Studies with tryptophan metabolites in vitro. Kynurenine metabolism in kidneys of mice infested with Schistosoma mansoni

The conversion in vitro of kynurenine into kynurenic acid and anthranilic acid in both normal kidneys and those obtained from mice infested with Schistosoma mansoni was investigated. Normal mouse kidneys seem to possess an excess of functional pyridoxal phosphate over those obtained from infested mice. Kynureninase and kynurenine transaminase in the latter kidneys are more easily inhibited by deoxypyridoxal phosphate and tartar emetic, indicating low stores of active pyridoxal phosphate. The possible implication of these findings in relation to the role of the kidneys in producing abnormal patterns of tryptophan metabolism and possibly contributing to the production of bladder tumours in bilharzial patients is discussed.     

Tetramethylpyrazine Ameliorates Rotenone-Induced Parkinson’s Disease in Rats: Involvement of Its Anti-Inflammatory and Anti-Apoptotic Actions

Parkinson’s disease (PD) is a slowly progressive neurodegenerative movement disorder. Apoptosis, neuroinflammation, and oxidative stress are the current hypothesized mechanisms for PD pathogenesis. Tetramethylpyrazine (TMP), the major bioactive component of Ligusticum wallichii Franchat (ChuanXiong), Family Apiaceae, reportedly has anti-apoptotic, anti-inflammatory and antioxidant effects. This study investigated the role of ‘TMP’ in preventing rotenone-induced neurobiological and behavioral sequelae. A preliminary dose–response study was conducted where rats received TMP (10, 20, and 40 mg/kg, i.p.) concomitantly with rotenone (2 mg/kg, s.c.) for 4 weeks. Catalepsy, locomotor activity, striatal dopamine content, and tyrosine hydroxylase “TH” and α-synuclein immunoreactivity were evaluated. The selected TMP dose (20 mg/kg) was used for western blot analysis of Bax, Bcl2, and DJ-1, immunohistochemical detection of nuclear factor kappa B (NF-кB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and glial fibrillary acidic protein (GFAP) expression, in addition to biochemical analysis of caspase-3 activity, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) levels. Results showed that TMP (20 mg/kg) significantly improved midbrain and striatal TH expression and striatal dopamine content as well as the motor deficits, compared to rotenone-treated group. These results were correlated with reduction in caspase-3 activity and α-synuclein expression, along with improvement of midbrain and striatal Bax/Bcl2 ratio compared to rotenone-treated group. TMP also attenuated rotenone-induced upregulation of Nrf2/HO-1 pathway. Furthermore, TMP downregulated rotenone-induced neuroinflammation markers: NF-кB, iNOS, COX2, and GFAP expression in both the midbrain and striatum. Taken together, the current study suggests that TMP is entitled to, at least partially, preventing PD neurobiological and behavioral deficits by virtue of its anti-apoptotic, anti-inflammatory, and antioxidant actions.     

Targeting Hormone-Related Pathways to Improve Grain Yield in Rice: A Chemical Approach

Sink/source relationships, regulating the mobilization of stored carbohydrates from the vegetative tissues to the grains, are of key importance for grain filling and grain yield. We used different inhibitors of plant hormone action to assess their effects on grain yield and on the expression of hormone-associated genes. Among the tested chemicals, 2-indol-3-yl-4-oxo-4-phenylbutanoic acid (PEO-IAA; antagonist of auxin receptor), nordihydroguaiaretic acid (NDGA; abscisic acid (ABA) biosynthesis inhibitor), and 2-aminoisobutyric acid (AIB; ethylene biosynthesis inhibitor) improved grain yield in a concentration dependent manner. These effects were also dependent on the plant developmental stage. NDGA and AIB treatments induced an increase in photosynthesis in flag leaves concomitant to the increments of starch content in flag leaves and grains. NDGA inhibited the expression of ABA-responsive gene, but did not significantly decrease ABA content. Instead, NDGA significantly decreased jasmonic acid and jasmonic acid-isoleucine. Our results support the notion that the specific inhibition of jasmonic acid and ethylene biosynthesis resulted in grain yield increase in rice.     

Stress-Induced Cytokinin Synthesis Increases Drought Tolerance through the Coordinated Regulation of Carbon and Nitrogen Assimilation in Rice

The effects of water deficit on carbon and nitrogen metabolism were investigated in flag leaves of wild-type and transgenic rice (Oryza sativa japonica ‘Kitaake’) plants expressing ISOPENTENYLTRANSFERASE (IPT; encoding the enzyme that mediates the rate-limiting step in cytokinin synthesis) under the control of P_SARK, a maturation- and stress-induced promoter. While the wild-type plants displayed inhibition of photosynthesis and nitrogen assimilation during water stress, neither carbon nor nitrogen assimilation was affected by stress in the transgenic P_SARK::IPT plants. In the transgenic plants, photosynthesis was maintained at control levels during stress and the flag leaf showed increased sucrose (Suc) phosphate synthase activity and reduced Suc synthase and invertase activities, leading to increased Suc contents. The sustained carbon assimilation in the transgenic P_SARK::IPT plants was well correlated with enhanced nitrate content, higher nitrate reductase activity, and sustained ammonium contents, indicating that the stress-induced cytokinin synthesis in the transgenic plants played a role in maintaining nitrate acquisition. Protein contents decreased and free amino acids increased in wild-type plants during stress, while protein content was preserved in the transgenic plants. Our results indicate that the stress-induced cytokinin synthesis in the transgenic plants promoted sink strengthening through a cytokinin-dependent coordinated regulation of carbon and nitrogen metabolism that facilitates an enhanced tolerance of the transgenic plants to water deficit.Plant hormones control many aspects of plant growth and development and the responses of plants to abiotic and biotic stresses. Cytokinins (CKs) have been shown to regulate plant cell differentiation, leaf senescence, and other key developmental processes (Sakakibara, 2006). It has also been shown that CKs regulate assimilate partitioning (Ronzhina and Mokronosov, 1994), sink strength (Kuiper, 1993), and source/sink relationships (Roitsch, 1999). The localized expression in tobacco (Nicotiana tabacum) of a promoterless ISOPENTENYLTRANSFERASE (IPT), a gene encoding the enzyme that catalyzes the rate-limiting step in CK synthesis, enhanced the local sink strength and quickly mobilized nutrients to the tissues with elevated CK (Guivarc’h et al., 2002). Changes in sink/source relationships were also observed in CK-deficient tobacco shoots and roots (Werner et al., 2008). Elevated CK levels enhanced the survival of plants under water-stress conditions (Rivero et al., 2007). The overexpression of IPT under the control of SENESCENCE-ASSOCIATED RECEPTOR KINASE (SARK; a maturation- and stress-induced promoter) improved the drought tolerance of both eudicots (Rivero et al., 2007; Qin et al., 2011) and monocots (Peleg et al., 2011). After a water-stress episode during the reproductive stages (pre and post anthesis), transgenic P_SARK::IPT rice (Oryza sativa) plants displayed higher grain yield than the wild type (Peleg et al., 2011). The transgenic P_SARK::IPT rice exhibited a differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These results suggested that changes in hormone homeostasis induced the modification of source/sink relationships in the transgenic plants, resulting in higher grain yields under stress conditions (Peleg et al., 2011).The maintenance of carbon (C) and nitrogen (N) assimilation is of paramount importance to ensure sink strength and improve stress tolerance without yield penalties. The interactions between C and N metabolism are vital for plant growth and development, and complex mechanisms operate in the plant to coordinate C assimilation with N metabolism (Nunes-Nesi et al., 2010). Thus, plants respond to changes in C and N metabolites through the regulation of translation and posttranslational modification mechanisms. C and N metabolites activate signaling pathways that regulate enzyme and transporter activities that control C and N fluxes, optimizing the plant response to developmental and environmental cues changing source/sink relationships (Coruzzi and Zhou, 2001).Plant hormones affect, either directly or indirectly, these pathways and can act antagonistically or synergistically when responding to environmental stress (Wilkinson et al., 2012). The exposure of plants to water-limiting conditions results in abscisic acid (ABA) synthesis that induces ABA-dependent gene expression (Yamaguchi-Shinozaki and Shinozaki, 2006), triggering the closure of stomata and reducing water loss during drought (Wilkinson and Davies, 2010). Other hormones, in particular CK, salicylic acid, ethylene, and jasmonic acid, also play direct or indirect roles in the plant responses to abiotic stress (Peleg and Blumwald, 2011). Under drought stress, plant CK content decreases, and the reduction in CK increases the plant responses to increasing ABA (Davies and Zhang, 1991), inducing stomata closure and inhibiting photosynthesis (Rivero et al., 2010). Our previous results suggested that the stress-induced CK synthesis, driven by a stress-induced promoter, protected against the deleterious effects of water deficit on the photosynthetic apparatus, allowing higher photosynthetic rates and higher yields after water deficit in tobacco (Rivero et al., 2009) and cotton (Gossypium hirsutum; Kuppu et al., 2013) plants grown in the greenhouse and peanut (Arachis hypogaea) plants grown under field conditions (Qin et al., 2011).Here, we analyzed gene expression profiles, metabolites, and enzymatic and photosynthetic activities of flag leaves of wild-type and transgenic rice expressing P_SARK::IPT exposed to water deficit during the reproductive stage and identified metabolic processes associated with the enhanced tolerance of the transgenic plants to water deficit. Our results indicate that the stress-induced CK synthesis in the transgenic plants promoted sink strengthening through the maintenance and coordination of N and C assimilation during water stress.     

Synergistic larvicidal and repellent effects of essential oils of three Origanum species on Rhipicephalus annulatus tick

Ticks are of great economic importance worldwide, both because they represent major obstacles to livestock productivity and because of their ability to transmit diseases to humans and animals. Although synthetic acaricides are the most common method for tick control, their overuse has led to the development of resistance as well as unacceptable residual levels in animal products and in the environment in general. There is therefore an urgent need to identify alternative treatments. Among such alternative approaches for tick control is plant essential oil (EO) therapy. In the present study, we investigated the synergistic effect of EOs of three oregano species—Origanum onites, O. majorana and O. minutiflorum—against Rhipicephalus annulatus larvae. Gas chromatography–mass spectrometry profiles of the three EOs revealed that carvacrol was their major component, with a concentration of 86.2% in O. majorana, 79.1% in O. minutiflorum and 77.4% in O. onites. The results of larvicidal assays revealed that the doses that lead to the death of 50% of the ticks (LC₅₀) were 22.99, 25.08 and 27.06 µL/mL for O. majorana, O. minutiflorum and O. onites EOs, respectively, whereas the doses that lead to the death of 99% (LC₉₉) were 41.26, 43.62 and 48.96 µL/mL. In addition, the LC₅₀ and LC₉₉ of the three oils combined was lower (viz., 4.01 and 6.97 µL/mL) than that of each oil alone. The tested EOs were also able to repel larvae of R. annulatus to varying degrees, with O. onites oil exhibiting the greatest repellent effect, as shown by the lowest RC₅₀ dose, followed by O. minutiflorum and O. majorana. Interestingly, this means that the oil that was least effective in killing the larvae was the most effective in repelling them. The calculated synergistic factor of any combination was higher than 1 which means that combinations have a synergistic effect. In conclusion, the combination of all three oils showed higher toxic and repellent activities than either oil separately or combinations of any two oils, suggesting synergistic effects with low doses. Further studies including field trials and the establishment of the mode of action and side effects are urgently needed to expand on these findings, and other tick stages such as adults should also be tested.

     

Studies with tryptophan metabolites in vitro: Kynurenine metabolism in liver homogenates of normal and Schistosoma mansoni-infested mice

The conversion of kynurenine into kynurenic acid and anthranilic acid in both normal and Schistosoma mansoni-infested mouse liver was investigated. It was found that in the S. mansoni-infested mouse liver there is probably a deficiency of pyridoxal phosphate that resulted in an inhibition of kynurenine transaminase and a low production of kynurenic acid. Deoxypyridoxine and its phosphorylated derivative inhibited kynurenine transaminase in the normal liver in a pattern qualitatively similar to that observed with infested liver. The lowered concentration of pyridoxal phosphate in the infested liver is discussed in view of the possibility of two combined mechanisms: (a) an antimetabolite being secreted by the infesting worms or present in its eggs that partially inhibited the phosphorylation of pyridoxal, and (b) concentration of pyridoxal phosphate by the worms, resulting in a lowered concentration of the cofactor in the host tissue.     

Protective Effects of Quercetin and Melatonin on Indomethacin Induced Gastric Ulcers in Rats

Background: Medications to prevent the development of NSAID-induced gastric ulcers have a large range of unpleasant side effects. Recent efforts have been focused on determining safer alternative nontoxic and natural forms of anti-ulcer treatments. Methods:Twenty-four male rats were divided into 4 groups: 1: control group that received no treatment; 2: the ndomethacin-treated group that received 20 mg/kg of indomethacin for 2 days to induce the development of gastric ulcers; 3: quercetin-treated group that in addition to the indomethacin treatment, received 50 mg/kg of quercetin 6 hours after and then daily for 14 days and; 4: the melatonin-treated group which received 20 mg/kg of melatonin 6 hours after each indomethacin treatment and then daily for 14 days. All drugs were administered orally. The following parameters were assessed in each group: mean ulcer index of gastric tissue, gastric acid volume and pH, oxidative stress markers: malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH), inflammatory markers: PGE-2, TNF-α, and IL-10, nitric oxide (NO) levels and the relative gene expression of BAX, BCL-2 and COX-2 by real time PCR.Results:Our findings revealed that the indomethacin-treated group had a significantly increased (p< 0.05) ulcer index, gastric acid volume, and elevated levels of stress, inflammatory, and apoptotic markers compared to controls. In the groups that received quercetin or melatonin, these factors were all significantly decreased (p< 0.05). Between quercetin and melatonin, there was no significant difference in their gastroprotective effect. Conclusion:Both quercetin and melatonin had protective antioxidant, anti-inflammatory and antiapoptotic activity against indomethacin-induced gastric ulcers.Key Words: Gastric ulcer, Indomethacin, Melatonin, Quercetin     

Novel Synthesis and Biological Evaluation of the First Pyrazole Thioglycosides as Pyrazofurin Analogues

This study reports a novel and efficient method for the synthesis of the first reported novel class of pyrazole thioglycosides 6a–h. These series of compounds were designed through the reaction of sodium 2-cyano-3-oxo-3-(4-substitutedphenylamino)prop-1-ene-1,1-bis(thiolate) salts 2 with hydrazine hydrate in ethanol at room temperature to give the corresponding sodium 5-amino-4-(substitutedphenylcarbamoyl)-1H-pyrazole-3-thiolates 3a–d. The latter compounds were treated with protected α-D-gluco- and galacto-pyranosyl bromides 4a,b in DMF at ambient temperature to give in a high yields the corresponding pyrazole thioglycosides 6a–h. Treatment of pyrazole salts 3a–d with hydrochloric acid at amobient temperature afforded the corresponding 3-mercaptopyrazole derivatives 5. The latter compounds were treated with peracetylated sugars 4 in sodium hydride in ethanol at ambient temperature to tolerate the S-glycosyl 6a–h compounds. Ammonolysis of the pyrazole thioglycosides 6a–h afforded the corresponding free thioglycosides 7a–h. The toxicity and antitumor activities of the synthesized compounds were studied.