Synthesis, crystal structure and luminescent behaviour of coordination complexes of copper with bi- and tridentate amines and phosphonic acids

The synthesis and crystal structure of four new copper(I) and copper(II) supramolecular amine, and amine phosphonate, complexes is reported. Reaction of copper(I) with 2-,9-dimethyl-1-10-phenanthroline (dmp) produced a stable 4-coordinate Cu(I) species, [Cu^(I)(dmp)₂]Cl · MeOH · 5H₂O (2), i.e., the increased steric hindrance in the ‘bite’ area of dmp did not prevent interaction with the metal and provided protection against oxidation which was not possible for the phen analogue [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36]. Subsequent addition of phenylphosphonic acid to (2) produced two structures from alternative synthetic routes. An ‘in situ’ process yielded red block Cu(I) crystals, [Cu^(I)(dmp)₂] · [C₆H₅PO₃H₂ · C₆H₅PO₃H] (4), whilst recrystallisation of (2) prior to addition of the acid (‘stepwise’ process) produced a green, needle-like Cu(II) complex, [Cu^(II)(dmp) · (H₂O)₂ · C₆H₅PO₂(OH)] [C₆H₅PO₂(OH)] (3). However, addition of excess dmp during the ‘stepwise’ process forced the equilibrium towards product (4) and resulted in an optimum yield (99%). The structure of (4) was similar to the phen analogue, [Cu^(II)Cl(phen)₂] · [C₆H₅PO₂(OH) · C₆H₅PO(OH)₂] (1) [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36], but the presence of dmp exerted some influence on global packing, whilst (3) exists as a polymeric layered material. In contrast, reaction of copper(I) with di-2-pyridyl ketone (dpk), followed by phenylphosphonic acid produced purple/blue Cu(II) species, [Cu^(II)(dpk · H₂O)₂] Cl₂ · 4H₂O (5), and [Cu^(II)(dpk · H₂O)₂] · [C₆H₅PO₂(OH)₂ · C₆H₅PO(OH)₂] (6), respectively, i.e., in both cases oxidation of copper occurred. Solid-state luminescence was observed in (2) and (4). The latter showing a 5-fold enhancement in intensity.     

Antifeedant and larvicidal activities of Rhein isolated from the flowers of Cassia fistula L.

Antifeedant and larvicidal activities of rhein (1,8-dihydroxyanthraquinone-3-carboxylic acid) isolated from the ethyl acetate extract of Cassia fistula flower were studied against lepidopteron pests Spodoptera litura and Helicoverpa armigera. Significant antifeedant activity was observed against H. armigera (76.13%) at 1000 ppm concentration. Rhein exhibited larvicidal activity against H. armigera (67.5), S. litura (36.25%) and the LC₅₀ values was 606.50 ppm for H. armigera and 1192.55 ppm for S.litura. The survived larvae produced malformed adults.     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide,but not to isonicotinic acid: Differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (M_r, 137) to isonicotinamide (M_r, 122), not to isonicotinic acid (M_r, 123), in the presence or absence of H₂O₂. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.     

The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli

In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH₃Hg(I) and Hg(II), and took up significantly more CH₃Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH₃Hg(I) and Hg(II) across the bacterial membrane.     

Effect of hypochlorous acid solution on the eradication and prevention of Pseudomonas aeruginosa infection,serum biochemical variables,and cecum microbiota in rats

In this study, hypochlorous acid solution, a weak acid, provided as drinking water to rats, was evaluated for its ability to eradicate and prevent Pseudomonas aeruginosa infection, while monitoring its simultaneous effect on serum biochemical variables and microbiota in the rat cecum. The results suggest that the solution could not eliminate the bacteria in the experimentally infected rats; however, the administration of a 10-parts-per-million (ppm) hypochlorous acid solution as drinking water was effective in inhibiting horizontal spread of P. aeruginosa infection among cage mates. Additionally, exposure to hypochlorous solution did not have any effect on serum biochemical variables of the rat including levels of total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total bilirubin, lipase, amylase, urea nitrogen, total protein, calcium (Ca), phosphorus (P), sodium (Na), chlorine (Cl), except for potassium (K) levels. The most frequently isolated bacteria in the rat cecum included species belonging to Bacteroidales, Lactobacillus, Clostridiales, Erysipelotrichaceae, Akkermansia, Coriobacteriales, and Firmicutes. The ratio of the terminal restriction fragment length polymorphism (T-RFLP) peaks did not differ across rats administered with 5 and 10 ppm weak acid solution as compared to the control group for any of the bacteria, except for Erysipelotrichaceae and Firmicutes, where the ratio of T-RFLP peaks was higher in the 5 ppm group for Erysipelotrichaceae and in the 10 ppm group for Firmicutes than that in the control group (P<0.01). The results suggest that the weak acid hypochlorous solution could not eradicate P. aeruginosa completely from rats. The solution was effective in preventing infection without affecting serum biochemical variables; however, some of bacterial microbiota may have changed due to administration of the solution.     

Effects of the Integration of Sunn Hemp and Soil Solarization on Plant-Parasitic and Free-Living Nematodes

Sunn hemp (SH), Crotolaria juncea, is known to suppress Rotylenchulus reniformis and weeds while enhancing free-living nematodes involved in nutrient cycling. Field trials were conducted in 2009 (Trial I) and 2010 (Trial II) to examine if SH cover cropping could suppress R. reniformis and weeds while enhancing free-living nematodes if integrated with soil solarization (SOL). Cover cropping of SH, soil solarization, and SH followed by SOL (SHSOL) were compared to weedy fallow control (C). Rotylenchulus reniformis population was suppressed by SHSOL at the end of cover cropping or solarization period (Pi) in Trial I, but not in Trial II. However, SOL and SHSOL did not suppress R. reniformis compared to SH in either trial. SH enhanced abundance of bacterivores and suppressed the % herbivores only at Pi in Trial II. At termination of the experiment, SH resulted in a higher enrichment index indicating greater soil nutrient availability, and a higher structure index indicating a less disturbed nematode community compared to C. SOL suppressed bacterivores and fungivores only in Trial II but not in Trial I. On the other hand, SHSOL enhanced bacterivores and fungivores only at Pi in Trial I. Weeds were suppressed by SH, SOL and SHSOL throughout the experiment. SHSOL suppressed R. reniformis and enhanced free-living nematodes better than SOL, and suppressed weeds better than SH.     

Enhanced biological removal of NOχ from flue gas in a biofilter by Fe(II)Cit/Fe(II)EDTA absorption

A mixed absorbent had been proposed to enhance the chemical absorption-biological reduction process for NO_x removal from flue gas. The mole ratio of the absorbent of Fe(II)Cit to Fe(II)EDTA was selected to be 3. After the biofilm was formed adequately, some influential factors, such as the concentration of NO, O₂, SO₂ and EBRT were investigated. During the long-term running, the system could keep on a steady NO removal efficiency (up to 90%) and had a flexibility in the sudden changes of operating conditions when the simulated flue gas contained 100-500 ppm NO, 100-800 ppm SO₂, 1-5% (v/v) O₂, and 15% (v/v) CO₂. However, high NO concentration (>800 ppm) and relative short EBRT (<100 s) had significant negative effect on NO removal. The results indicate that the new system by using mixed-absorbent can reduce operating costs in comparison with the single Fe(II)EDTA system and possesses great potential for scale-up to industrial applications.     

Sequence Diversity in MIC6 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.     

Purification and inflammatory edema induced by two PLA2 (Anch TX-I and Anch TX-II) from sea anemone Anthothoe chilensis (Actiniaria: Sagartiidae)

The Anch TX-I and II PLA₂ were purified from Anthothoe chilensis (Lesson, 1830) from the extract of the anemone after only two chromatographic step using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on μ-Bondapak C18 column. Both PLA₂ showed a molecular mass of ~ 14 kDa determined by MALDI-TOF mass spectrometry and showed a high catalytic activity (data not showed). Although homologous with mammalian or snake venom group I PLA₂s, Anch TX-I and II is sufficiently structurally different for the question of its placement into the existing PLA₂ classification scheme to arise. In addition, Anch TX-I and II, despite possessing many common structural features, also differ in some important structural properties. The amino acid sequence of both PLA₂ (Anch TX-I and III) showed high amino acid sequence identity with PLA₂Rhopilema nomadica and Bunodosoma caissarum Cnidaria and PLA₂ of group III protein isolated from the Mexican lizard Heloderma horridum horridum and Heloderma suspectum. In addition, Anch TX-I and Anch TX-II showed high amino acid sequence identity with PLA₂ from group III also showed significant overall homology to bee Apis dorsata, Bombus terrestris and Bombus pennsylvanicus and PLA₂. We also investigated the in vivo edematogenic activity of Anch TX-I and Anch TX-II in a model of paw and skin edema in rats and observed that both are able to induce dose-dependent edema.     

Synthesis and NMR properties of novel 5,6-dihydroborauracil derivatives

Novel boron compounds - 5,6-saturated borauracil derivatives (4-bromo-5,6-dihydroborauracil, 4-hydroxy-5,6-dihydroborauracil and 4-methoxy-5,6-dihydroborauracil) are presented along with other boron compounds obtained from N-vinylurea: N-substituted β-boronic amino acid - 2-{[(dihydroxyborano-amino)(dihydroxyboranooxy)methyl]-amino}ethylboronic acid and substituted methoxy-borane O-[(1-amino-1-N-vinylamino)methyl]dihydroxyboronate.     

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10⁶ spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Dormancy and germination in Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee seeds: Effects of short-time moist chilling and plant growth regulators

We investigated the germination requirements of the species Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee (Lamiaceae). We studied the effects of scarification, short-time moist chilling (+4 °C) for 15 and 30 days, and various doses of gibberellic acid (GA₃; 0, 100, 150 and 250 ppm), Kinetin (KIN; 50 ppm) and a combination of 250 ppm GA₃ and 50 ppm KIN. The hormone and moist chilling treatments were carried out under both continuous darkness (20 °C) and photoperiodic (20/10 °C; 12/12 h, respectively) conditions. Seeds failed to germinate in response to short-time moist chilling treatments with distilled water under both continuous darkness and photoperiodic conditions. Seeds were found to have dormancy. Treatments with GA₃ or a combination of GA₃ and KIN were successful at breaking seed dormancy. A maximum of 37% of the seeds germinated after GA₃ application in all series. When only KIN was applied at a 50 ppm concentration, germination (12%) was found only with moist chilling for 30 days under continuous darkness. The highest germination rates were found in seeds treated with combination of 250 ppm GA₃ and 50 ppm KIN. In the combination treatments, while the moist chilling treatments for 15 days resulted in 68 and 73% germination, respectively, these rates were up to 95% in the moist chilling treatments for 30 days under continuous darkness and photoperiodic conditions. Mean germination time (MGT) in GA₃ and KIN combinations was lower than in other treatments. Scarification with 80% sulphuric acid did not promote germination. The characteristics of physiological dormancy of S. germanica ssp. bithynica seeds are consistent with conditions of existence in the in alpine habitat of this species.     

N-(1-Carboxyethyl)alanine (alanopine), a new non-sugar component of lipopolysaccharides of Providencia and Proteus

O-Polysaccharides were released by mild acid degradation of lipopolysaccharides of Providencia alcalifaciens O35 and Proteus vulgaris O76 and were studied by 1D and 2D ¹H and ¹³C NMR spectroscopies, including HMBC and NOESY (ROESY) experiments. Both polysaccharides were found to contain N-(1-carboxyethyl)alanine (alanopine) that is N-linked to 4-amino-4,6-dideoxyglucose. Analysis of published data [Vinogradov, E.; Perry, M. B. Eur. J. Biochem.2000, 267, 2439-2446] shows that alanopine is present also on the same sugar in the lipopolysaccharide core of Proteus mirabilis O6 and O57.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Phylogenetic differences in cardiac activity, metal accumulation and mortality of limpets exposed to copper: A prosobranch-pulmonate comparison

Taxonomic relationships for pollutant tolerance in marine invertebrates are surprisingly poorly known, despite being potentially useful for pollution biomonitoring. A popular view is that cellular and molecular adaptations for natural stress may be important in tolerating pollution. We compared the physiological and mortality responses to copper (Cu²⁺) of limpets from two different lineages: the Prosobranchia (Patellogastropoda: Helcion concolor and Cellana capensis) and the Pulmonata (Siphonaria serrata and Siphonaria capensis). Copper tolerance was apparently more closely related to phylogenetically-based physiology, than to tolerance of desiccation and or heat. The Siphonaria limpets were nearly an order of magnitude more tolerant of copper than the patellogastropod limpets, even though S. serrata has the lowest intertidal distribution. Initial copper exposure (0.25 ppm Cu²⁺ for 2 h) induced heart rate depression in Siphonaria (to around 50% of the baseline rate), while their tissue copper concentrations remained at the relatively high control levels. Copper exposure (0.25 ppm Cu²⁺ for 2 h) had no effect on heart rate of the patellogastropod limpets, but led to a significant increase in tissue copper. These results suggest that enhanced copper tolerance by Siphonaria relates to cardiac depression and a concomitant metabolic depression. Such physiological attributes are implicated in prolonged behavioural isolation, involving pneumostome closure and shell clamping, which is likely to reduce the uptake of copper. Furthermore, better regulation of internal copper levels by Siphonaria, is suggested by their exclusive possession of blood haemocyanin. Dependence on relatively high aerobic metabolism by the patellogastropod limpets, would limit their capacity for isolation and pollutant avoidance.     

Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide δ-l-α-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme. δ-l-α-Aminoadipoyl-l-cysteinyl-d-2-amino-3,3-dideuteriobutyrate (lld-ACd₂Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer δ-l-α-aminoadipoyl-l-cysteinyl-l-2-amino-3,3-dideuteriobutyrate (lll-ACd₂Ab) has the opposite configuration at its third amino acid. lll-ACV has previously been shown to inhibit IPNS turnover of its substrate lld-ACV; the all-protiated tripeptide δ-l-α-aminoadipoyl-l-cysteinyl-d-2-aminobutyrate (lld-ACAb) is a substrate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the crystal structures of the IPNS:Fe(II):lld-ACd₂Ab and IPNS:Fe(II):lll-ACd₂Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.     

Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in controlling Anopheles arabiensis and Culex quinquefasciatus mosquitoes

The present study aimed to investigate, the larvicidal, adult emergence inhibition and oviposition deterrent activity of aqueous leaves extract of Calotropis procera against Anopheles arabiensis and Culex quinquefasciatus as natural mosquito larvicide. The larvicidal activity was monitored against 2nd, 3rd and 4th instar larvae of each mosquito species 24 h post-treatment. Adult emergence inhibition activity was tested by exposing 3rd instar larvae of each mosquito species to different concentrations of extracts (200, 400, 600, 800 and 1000 ppm for An. arabiensis and 100, 200, 300, 400, 500 and 600 ppm for Cx. quinquefasciatus). Probit analysis was used to analyze data from bioassay experiments. The oviposition deterrent activity was tested by using three different concentrations of extracts (1000, 500 and 200 for An. arabiensis, and 1000, 500 and 100 for Cx. quinquefasciatus) that caused high, moderate and low larval mortality in the larvicidal experiment against 3rd instar larvae. It was found that, LC50–LC90 values calculated were 273.53–783.43, 366.44–1018.59 and 454.99–1224.62 ppm for 2nd, 3rd and 4th larval instars, respectively, of An. arabiensis and 187.93–433.51, 218.27–538.27 and 264.85–769.13 ppm for 2nd, 3rd and 4th larval instars, respectively, of Cx. quinquefasciatus. Fifty percent of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively. The pupal stage was not affected till a concentration of 5000 ppm. The extract showed oviposition deterrence and effective repellence against both mosquito species at different concentrations, with the observation on that maximal eggs were laid in low concentration of extract. These results suggest that the leaves extract of C. procera possess remarkable larvicidal, adult emergence inhibitor, repellent and oviposition deterrent effect against both An. arabiensis and Cx. quinquefasciatus, and might be used as natural biocides for mosquito control.