Effects of 5-aminolevulinic acid on the inflammatory responses and antioxidative capacity in broiler chickens challenged with lipopolysaccharide

5-Aminolevulinic acid (5-ALA) is an intermediate in haem biosynthesis and has anti-apoptotic, anti-inflammatory, antioxidant, and other pharmacological effects. This study aimed to investigate the effect of dietary supplementation with 5-ALA on growth performance, antioxidant capacity, and inflammatory response of the lipopolysaccharide (LPS)-challenged broiler chickens. The experiment was designed as a 2 × 2 factorial arrangement with dietary 5-ALA (0 or 60 mg/kg) and LPS (injection of saline or 0.5 mg/kg BW) levels as treatments. A total of 240 one-day-old Arbor Acres broilers were distributed into four treatments consisting of six replicates of 10 birds. All the experimental broilers were intraperitoneally injected with LPS or sterile saline at 16, 18, and 20 days of age. Our results showed that dietary 5-ALA supplementation reduced (P < 0.05) the feed to gain before broilers were stimulated with LPS (days 1–15). LPS challenge decreased (P < 0.05) the catalase (CAT), total superoxide dismutase activities and increased the content of malondialdehyde (MDA) in the serum of broiler chickens. However, 5-ALA supplementation had a tendency to increase (P = 0.08) the activity of CAT and decreased (P < 0.05) the content of MDA. LPS challenge showed higher (P < 0.05) interleukin (IL)-1β, IL-6, and IL-10 concentrations in the serum, whereas dietary 5-ALA supplementation decreased (P < 0.05) the levels of IL-1β and IL-6. Additionally, dietary 5-ALA supplementation significantly attenuated (P < 0.05) the upregulation of mRNA expression levels of hepatic toll-like receptor 4 (TLR4), IL-1β, and IL-2 induced by LPS challenge. Moreover, dietary 5-ALA supplementation also enhanced the mRNA expression of 5-aminolevulinate dehydratase, ferrochelatase, and haem oxygenase-1 (HO-1) as compared to the unsupplemented groups. In conclusion, our results suggested that supplementation of 60 mg/kg 5-ALA exhibited LPS-induced anti-inflammatory and antioxidant properties by enhancing the HO-1 expression and inhibiting the TLR4/NF-κB signalling pathway.     

Dinuclear macrocyclic polyamine zinc(II) complexes linked with flexible spacers: synthesis, characterization, and DNA cleavage

Dinuclear macrocyclic polyamine zinc(II) complexes, which have two cyclen groups linked by flexible spacers, have been synthesized as DNA cleavage agents. The structures of these new dinuclear complexes are consistent with the data obtained from elemental analysis, MS and ¹H NMR spectroscopy. The catalytic activity of these dinuclear complexes on DNA cleavage was studied. The results showed that the dinuclear zinc(II) complexes can catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II).     

Demecolcine-induced enucleation of sheep meiotically maturing oocytes

The objective of this study was to investigate the possible effect of demecolcine, a microtubule-disrupting reagent, on induced enucleation (IE) of sheep meiotically maturing oocytes. Immunofluorescent staining with anti-tubulin antibodies was used to examine the spindle status of the oocytes. When the oocytes with intact germinal vesicles (GV) were cultured in the medium containing various concentrations of demecolcine (0.01 to 0.4 microg.mL-1) for 20 to 22 h, the spindle microtubule organization and first polar body (PB1) extrusion were inhibited by demecolcine in a dose-dependent manner. The highest IE rate (58.1%) was from the treatment with 0.04 microg.mL-1 demecolcine. Demecolcine treatment applied after germinal vesicle breakdown (GVBD) or at metaphase (M) yielded a PB1 extrusion rate and IE efficiency similar to the treatment applied at the onset of maturation. Analysis by immunofluorescence showed that both nonspindle microtubules and spindle microtubules were significantly disorganized by demecolcine. Combination treatment with demecolcine and cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) led to single pronuclear formation rather than PB1 extrusion. When demecolcine-treated oocytes were transferred into demecolcine-free medium, the ability to extrude PB1 was quickly restored and a 72.1% IE rate was obtained following such treatment. These results demonstrate that demecolcine can be used as a potential reagent for induced enucleation of sheep meiotically maturing oocytes and may greatly facilitate research in nuclear transfer.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system.

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.     

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET_A and ET_B) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET_A and ET_B receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC α, β2, δ, ε and ζ were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.     

NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the rat

Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.     

Isolation,Diversity, and Antimicrobial Activity of Rare Actinobacteria from Medicinal Plants of Tropical Rain Forests in Xishuangbanna,China

Endophytic actinobacteria are relatively unexplored as potential sources of novel species and novel natural products for medical and commercial exploitation. Xishuangbanna is recognized throughout the world for its diverse flora, especially the rain forest plants, many of which have indigenous pharmaceutical histories. However, little is known about the endophytic actinobacteria of this tropical area. In this work, we studied the diversity of actinobacteria isolated from medicinal plants collected from tropical rain forests in Xishuangbanna. By the use of different selective isolation media and methods, a total of 2,174 actinobacteria were isolated. Forty-six isolates were selected on the basis of their morphologies on different media and were further characterized by 16S rRNA gene sequencing. The results showed an unexpected level of diversity, with 32 different genera. To our knowledge, this is the first report describing the isolation of Saccharopolyspora, Dietzia, Blastococcus, Dactylosporangium, Promicromonospora, Oerskovia, Actinocorallia, and Jiangella species from endophytic environments. At least 19 isolates are considered novel taxa by our current research. In addition, all 46 isolates were tested for antimicrobial activity and were screened for the presence of genes encoding polyketide synthetases and nonribosomal peptide synthetases. The results confirm that the medicinal plants of Xishuangbanna represent an extremely rich reservoir for the isolation of a significant diversity of actinobacteria, including novel species, that are potential sources for the discovery of biologically active compounds.The class Actinobacteria accounts for a high proportion of soil microbial biomass and contains the most economically significant prokaryotes, producing more than half of the bioactive compounds in a literature survey (46), including antibiotics (6), immunosuppressive agents (55), antitumor agents (18), and enzymes (64). Actinobacteria belonging to the genus Streptomyces, in particular, are excellent producers. The emergence of drug resistance in many bacterial pathogens and the current increase in the number of fungal infections has caused a resurgence of interest in finding new reserves of biologically active compounds (63). As the search for novel natural products continues, it becomes apparent that the rate of discovery of new compounds from soil streptomycetes has decreased, whereas the rate of reisolation of known compounds has increased (28). Recently, evidence has accumulated that rare actinomycete species, which are often very difficult to isolate and cultivate, might represent a unique source of novel biologically active compounds (4). On the other hand, new microbial habitats need to be examined in the search for novel bioactive compounds. One biologically important but relatively overlooked niche is the inner tissues of higher plants. Early studies have demonstrated that some actinobacteria can form intimate associations with plants and colonize their inner tissues. Frankia species and Streptomyces scabies can penetrate their hosts and establish either pathogenic or endophytic associations (5, 24). The actinomycetes that reside in the tissues of living plants and do not visibly harm the plants are known as endophytic actinobacteria (37). These actinobacteria are relatively unstudied and are potential sources of novel natural products for exploitation in medicine, agriculture, and industry (73).Endophytic actinobacteria have attracted attention in recent years, with increasing reports of isolates from a range of plant types, including crop plants (cereals, such as wheat and rice, as well as potatoes, carrots, tomatoes, and citrus) (2, 16, 62, 71, 74, 80) and medicinal plants (75, 88). The culturable endophytic actinobacteria from these plants were found to fall within a narrow species distribution: Streptomyces spp. were the predominant species, and Microbispora, Micromonospora, Nocardioides, Nocardia, and Streptosporangium were the common genera. Endophytic actinobacteria have been demonstrated to improve and promote the growth of host plants as well as to reduce disease symptoms caused by plant pathogens through various mechanisms, including the production of secondary metabolites, which are used in direct antagonism against pests and diseases (9, 10, 12), changes in host physiology (42), and the induction of systemic acquired resistance in plants (15). Another significant function found for these actinobacteria was antibiotic activity, suggesting that endophytic actinobacteria can be an interesting source for bioprospecting. New antibiotics from endophytic Streptomyces spp.—alnumycin, munumbicins A to D, and coronamycins—have been reported (7, 11). Recently, two novel antitumor anthraquinones, lupinacidins A and B, were isolated from a new endophytic Micromonospora sp. (43). Moreover, new species of endophytic actinobacteria have been increasingly reported (25, 35). Thus, endophytic actinobacteria are expected to be potential sources of new species and new bioactive agents.Of the myriad ecosystems on earth, those with the greatest general biodiversity seem also to have the greatest number and the greatest diversity of endophytes (73). Tropical and temperate rain forests are the most biologically diverse terrestrial ecosystems on earth and thus the greatest possible resource for the acquisition of novel microorganisms and their products (73). One area of enormous plant biodiversity is Xishuangbanna, located in the People''s Republic of China at the border with Myanmar. This area lies at the ecotone between the Asian tropics and subtropics and is dominated by tropical seasonal rain forests (87). Xishuangbanna contains more than 5,000 species of vascular plants, comprising 16% of China''s total plant diversity, and more than 3,000 are endemic species (53, 60), many of which have ethnobotanical histories. Until the present, little research was carried out to isolate endophytic actinobacteria and their secondary metabolites from Xishuangbanna (36, 86). In our long-term study of endophytic actinobacterial diversity and bioactive metabolites from tropical rain forest medicinal plants in Xishuangbanna, many bioactive endophytic Streptomyces spp. have been isolated (49). However, the work to date is insufficient to provide a general understanding of the diversity, distribution, and ecology of tropical rain forest endophytic actinobacteria and to facilitate further exploitation of the diverse functions of this novel microbial source.In the present study, the diversity of rare endophytic actinobacteria associated with medicinal plants from the tropical rain forest in Xishuangbanna was investigated by combining special culturing techniques. The selected isolates were also identified by 16S rRNA gene analysis. The overall aims of this study were (i) to analyze the actinobacterial community and reveal whether the rain forest investigated in Xishuangbanna represents a valuable source for abundant endophytic actinobacteria and new species, (ii) to evaluate the antimicrobial activities of these actinobacteria and the biosynthetic potential of related secondary metabolites, and (iii) to study the relationships between the taxa of these endophytic actinobacteria and the isolation methods applied.     

Biogenic Formation of As-S Nanotubes by Diverse Shewanella Strains

Shewanella sp. strain HN-41 was previously shown to produce novel, photoactive, As-S nanotubes via the reduction of As(V) and S₂O₃²⁻ under anaerobic conditions. To determine if this ability was unique to this bacterium, 10 different Shewanella strains, including Shewanella sp. strain HN-41, Shewanella sp. strain PV-4, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were examined for production of As-S nanotubes under standardized conditions. Of the 10 strains examined, three formed As-S nanotubes like those of strain HN-41. While Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly formed As-S precipitates in 7 days, strains S. alga BrY and S. oneidensis MR-1 reduced As(V) at a much lower rate and formed yellow As-S after 30 days. Electron microscopy, energy-dispersive X-ray spectroscopy, and extended X-ray absorption fine-structure spectroscopy analyses showed that the morphological and chemical properties of As-S formed by strains S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 were similar to those previously determined for Shewanella sp. strain HN-41 As-S nanotubes. These studies indicated that the formation of As-S nanotubes is widespread among Shewanella strains and is closely related to bacterial growth and the reduction rate of As(V) and thiosulfate.A number of bacterial strains have been shown to contribute to the formation of diverse arsenic minerals (4). If sulfide is present as a ligand for immobilization of arsenic, As-S precipitates often form. Desulfosporosinus auripigmentum, which can be isolated from lake sediments, reduces As(V) to As(III) and S(VI) to S(−II) during anaerobic respiration and forms a yellow arsenic sulfide precipitate (7). While Desulfovibrio strain Ben-RB also produces precipitated arsenic sulfide in culture media, As reduction was not correlated with energy conservation (6). Other taxonomically divergent microorganisms isolated from various arsenic-rich sites have also been shown to reduce As(V) to As(III) and form arsenic sulfide precipitates (1, 2).We previously reported that Shewanella sp. strain HN-41 produces an extensive extracellular network of filamentous arsenic-sulfide (As-S) nanotubes via its dissimilatory metal-reducing activity (4). The As-S nanotubes, which formed via the reduction of As(V) and S₂O₃²⁻, were initially amorphous As₂S₃ but evolved with increasing incubation time toward polycrystalline phases of the chalcogenide minerals realgar (AsS) and duranusite (As₄S). Because the Shewanella As-S nanotubes behaved both as metals and as semiconductors, in terms of their electrical and photoconductive properties, respectively, it was postulated that they may provide useful materials for novel nano- and optoelectronic devices (4).While several bacterial species have been shown to produce amorphous and particulate As-S precipitates (1, 2, 4, 7), the formation of the As-S nanotubes by other bacteria has not yet been described, suggesting that this may be a unique property of Shewanella strains. To test this hypothesis, 10 different Shewanella strains, including Shewanella sp. strains PV-4 and HN-41, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were inoculated into HEPES-buffered basal medium (3, 5) containing 10 mM sodium dl-lactate as the electron donor and 5 mM arsenate (Na₂HAsO₄·7H₂O) and 5 mM thiosulfate (Na₂S₂O₃·5H₂O) as the electron acceptors. All chemicals and methods for sample preparation and characterization used in this study were previously described (4).Of the 10 different Shewanella strains examined, only four strains, Shewanella sp. strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1, produced As-S yellow precipitates in culture medium following incubation in the presence of arsenate and thiosulfate. Shewanella sp. strain HN-41 and S. putrefaciens CN-32 produced yellow precipitates of As-S after 7 days of incubation, whereas S. alga BrY and S. oneidensis MR-1 produced only a small amount of visible precipitate after 30 days of incubation. The remainder of the tested Shewanella strains failed to produce yellow precipitates, regardless of incubation time.The culture medium of the strains tested was periodically sampled during the bacterial incubation period to determine the concentrations of lactate, acetate, arsenic, and sulfide in the aqueous solution. Among the 10 strains examined, Shewanella strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 metabolized lactate in growth medium containing arsenate and thiosulfate (Table ​(Table1).1). Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly consumed lactate both as an electron donor and as a carbon source (see Fig. S1 in the supplemental material). Cultures of S. alga BrY and S. oneidensis MR-1 consumed ∼1.4 mM lactate after 7 days, while Shewanella sp. strain HN-41 and S. putrefaciens CN-32 consumed 1.7 mM and 2.3 mM lactate, respectively. Although S. putrefaciens CN-32 reduced As(V) in the culture medium supplemented with 5 mM As(V) as the sole electron acceptor, Shewanella sp. strain HN-41, S. alga BrY, and S. oneidensis MR-1 did not reduce As(V) and did not oxidize lactate to acetate (data not shown). Consequently, the latter three strains could not utilize As(V) as an electron acceptor for respiratory metabolism.

TABLE 1.

Influence of thiosulfate on the consumption of lactate, reduction of As(V), and formation of As-S nanotubes by Shewanella strains in medium containing lactate and 5 mM As(V)