous sand indicates long storage times for carbonate grains, which tend to decrease in size with age, such that the entire period of relative sea-level inundation (∼5000 years) is represented in the sediment. Despite an apparently healthy modern coral ecosystem, the surficial sand pool of Kailua Bay is dominated by sand reflecting an antecedent system, possibly one that existed under a +1–2 m sea-level high stand during the mid- to late Holocene. Accepted: 20 December 1999     

Interactions of Amyloid β Peptide 1–40 and Cerebrosterol

Amyloid β peptides appear to play a role in physiological processes; however, they are also involved in the pathogenesis of Alzheimer disease. Their actions under normal conditions are probably mediated by soluble monomeric l-isoforms at low concentrations, perhaps via highly specific interactions. On the contrary, toxic effects of aggregated natural l-isoforms/synthetic d-isoforms on membranes are very similar, but synthetic reverse/random l-isoforms without pronounced aggregation properties are not toxic. Our previous work reported interactions of non-aggregated/aggregated l-isoforms of amyloid β peptides 1–40/1–42 with racemic 24-hydroxycholesterol. In this study, stereospecificity in the interactions of natural 24(S)hydroxycholesterol (cerebrosterol) or synthetic 24(R)hydroxycholesterol with soluble fragment 1–40 was evaluated by means of an in vitro test based on increased vulnerability of the hemicholinium-3 sensitive high-affinity choline uptake system in rat hippocampal cholesterol-depleted synaptosomes to the actions of amyloid β; computational simulations were also performed. Our results suggest that: (1) 24(S)hydroxycholesterol interacts with l-peptide 1–40 but not with the reverse l-peptide 40–1, (2) 24(R)hydroxycholesterol does not interact with l-peptide 1–40 or reverse 40–1, and (3) both enantiomers can probably interact with d-peptide 1–40. Therefore, the binding of 24(S)hydroxycholesterol is not fully stereospecific and the interaction could not reflect a physiological mechanism. Data from the computational simulation indicate that the hydrophobic core of the amyloid β molecule interacts with the hydrophobic part of 24(S)hydroxycholesterol, but no hydrogen bonds with high stability were found. Using this procedure, globular amyloid β could retain 24(S)hydroxycholesterol and thus contribute to its pathological accumulation in the brains of patients with Alzheimer disease.     

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of β-d-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid β-d-galactosidase with a low mobility and maximal activity atpH 3–5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-d-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 → 4)-lactone. (2) Lactose-hydrolyzing β-d-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active atpH 5–6 and strongly inhibited by glucono-(1 → 5)-lactone but not much affected by galactono-(1 → 4)-lactone. (3) Neutral β-d-galactosidase with a fast mobility and maximal activity atpH 6–8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-β-d-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 → 5)-lactone and (less) by galactone-(1 → 4)-lactone. Neutral β-d-galactosidase and neutral β-d-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral β-d-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation. This investigation was supported in part by Public Health Service Grant RR-00251 from the Division of Research Resources and by funds of the University of Utrecht.     

Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system

The effects of l-arginine, and its analogues N ^ω-nitro-l-arginine methyl ester and N ^ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation. l-Arginine, at 10^–8 mol · l^–1induced a slight vasodilatory effect (max 10%). N ^ω-nitro-l-arginine methyl ester and N ^ω-Nitro-l-arginine in the range 10^–8–10^–4 mol · l^–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10^–4 mol · l^–1 N ^ω-nitro-l-arginine or N ^ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l^–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced a strong vasoconstriction (already significant at 10^–5 mol · l^–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine induced a biphasic response with vasodilation at low concentration (max 15% at 10^–8 mol · l^–1). Serotonin displayed a dose-response vasodilation in the range 10^–8–10^–4 mol · l^–1 (max 20%). These vasodilative effects were reduced or abolished by 10^–4 mol · l^–1 l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system. Accepted: 1 July 1996     

Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Received: 7 February 1997 / Accepted: 6 June 1997     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

Variations of two pools of glycogen and carbohydrate in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> grown with various ethanol concentrations

Glycogen, a major reservoir of energy in Saccharomyces cerevisiae, is found to be present as soluble and membrane-bound insoluble pools. Yeast cells can store excess glycogen when grown in media with higher concentration of sugar or when subjected to nutritional stress conditions. Saccharomyces cerevisiae NCIM-3300 was grown in media having ethanol concentrations up to 12% (v/v). The effects of externally added ethanol on glycogen and other carbohydrate content of yeast were studied by using alkali digestion process. Fermentative activities of cells grown in the presence of various ethanol concentrations (2–8% v/v) exhibited increase in values of glycogen and other carbohydrate, whereas cells grown with higher concentrations of ethanol (10–12% v/v) exhibited depletion in glycogen and carbohydrate content along with decrease in cell weight. Such inhibitory effect of ethanol was also exhibited in terms of reduction in total cell count of yeast grown in media with 2–16% (v/v) ethanol and 8% (w/v) sugar. These data suggest that, as the plasma membrane is a prime target for ethanol action, membrane-bound insoluble glycogen might play a protective role in combating ethanol stress. Elevated level of cell-surface α-glucans in yeast grown with ethanol, as measured by using amyloglucosidase treatment, confirms the correlation between ethanol and glycogen.     

Edwardsiella hoshinae,a new species of enterobacteriaceae

Eight strains isolated from birds, reptiles, and water constitute a new DNA hybridization group that is 37–58% related toEdwardsiella tarda and less than 10% related to other species of Enterobacteriaceae (SI nuclease method). This homogeneous group (78–100% relatedness within the group) constitutes a new species that is namedEdwardsiella hoshinae sp. nov. (type strain, CIP 78.56 ATCC 33379). Strains of this species produce acid fromd-mannitol, sucrose,d-trehalose, and salicin, and give a positive malonate test. Seven other strains that produced acid fromd-mannitol and sucrose (but not fromd-trehalose and salicin) and were malonate negative were found to belong to theEdwardsiella tarda DNA hybridization group. The base composition of the DNAs ofE. tarda andE. hoshinae is 55–58 mol% G+C.     

Production of Bioactive γ-Glutamyl Transpeptidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Using SUMO Fusion Partner and Application of the Recombinant Enzyme to <Emphasis Type="SmallCaps">l</Emphasis>-Theanine Synthesis

The amino acid l-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of l-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni–NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of l-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of l-theanine.     

Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA₃-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA₃-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.     

Increased Lipid Peroxidation and Ascorbic Acid Utilization in Testis and Epididymis of Rats Chronically Exposed to Lead

The hypothesis has been recently presented that lead may exert its negative effect at least partially through the increase of reactive oxygen species (ROS) level in tissues. However, little is known about the influence of lead intoxication on equilibrium between generation and elimination of ROS in the male reproductive system. Sexually mature male Wistar rats were given ad libitum 1% of aqueous solution of lead acetate (PbAc) for 9 months. Significantly higher lead concentrations were found in blood [median 7.03 (Q25–Q75: 2.99–7.65) versus 0.18 (0.12–0.99) μg dl⁻¹, P < 0.01], caput epididymis [median 5.51 (Q25–Q75: 4.31–7.83) versus 0.51 (0.11–0.80) μg g⁻¹ d.m., P < 0.001], cauda epididymis [median 5.88 (Q25–Q75: 4.06–8.37) versus 0.61 (0.2 – 1.08) μg g⁻¹ d.m., P < 0.001] and testis [median 1.81 (Q25–Q75: 0.94–2.31) versus 0.17 (0.03–0.3) μg g⁻¹ d.m., P < 0.01] of lead-intoxicated rats when compared to the control. The concentration of ascorbyl radical, generated in vitro from l-ascorbic acid (present in tissues in vivo) was measured by means of Electron Paramagnetic Resonance (EPR) spectroscopy. The EPR signal of ascorbyl radical in caput epididymis, cauda epididymis, testis and liver of lead acetate-treated animals revealed a significant decrease by 53%, 45%, 40% and 69% versus control tissues, respectively. Plasma l-ascorbic acid content measured by high performance liquid chromatography (HPLC) method and total antioxidant status (TAS) measured by means of spectrophotometry were also significantly lower in the intoxicated versus control animals (28% and 21%, respectively). In the group exposed to lead the concentration of lipid peroxide in homogenates of the reproductive system organs was significantly elevated versus control group. It can be assumed that the lower EPR signal was caused by decreased tissue concentrations of l-ascorbic acid. The latter may have resulted from consumption of ascorbic acid for scavenging of ROS excess in tissues of animals chronically exposed to lead.     

Some biochemical changes during leaf rolling in Ctenanthe setosa (Marantaceae)

The changes in the levels of proline, sugar and soluble protein during leaf rolling and its relationship to osmotic adjustment were studied in laboratory conditions. Upon irrigation of plants which have rolled leaves, many sugar crystals occurred on the abaxial surface of the leaves in Ctenanthe setosa (Rosc.) Eichler. The sugar crystals were determined to have sucrose, glucose and fructose. The levels of reducing sugars and proline are higher in rolled leaves while soluble protein levels in rolled leaves are lower than those of unrolled leaves. It was found 1–3, 9–13, 16–21 and 24–28 crystals at degree of leaf rolling 23, 28, 47 and 52%, respectively. Finally, we found a significant correlation between the crystal number and degree of leaf rolling in Ctenanthe setosa. In addition, soluble sugars are found predominant accumulating solute in the plant and are of major importance as a contributor to osmotic adjustment during leaf rolling.     

Roles of Soluble Sugars in Protecting Phytochrome- and Gibberellin A3-Mediated Germination Control in Skotodormant Lettuce Seeds

Skotodormant seeds of Lactuca sativa Grand Rapids imbibed in darkness for 10 days (10-day DS) germinated poorly upon terminal treatment with red light (R) or gibberellin A₃ (GA₃). Soluble sugars in the imbibition solutions influenced the depth of skotodormancy. Ten-day DS seeds, imbibed in 50–500 mm sucrose or 100–500 mm glucose and given terminal GA₃ germinated completely and germinated about 80% when imbibed in 100 mm galactose, mannose, lactose, or maltose. In contrast, terminal R applied to 10-day DS seeds caused only 20–50% germination. If given R at day 0 and imbibed for 10 days in darkness in 500 mm sucrose or glucose, seeds washed free of exogenous glucose or sucrose then germinated about 50% in darkness in water. These seeds responded to terminal R or GA₃ with complete germination. When seeds were given FR at day 0, germination responses following terminal R or GA₃ were significantly lower when the duration of DS was increased from 7–10 day DS to 15 days. In 10-day DS seeds given initial FR and imbibed in either solutions of 50 or 100 mm sucrose and KNO₃, either terminal R or GA₃ treatment gave complete or near complete germination. It is concluded that seed exposure to certain soluble sugars and/or nitrate during a 10-day DS protected certain substrates and thereby extended the sensitivity of the seeds to terminal R or GA₃ treatment. The study provides substantial evidence for nonhormonal factors associated with light and GA action in the control of seed skotodormancy. Received October 30, 1996; accepted April 22, 1997     

Catalysis on dinuclear Mn(II) centers: hydrolytic and redox activities of rat liver arginase

 Rat liver arginase contains a dinuclear Mn₂(II,II) center in each subunit having EPR properties similar to those observed in Mn-catalases. The principal physiologic role of arginase is catalyzing the hydrolytic cleavage of l-arginine to produce l-ornithine and urea. Here we demonstrate that arginase catalyzes the disproportionation of hydrogen peroxide by a redox mechanism analogous to Mn-catalases, but at rates that are 10^–5 to 10^–6 of k _cat for the Mn-catalases, and also exhibits peroxidase activity. The dinuclear Mn₂(II,II) center is essential for maximal catalase activity, since both the H101N and H126N mutant arginases containing only one Mn(II)/subunit have catalase activities that are <3% of that for the wild-type enzyme. Like the Mn-catalases, the catalase activity of arginase is not inhibited by millimolar concentrations of CN^–, the most potent inhibitor of heme catalases, or by EDTA, a chelator of free metal ions. The catalase activity of arginase is not significantly inhibited by Cl^– or F^–, in contrast to Mn-catalases, while potent inhibitors of the hydrolytic activity are also effective inhibitors of the catalase activity. These results suggest that lower affinity of hydrogen peroxide to the active site of arginase contributes to the lower catalase activity. EPR spectroscopy reveals that potent inhibitors of the hydrolytic reaction, including N ^ω-hydroxy-l-arginine, l-lysine, and l-valine, decouple the electronic interaction between the Mn²⁺ ions, most probably by removing a μ-bridging ligand or by increasing the intermanganese separation. The capacity for arginase to deliver a hydroxide ion to hydrolyze the l-arginine substrate is suggested to arise from a "dinuclear effect", wherein the two metal ions contribute more or less equivalently in deprotonation of metal-bound water molecule. Structure-reactivity analyses of these reactions will provide insights into the factors that control redox versus hydrolytic function in dimanganese clusters. Received: 18 November 1996 / Accepted: 7 April 1997     

Zhihengliuella salsuginis sp. nov., a moderately halophilic actinobacterium from a subterranean brine

A novel moderately halophilic, alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic, coccus-shaped, Gram-positive bacterium, designated strain JSM 071043^T, was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Growth occurred with 0.5–20% (w/v) NaCl (optimum 5–10%) at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl₂·6H₂O (optimum 5–10%). The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose. The major cellular fatty acids were anteiso-C_15:0, iso-C_16:0 and anteiso-C_17:0. Strain JSM 071043^T contained MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content was 67.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 071043^T was a member of the suborder Micrococcineae, and was most closely related to Zhihengliuella halotolerans YIM 70185^T (sequence similarity 98.9%) and Zhihengliuella alba YIM 90734^T (98.2%), and the three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 071043^T represents a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed. The type strain is JSM 071043^T (= DSM 21149^T = KCTC 19466^T).