Biochemical Characterization of Allantoinase from <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

Bacterial allantoinase (ALLase; EC 3.5.2.5), which catalyzes the conversion of allantoin into allantoate, possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. Here, we characterized ALLase from Escherichia coli BL21. Purified recombinant ALLase exhibited no activity but could be activated when preincubating with some metal ions before analyzing its activity, and was in the order: Mn²⁺- ≫ Co²⁺- > Zn²⁺- > Ni²⁺- > Cd²⁺- ~Mg²⁺-activated enzyme; however, activity of ALLase (Mn²⁺-activated form) was also significantly inhibited with 5 mM Co²⁺, Zn²⁺, and Cd²⁺ ions. Activity of Mn²⁺-activated ALLase was increased by adding the reducing agent dithiothreitol (DTT), but was decreased by treating with the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Inhibition of Mn²⁺-activated ALLase by chelator 8-hydroxy-5-quinolinesulfonic acid (8-HQSA), but not EDTA, was pH-dependent. Analysis of purified ALLase by gel filtration chromatography revealed a mixture of monomers, dimers, and tetramers. Substituting the putative metal binding residues His59, His61, Lys146, His186, His242, and Asp315 with Ala completely abolished the activity of ALLase, even preincubating with Mn²⁺ ions. On the basis of these results, as well as the pH-activity profile, the reaction mechanism of ALLase is discussed and compared with those of other cyclic amidohydrolases.     

Influence of chroline substitution pattern on the degradation of polychlorinated biphenyls by eight bacterial strains

We compared the metabolism of eight di- and trichlorobiphenyls by eight bacterial strains chosen to represent a broad range of degradative activity against polychlorinated biphenyls (PCBs). The PCB congeners used were 2,3-, 2,3′-, 2,4′-, 3,3′-, 2,3,3′-, 2,4,4′-, 2,5,3′-, and 3,4,2′-chlorobiphenyl. The bacterial strains used wereCorynebacterium sp. MB1,Alcaligenes strainsA. eutrophus H850 andA. faecalis Pi434, andPseudomonas strains LB400 and H1130,P. testosteroni H430 and H336, andP. cepacia H201. The results indicated that both the relative rates of primary degradation of PCBs and the choice of the ring attacked were dependent on the bacterial strain used. The bacterial strains exhibited considerable differences in their relative reactivity preferences for attack on mono- and dichlorophenyl groups and in the degree to which the attack was affected by the chlorine substitution pattern on the nonreacting ring. For MB1 the reactivity pattern was 3-≥4-≫2-chlorophenyl with no attack on 2,4- or 2,5-chlorophenyl groups. This strain was relatively insensitive to the chlorine substitution pattern on the nonreacting ring. Strains H1130, H430, H201, and Pi434 exhibited the same reactivity preferences as MB1, but for these strains (and for all others tested) the chlorination pattern on the nonreacting ring had a strong effect. For strain H336 the reactivity preference was 4-≥2->2,4-≥3-chlorophenyl, with no evidence of attack on 2,5-chlorophenyl rings. For strains H850 and LB400 the relative reactivity was 2->2,5->3-≫2,4->4-chlorophenyl. On this basis we propose that the eight bacterial strains represent four distinct classes of biphenyl/PCB-dioxygenase activity. The types of products formed were largely strain-independent and were determined primarily by the chlorine substitution pattern on the reacting ring. When the reacting ring was an unsubstituted phenyl or a 2-chlorophenyl group, the products were chlorobenzoic acids in high yields; for a 3-chlorophenyl ring, both chlorobenzoic acids and chloroacetophenones in moderate yields; and for a 4- or 2,4-chlorophenyl group, chlorobenzoic acids in low yields with an apparent accumulation ofmeta ring-fission product. Strains H850 and LB400 were able to degrade the 3-chlorobenzoic acid that they produced from the degradation of 2,3′-chlorobiphenyl. We conclude that despite differences among strains in the specificity of the initial dioxygenase, the specificities of the enzymes responsible for the subsequent degradation to chlorobenzoic acid and/or chloroacetophenone are quite similar for all strains.     

Substituted phenols as repellents for male Costelytra zealandica (Goleoptera: Scarabaeidae) in mating flights

Methyl‐ and halogen‐substituted phenols were found to be strongly repellent to male Costelytra zealandica (White) beetles in mating flights. Phenols substituted in the 2‐position were stronger repellents than those substituted in the 3‐ or 4‐position. Some of these compounds reduced the effectiveness of a potent sex attractant source by > 90 %.     

Stimulation of natural killer cells by homoeopathic complexes: An in vitro and in vivo pilot study in advanced cancer patients

The present study was designed in order to evaluate the effects of five homoeopathic complex preparations on functional activity natural killer cells (NKCs) in advanced cancer patients. We examined the effects of Coenzyme Compositum®, Ubichinon Compositum®, Glyoxal Compositum®, Katalysatoren® and Traumeel® on the functional activity of NKCs. Experimental procedures included in vitro and in vivo trials. The in vitro trials were performed in NKCs isolated from 12 healthy volunteers (aged 44 ± 4 years) and incubated with the five homoeopathic complex preparations. The in vivo trials were performed in 15 advanced cancer patients (aged 55 ± 12 years) supplemented for 3 months with the homoeopathic preparations. All five homoeopathic preparations significantly increased the cytotoxic activity of the NKCs at the lowest NKCs/target cell ratio 12:1 (p < 0·05). The order of activity was: Ubichinon Compositum® > Glyoxal Compositum® > Katalysatoren® > Traumeel® > Coenzyme Compositum®. In the advanced cancer patients, the homoeopathic preparation significantly increased NKCs cytotoxic activity (p < 0·05). The homoeopathic complex preparations tested in this study can be used as an adjuvant immunotherapy in advanced cancer patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulation of Norepinephrine Release from Isolated Bovine Irides by Histamine

In the present study, we investigated the effect of histamine on sympathetic neurotransmission from isolated, superfused bovine irides. We also studied the pharmacology of prejunctional histamine receptors that regulate the release of norepinephrine (NE) from this tissue. The effect of exogenous histamine and various histamine receptor agonists was examined on the release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation using the Superfusion Method. Histamine receptor agonists caused a concentration-dependent inhibition of field-stimulated [³H]NE overflow with the following rank order of potency: imetit > histamine > R-α-methylhistamine. In all cases, the inhibitory action of histamine receptor agonists was attenuated at high concentrations of these compounds. The histamine receptor antagonists, clobenpropit (H₃-antagonist/H₄-agonist) and thioperamide (H₃-antagonist) blocked the inhibitory response elicited by R-α-methylhistamine and imetit, respectively. Inhibitory effects of R-α-methylhistamine and clonidine were not additive suggesting that prejunctional H₃- and α₂-adrenoceptors coexist at neurotransmitter release sites. We conclude that histamine produces an inhibitory action on sympathetic neurotransmission in the bovine iris, an effect mimicked by selective H₃-receptor agonists and blocked by H₃-antagonists.     

Modelling antisepsis using defined populations of facultative and anaerobic wound pathogens grown in a basally perfused biofilm model

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.     

Enzymatic synthesis of isopentenyl pyrophosphate in sweet potato root tissue in response to infection by black rot fungus

Cell-free systems were prepared from fresh, cut and black rot-diseasedtissues of sweet potato roots. When incubated in the presenceof mevalonate, all these systems were capable of synthesizing5-phosphomevalonate, mevalonate-5-pyrophosphate and isopentenylpyrophosphate. The time courses for the appearance of the ¹⁴C-labeledmetabolites suggested the following order of synthesis: mevalonate 5-phosphomevalonate mevalonate-5-pyrophosphate isopentenylpyrophosphate. It was also shown; on prolonged incubation, thatisopentenyl pyrophosphate was converted slowly to isopentenylmonophosphate. The activity for synthesis of isopentenyl pyrophosphatefrom mevalonate was higher in diseased tissue than in cut andhealthy tissues. ¹This paper constitutes Part 78 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received June 18, 1969; )     

桂北不同林龄桉树人工林土壤碳库管理指数和碳组分的变化特征

采用时空互代法,以广西北部低山丘陵地区不同林龄(1、2、3、4、5和8 a)桉树人工林为研究对象,探讨林龄对桉树人工林地土壤碳库管理指数的影响及其规律。结果表明:(1)随着林龄的增加,土壤有机碳总体表现为增加的趋势,1～8 a桉树土壤有机碳范围在5.79～15.57 g·kg^-1之间,随着土层的加深而降低; 0～40 cm土层土壤有机碳平均含量表现为8 a>5 a>3 a>4 a>2 a>1 a。(2)土壤非活性有机碳、碳储量随林龄和土层的变化规律与土壤有机碳基本一致。土壤活性有机碳含量大小依次表现为8 a>5 a>4 a>3 a>2a> 1 a,占土壤有机碳的比例随林龄变化无明显规律,8 a和其他林龄间均具有显著差异。(3)碳库管理指数随林龄增加整体呈上升趋势,8 a桉树人工林土壤碳组分含量及碳库管理指数均高于10 a对照马尾松林。碳库管理指数与土壤有机碳、非活性有机碳、活性有机碳、碳储量、碳库活度、全氮、容重呈极显著或显著的相关性,不同林龄和土层间碳库管理指数有差异性。适当延长桉树人工林的轮伐周期,减...     

Characterization of the Hydrolytic Activity of Avocado Cellulase

The cellulase produced by ripening avocado fruits (Persea americanaMill cv. Fuerte) was isolated and purified using chromatofocusing(pH 7–4) and gel filtration on a Bio- Gel P-100 column.Characteristics of the cellulase were assessed by using, assubstrates, a range of polysaccharides containing various sugarresidues and varying types of linkages between the residues.Only those substrates containing (14)-ß-glucosyl linkageswere hydrolyzed by the purified enzyme. Two polysaccharidesthat were extensively hydrolyzed by the cellulase were carboxymethylcelluloseand (13),(14)-ß-D-glucans such as from Avena endospermcell walls. Characterization of the activity in the degradationof the mixed linked glucan of Avena and cellodextrins indicatedthat the enzyme has a limit recognition-hydrolytic site of four(l4)-ß linked glucose residues. It was also foundthat the enzyme could cleave only (14)-ß-linkagesthat were adjacent to other (l4)-ß-D-glucosyl linkages.Activity of the cellulase against isolated avocado fruit cellwalls indicated that the purified enzyme was incapable of appreciablysolubilizing the cellulosic components of these walls. ¹Supported in part by National Science Foundation Research GrantPCM 7818588. ²USDA-ARS, Dairy Forage Research Center, University of Wisconsin,Madison, WI 53706. ³Department of Vegetable Crops, University of California, Davis,CA 95616. (Received September 14, 1985; Accepted February 12, 1986)     

Effect of brown algae metabolites on the synthesis of O-glycosyl hydrolases by bacteria degrading the thallus of <Emphasis Type="Italic">Fucus evanescens</Emphasis>

The effect of fucoidan, extractive substances from Fucus evanescens and the Laminaria cichorioides protein (an inhibitor of endo-1→3-β-D-glucanase) on the degradation of F. evanescens thallus and on the growth of bacteria involved in this process was studied. The complex of O-glycosyl hydrolases and the level of their enzymatic activity in bacteria cultivated under various conditions depended significantly on the composition of the growth medium. The highest taxonomic diversity was observed for bacteria isolated from the thallus degraded in the control medium (sea water). These bacteria were characterized by very low levels of activity of the enzymes degrading polysaccharides (fucoidan, laminaran, and pustulan). In the presence of 1→3-β-D-glucanase inhibitors, the taxonomic diversity of the microorganisms degrading F. evanescens thallus was decreased, and the activity of O-glycosyl hydrolases (particularly, of fucoidan hydrolases) was increased.     

The seasonal abundance and vertical distribution of the <3-μm phytoplakton in the north basin of Lake Biwa

The seasonal changes in the size-fractionated chlorophylla concentrations (<3 μm, 3 to 25 μm, and >25 μm) were investigated at a pelagic site of the north basin of Lake Biwa during June to December 1985. Autofluorescing plankton cells in the <3-μm fractions were also examined using the fluorescein isothiocyanate staining epifluorescence microscopic technique. The <3-μm phytoplankton (usually dominated by chroococcoid cyanobacteria except for a few cases dominated by small eukaryotes) showed a clearly different pattern of seasonal change compared with the larger fractions. That is, from August to early September, chlorophylla of the larger fractions declined considerably, while the <3-μm chlorophylla did not decrease significantly. Moreover, cyanobacterial cell density in the <3-μm fraction showed a maximum value (2–3.5×10⁵ cells·ml⁻¹) during this period. The relative contribution of the <3-μm chlorophylla to the total chlorophylla increased from <5% to 45% during the course of this change. No clear vertical trend in the distribution and composition of the <3-μm phytoplankton was found, except that relatively large cyanobacteria (>4 μm³) appeared at a depth of 15m but not at 0,5 and 10 m from late July to August. These large cells were also found in November and December. The drastic seasonal change of phytoplankton size structure occurring in this basin was discussed in relation to grazing, nutrient depletion and sinking. Contribution from Otsu Hydrobiological Station, Kyoto Univeristy (No. 308, foreign language series).     

Effects of side chain modification on the activity of propyl cyclohexaneacetate as an attractant to the German cockroach

Propyl cyclohexaneacetate, a synthetic attractant to the German cockroach, Blattella germanica, appears to consist of a head (cyclohexane ring) and a tail (ester linkage). The tail was modified as regards a number of structural parameters, and the change in activity was interpreted in terms of the corresponding receptor site.Irrespective of the position and direction of the ester linkage, six atoms were optimum for the side chain. The activity increased when the terminal methyl group was replaced with chlorine, and decreased when changed into methylene. The methyl branch in the alcohol unit depressed the activity. The order of attraction among the esters with six atom side chain was as follows: propyl cyclohexaneacetate > cyclohexylmethyl butanoate ≧ ethyl 3-cyclohexylpropanoate ≧ cyclohexyl pentanoate > butyl cyclohexanecarboxylate = 2-cyclohexylethyl propanoate.Ethers, ketones and hydrocarbons which were derived from the esters with six atom side chains by replacing either or both of the carbonyl groups and ether oxygen with methylene(s) were inferior to the parent esters. Their relative activities were in the following order: pentyl cyclohexyl ether > propyl 2-cyclohexylethyl ether > butyl cyclohexyl-methyl ether = pentyl cyclohexyl ketone = butyl cyclohexylmethyl ketone = 6-cyclohexylhexane. The SAR in respect of the ester group resembled that in the muscarinic activity of acetylcholine.     

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Δ⁵ to Δ⁷ or to Δ^5,7. Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24α-ethyl = trans-Δ²²-24α-ethyl > trans-Δ²²-24β-ethyl = 24α-E-ethylidene = 24α-methyl > 24β-methyl = trans-Δ²²-24β-methyl = 26-methyl = saturated C₇ side chain and C-20 R (17-αH, 20-αH, right-handed conformer) = cis-Δ²²-C₇ side chain and C-20 R > saturated C₇ side chain and C-20 S (17-αH, 20-βH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20α-hydroxycholesterol = 24ξ-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20α-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.     

Polymorphism analysis of prion protein gene in 11 Pakistani goat breeds

The association between caprine PrP gene polymorphisms and its susceptibility to scrapie has been investigated in current years. As the ORF of the PrP gene is extremely erratic in different breeds of goats, we studied the PrP gene polymorphisms in 80 goats which belong to 11 Pakistani indigenous goat breeds from all provinces of Pakistan. A total of 6 distinct polymorphic sites (one novel) with amino acid substitutions were identified in the PrP gene which includes 126 (A -> G), 304 (G -> T), 379 (A -> G), 414 (C -> T), 428 (A -> G) and 718 (C -> T). The locus c.428 was found highly polymorphic in all breeds as compare to other loci. On the basis of these PrP variants NJ phylogenetic tree was constructed through MEGA6.1 which showed that all goat breeds along with domestic sheep and Mauflon sheep appeared as in one clade and sharing its most recent common ancestors (MRCA) with deer species while Protein analysis has shown that these polymorphisms can lead to varied primary, secondary and tertiary structure of protein. Based on these polymorphic variants, genetic distance, multidimensional scaling plot and principal component analyses revealed the clear picture regarding greater number of substitutions in cattle PrP regions as compared to the small ruminant species. In particular these findings may pinpoint the fundamental control over the scrapie in Capra hircus on genetic basis.     

Theoretical studies on identity SN2 reactions of lithium halide and methyl halide: A microhydration model

Reactions of lithium halide (LiX, X = F, Cl, Br and I) and methyl halide (CH₃X, X = F, Cl, Br and I) have been investigated at the B3LYP/6-31G(d) level of theory using the microhydration model. Beginning with hydrated lithium ion, four or two water molecules have been conveniently introduced to these aqueous-phase halogen-exchange S_N2 reactions. These water molecules coordinated with the center metal lithium ion, and also interacted with entering and leaving halogen anion via hydrogen bond in complexes and transition state, which to some extent compensated hydration of halogen anion. At 298 K the reaction profiles all involve central barriers ΔE _cent which are found to decrease in the order F > Cl > Br > I. The same trend is also found for the overall barriers (ΔE _ovr ) of the title reaction. In the S_N2 reaction of sodium iodide and methyl iodide, the activation energy agrees well with the aqueous conductometric investigation.     

Physico-chemical Property of Rare Earths—Effects on the Energy Regulation of Photosystem II in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Photosystem II (PSII) from Arabidopsis thaliana treated by lanthanum (La³⁺), cerium (Ce³⁺), and neodymium (Nd³⁺) were isolated to investigate the effects of 4f electron characteristics and alternation valence of rare earth elements (REEs) on PSII function regulation comparatively. Results showed that REE treatment could induce the generous expression of LhcII b in A. thaliana and increase the content of light-harvesting complex II and its trimer on the thylakoid membrane significantly. Meanwhile, the light absorption in the red and blue region and fluorescence quantum yield near 683 nm were obviously increased; oxygen evolution rate was greatly improved too, suggesting that REEs could enhance the efficiency of light absorption, regulate excitation energy distribution from photosystem I (PSI) to PSII, and thus increase the activity of photochemical reaction and oxygen evolution accordingly. The efficiency order of the four treatments was Ce³⁺ > Nd³⁺ > La³⁺ > control.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

Antioxidant activity of d-α-, dl-β-, d-γ- and d-δ-tocopherol was investigated with fatty acid methylester of milk fat from which unsaponifiable matter had been removed. Autoxidation was carried out at 50°C and its degree was indicated by peroxide value, α- or β-Tocopherol was more effective at lower concentrations (0.003 and 0.01%) than at higher concentrations (0.05, 0.1 and 0.5%). The antioxidant activity of γ- and δ-tocopherol was increased with the increase of tocopherol concentration within the range of 0.001 to 0.5%. The order of antioxidant activity of these tocopherols, which was compared in terms of the time to reach 30 meq of peroxide value, varied with the concentration; γ > β > δ > α at 0.001%, α > γ > β > δ at 0.003%, γ > δ > β > α at 0.01%, and δ > γ > β > α at the concentrations more than 0.05%. α-Tocopherol at the concentration of 0.003%, which corresponded to the concentration in original milk fat, was more effective than other tocopherols at the same concentration and α-tocopherol at other concentrations. Synergism due to the combination of β-, γ-, or δ-tocopherol with 0.003% of α-tocopherol was not observed.     

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure–activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3–5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 μM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10–100 μM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15–55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.     

Increase in XET activity in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) cells habituated to dichlobenil

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 μM. The 2-μM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.