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Superoxide free radicals are produced in glyoxysomes.

The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O(2) (-) radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O(2) (-) radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism.

Journal Article↗

Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxysomes and leaf peroxisomes).

The intraorganellar distribution of superoxide dismutase (SOD) (EC 1.15.1.1) in two types of plant peroxisomes (glyoxysomes and leaf peroxisomes) was studied by determinations of SOD latency in intact organelles and by solubilization assays with 0.2 molar KCl. Glyoxysomes were purified from watermelon (Citrullus vulgaris Schrad.) cotyledons, and their integrity, calculated on the basis of glyoxysomal marker enzymes, was about 60%. Under the same conditions, the latency of SOD activity determined in glyoxysomes was 40%. The difference between glyoxysomal intactness and SOD latency was very close to the percentage of isozyme Mn-SOD previously determined in glyoxysomes (LM Sandalio, LA Del Río 1987 J Plant Physiol 127: 395-409). In matrix and membrane fractions of glyoxysomes, SOD exhibited a solubilization pattern very similar to catalase, a typical soluble enzyme of glyoxysomes. The analysis of the distribution of individual SOD isozymes in glyoxysomal fractions treated with KCl showed that Cu,Zn-SOD II, the major SOD isozyme in glyoxysomes, was present in the soluble fraction of these organelles, whereas Mn-SOD was bound to the glyoxysomal membrane. These data in conjunction with those of latency of SOD activity in intact glyoxysomes suggest that Mn-SOD is bound to the external side of the membrane of glyoxysomes. On the other hand, in intact leaf peroxisomes where only a Mn-containing SOD is present (LM Sandalio, JM Palma, LA Del Río 1987 Plant Sci 51: 1-8), this isozyme was found in the peroxisomal matrix. The physiological meaning of SOD localization in matrix and membrane fractions of glyoxysomes and the possibility of new roles for plant peroxisomes in cellular metabolism related to activated oxygen species is discussed.

Journal Article↗

Peculiar acute toxic colitis after ingestion of colocynth: a clinicopathological study of three cases.

We report three examples of toxic acute colitis which occurred after ingestion of colocynth (Citrullus colocynthis) for ritual purposes. The prominent clinical feature was dysenteric diarrhoea; colonoscopic changes included congestion and hyperaemia of the mucosa with abundant exudates but no ulceration or pseudopolyp formation. A causal relationship between colonic injury and the intake of colocynth was supported by the following features: (1) the pharmacology of the colocynth extract ingested; (2) the temporal relationship between colocynth intake and clinical onset (eight to 12 h); (3) the rapid recovery within three to six days, with normal endoscopy at day 14; (4) the absence of other possible causes for the observed patterns, except in one case, in which a concomitant intestinal infection with Clostridium perfringens Type A was discovered; (5) the specific pathological features. Colonic biopsies taken 27, 44, and 72 h after colocynth intake showed: erosions with fibrino-purulent exudate, early fibrosis of the lamina propria, hyaline thickening of the superficial epithelial basal membrane. These pathological features completely disappeared within 14 days in all three cases.

Acute Disease↗

Molecular phylogeny of Cucumis species as revealed by consensus chloroplast SSR marker length and sequence variation.

To investigate phylogenetic relationships in the genus Cucumis, 9 consensus chloroplast simple sequence repeat (ccSSR) primer pairs (ccSSR3, 9, 11, 13, 14, 17, 20, 21, and 23) were employed for DNA fragment length variation and 5 amplified fragments, ccSSR4, 12, 13, 19, and 20, were sequenced using total DNA from 13 accessions representing 7 African Cucumis species (x = 12), 3 Cucumis melo L. (x = 12) accessions, 2 Cucumis sativus L. (x = 7) accessions, and 1 Cucumis hystrix Chakr. (x = 12) accession. A Citrullus lanatus (Thunb.) Matsum. & Nakai (x = 11) accession was used as an outgroup. While fragment length analysis revealed the existence of 3 major species clusters (i.e., a group of African Cucumis species, a group composed of C. melo accessions, and a group containing C. sativus and C. hystrix species), sequence variation analysis identified 2 major species clusters (i.e., a group of African Cucumis species and a group composed of C. melo, C. sativus, and C. hystrix species). Comparative analysis using nuclear DNA (previous studies) and cpDNA sequence substitution data resulted in the placement of C. melo and C. sativus in different cluster groupings. Thus, both nuclear and cytoplasmic DNA should be employed and compared when a putative progenitor or specimens of an ancestral Cucumis species lineage is investigated. In addition, C. ficifolius (2x) and C. aculeatus (4x) of the African Cucumis species clustered together in this study. This result does not agree with reported isozyme analyses, but does agree with previously characterized chromosome homologies between these 2 species. Although African Cucumis species and C. hystrix do not share a close relationship, genetic affinities between C. sativus and C. hystrix are considerable. Combined evidence from previously published studies and data presented herein lend support to the hypothesis that C. hystrix is either a progenitor species of C. sativus or that they at least share a common ancestral lineage.

Cell Nucleus↗

Production of plant non-protein amino acids by recombinant enzymes of sequential biosynthetic reactions in bacteria.

We constructed the co-expression vector, pFK4, in which two cDNAs encoding serine acetyltransferase (SATase) and beta-(pyrazol-1-yl)-L-alanine/L-cysteine synthase (beta-PA/CSase) from Citrullus vulgaris (watermelon) were over-expressed under the transcriptional control of T7 promoter in Escherichia coli. Accumulation of both SATase and beta-PA/CSase in soluble extracts of E. coli was confirmed by immunoblotting. The high enzymatic activities of SATase and L-cysteine synthase (CSase) were detected in cell-free extracts of E. coli carrying pFK4. The activities of the formation of beta-PA and L-mimosine, plant non-protein amino acids, from O-acetyl-L-serine (OAS) and the precursor heterocyclic compounds, pyrazole and 3,4-dihydroxypyridine, were also found in the extracts. beta-PA was also produced in vivo from L-serine and pyrazole as precursors by E. coli cells transformed with pFK4. beta-PA was accumulated mainly in the extra-cellular culture medium. The pronounced accumulation of L-cysteine and L-methionine was observed in the cells transformed with pFK4. Additionally, we also constructed vectors which carried chimeric genes encoding fusion proteins of SATase and beta-PA/CSase. However, the fusion proteins tended to form insoluble inclusion bodies and thus to exhibit only weak enzymatic activities. The successful results of pFK4 shows the way to create a new sequential biosynthetic pathway of plant specific amino acids in bacterial cells by means of recombinant DNA technology.

Acetyltransferases↗

[Molecular genetics and biotechnology in medicinal plants: studies by transgenic plants].

The advances in molecular genetics and biotechnology in the field of medicinal plant research are discussed with focusing on the works using transgenic plants. Differentiated organ cultures and transgenic teratomas, incited by the infection with mutants of Agrobacterium Ti and Ri plasmids, were established in quinolizidine-alkaloid producing plants and Solanaceae plants. These cultured cells were used for the production and bioconversion of specific alkaloids produced in these plants. The methods of integration of foreign genes into medicinal plants were developed using an Ri binary vector. The mode of gene expression driven by TR1'-2' promoters was elucidated in transgenic medicinal plants, e.g., Nicotiana tabacum, Glycyrrhiza uralensis, Digitalis purpurea and Atropa belladonna. The genes for herbicide resistance, mammalian cytochrome P450 and bacterial beta-hydroxydecanoylthioester dehydrase were transferred and expressed in plants either to confer herbicide-resistant trait or to change the pattern of metabolites. The cDNA clones encoding cysteine synthase responsible for sulfur assimilation and biosynthesis of non-protein amino acids were isolated and characterized from Spinacea oleracea and Citrullus vulgaris. The functional lysine residue was identified by site-directed mutagenesis experiments. An over-expression system in Escherichia coli was constructed for the bacterial production of the plant specific non-protein amino acids. We made transgenic N. tabacum integrated with sense- and antisense-constructs of cysteine synthase cDNA driven by cauliflower mosaic virus 35S promoter for the purpose of genetic manipulation of biosynthetic flow of cysteine in plants. The future prospects of medicinal plant research are also discussed in the context of modern plant molecular biology.

Alkaloids↗

Effect of a phloxine B-cucurbitacin bait on diabroticite beetles (Coleoptera: Chrysomelidae).

Cucurbitacin E glycoside, extracted from a bitter mutant of Hawkesbury watermelon [Citrulls lanatus (Thunb.) Matsum. & Nakai (Syn. Citrullus vulgaris Schrad)] is the active ingredient of a feeding stimulant for the corn rootworm complex. It is the primary component of a water-soluble bait that can be combined with toxins for adult diabroticite control. Studies were conducted using phloxine B (D&C Red 28), a xanthene dye, as the toxin. This dye was efficacious against Diabrotica undecimpunctata howardi Barber, spotted cucumber beetle, and Acalymma vittatum (F.), striped cucumber beetle, in cucumber plots and could be recovered from cucumber leaves for 8 d after treatment. The average amount of dye recovered per dead spotted cucumber beetle at 8 d after treatment was 0.173 microg. Concentrated and sugar-free fermented forms of the watermelon extract were developed and compared with the fresh juice in field applications on cucumber plants. There was no significant difference in mortality of beetles from phloxine B-bait prepared with fresh, fermented, or concentrated extract, although in laboratory studies, fermented juice had higher feeding stimulant activity.

Animals↗

Relationship between dimedone concentration and formaldehyde captured in plant tissues.

In different parts of water-melon plants (Citrullus vulgaris L.) formaldehyde (HCHO), in dimedone adduct form (formaldemethone), and fully N-methylated substances were identified and determined by OPLC as well as HPLC, capillary GC and GC-MS methods using authentic substances. The HCHO captured originates from dynamic methylation and demethylation processes in which this simplest aliphatic aldehyde is bound in the form of highly reactive hydroxymethyl groups. Dimedone will react with the small quantity of HCHO in equilibrium with the hydroxymethyl groups and it follows from this that the rate of HCHO captured as the dimedone adduct will increase parallel to the increasing concentration of dimedone applied, as a methanolic solution, until a plateau is reached. The level of HCHO was very high in the roots of water-melon seedlings.

Chromatography, Gas↗

Regulation of oxylipin synthesis.

Two very common groups of oxylipins formed in plants involve the conversion of fatty acid hydroperoxides, such as hydroperoxy-octadecatrienoic acid, into further metabolites by allene oxide synthase and hydroperoxide lyase. Both of these oxylipin branch pathways appear to be ubiquitous or nearly so in plants, but the relative activities of these two branches vary among plant species. In most plants examined, including Arabidopsis, product formation from either of these pathways is minimal until elicited by wounding or some other means, upon which products from both pathways, such as jasmonic acid and C(6) aldehydes and alcohols, can increase by orders of magnitude. In some plant species such as Artemisia and Jasminum spp. oxylipin product formation is heavily skewed towards allene oxide synthase products. Others such as watermelon (Citrullus lanatus) produce 10-fold higher amounts or more of hydroperoxide lyase than allene oxide synthase products. Arabidopsis and tobacco are intermediate between these extremes. Artemisia and Jasminum are also unusual in that they do not require wounding or other types of induction for high oxylipin product formation. Release of non-esterified fatty acids appears to be correlated with oxylipin formation, but phospholipase A(2) appears not to be involved with oxylipin production, at least in the case of Artemisia leaves.

Arabidopsis↗

Effect of saponin on mortality and histopathological changes in mice.

We evaluated the acute toxicity and histopathological effects of saponin (extracted from the plant Citrullus colocynthis) on mice in order to assess its safety. The median lethal dose (LD50) of the saponin was 200 mg/kg. The histological changes were confined to the small intestine, liver and kidney, whereas the stomach, large intestine and heart appeared normal. The changes in the small intestine included haemorrhage and erosion of the mucosa. In addition, hepatorenal damage resulted from necrosis of liver cells and renal tubules.

Acute Disease↗

[Morphological studies on 18 species of gualoupi].

OBJECTIVE: To gain a clear idea of the drug Gualoupi and its adulterants. METHOD: Collecting data and spot investigating in 30 provinces, cities and autonomous regions in China. RESULT: Gualoupi used as drug in China consists of 15 species of Trichosanthes, 1 species of Momordica, 1 species of Citrullus and 1 species of Adenia(Passifloraceae). CONCLUSION: The main commodity is the rind of T. kirilowii or T. rosthornii, and the main adulterant is the rind of T. hupehensis.

Drug Contamination↗

[Identification of medicinal gualou (fruit-rind of Trichosanthes) and its mixed fruits in Sichuan].

Our investigation shows that in Sichuan Province there are 7 species of medicinal gualou (fruid-rind of Trichosanthes) and its confused fruits (6 species of Trichosanthes and 1 species of Citrullus), among which 2 species are included in the Chinese Pharmacopoeia. The drug and its confused fruits are similar in shape. This paper emphatically deals with the tissue identification of the above mentioned 7 species.

Drug Contamination↗

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants.

Seven trypsin inhibitors were isolated from the seeds of Cucurbitaceae plants: two from cucumber (Cucumis sativus) and red bryony (Bryonia diotica) and one from figleaf gourd (Cucurbita ficifolia), spaghetti squash (Cucurbita pepo var. (vegetable spaghetti) and water melon (Citrullus vulgaris). The inhibitors were purified by fractionation with ammonium sulphate, followed by ion-exchange chromatography and affinity chromatography using immobilized trypsin or anhydro-trypsin. The homogeneous inhibitors from cucumber and water melon are made up of 32 and 30 amino acid residues, respectively, whereas the remaining ones of 29 residues. All inhibitors contain three disulphide bridges and are free of threonine, phenylalanine and tryptophan. Inhibitors from spaghetti squash and CSTI IIb from cucumber are inactivated by acetylation of free amino groups whereas the remaining ones are inactivated by modification of arginine with 1,2-cyclohexanedione. Thus the P1 residues of the reactive sites of the inhibitors are lysine and arginine, respectively.

Amino Acids↗

[Microbiological quality of street sold fruits in San José, Costa Rica].

The sanitary quality of street sold fruits was analyzed during the period from march 1990 thru march 1993 in San Jose, Costa Rica. It looked for the presence of Salmonella spp. Shigella spp., Escherichia coli as well as fecal coliforms in natural refreshments, fruit salads and the fruits most frecuently expended on streets, either in slices as the pineapple (Ananas comosus), papaya (Carica papaya), non-ripe mangoe (Mangifera indica) and watermelon (Citrullus vulgaris) and those that can be eaten without peeling, like nances (Byrsonima crassifolia) and jocotes (Spondias purpurea). 25 samples of each fruit, 50 natural refreshments and 50 fruit salads were processed according to rinse solution method, and the bacteriological determination was based in the methodology described by Vanderzant & Splittstoesser and the Bacteriological Analytical Manual. In the same way, it was used the Most Probable Number for 5 tubes described in the Standar Methods of Water and Wastewater in orden to analyze 15 samples of ready to use water by the fruit hawker. The nutritional value was studied according to the food composition tables for Costa Rica, Latin America and USA. The results show that more than 30% of fruit samples, 70% of natural refreshments and 96% of fruit salad presented fecal coliforms. Same time, all of them present important contamination indexes with E. coli. Salmonella spp. and Shigella spp. were not isolated. The water analysis revelead that 53% contained fecal coliforms, probably due to the lack of hygiene in the utensils used to collect water. The nutritional evaluation shows that fruit portions (except watermelon) satisfy more than 100% of the diary recommendation of vitamin C (60 mg) and 4-7% of the recommended ingestion of dietetic fiber (30g).

Costa Rica↗

Proteomic analysis of rice leaves during drought stress and recovery.

Three-week old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 days of transpiration without watering, during which period the mid-day leaf water potential declined to approximately -2.4 MPa, compared with approximately -1.0 MPa in well-watered controls. More than 1000 protein spots that were detected in leaf extracts by proteomic analysis showed reproducible abundance within replications. Of these proteins, 42 spots showed a significant change in abundance under stress, with 27 of them exhibiting a different response pattern in the two cultivars. However, only one protein (chloroplast Cu-Zn superoxide dismutase) changed significantly in opposite directions in the two cultivars in response to drought. The most common difference was for proteins to be up-regulated by drought in CT9993 and unaffected in IR62266; or down-regulated by drought in IR62266 and unaffected in CT9993. By 10 days after rewatering, all proteins had returned completely or largely to the abundance of the well-watered control. Mass spectrometry helped to identify 16 of the drought-responsive proteins, including an actin depolymerizing factor, which was one of three proteins detectable under stress in both cultivars but undetectable in well-watered plants or in plants 10 days after rewatering. The most abundant protein up-regulated by drought in CT9993 and IR62266 was identified only after cloning of the corresponding cDNA. It was found to be an S-like RNase homologue but it lacked the two active site histidines required for RNase activity. Four novel drought-responsive mechanisms were revealed by this work: up-regulation of S-like RNase homologue, actin depolymerizing factor and rubisco activase, and down-regulation of isoflavone reductase-like protein.

Actin Depolymerizing Factors↗

Decoding microbial metabolic complementarity from individual traits to community structuring.

A fundamental challenge in microbiome research lies in elucidating the functional capacity of microbial communities through community membership and genomic data. As community structuring and emergent functional traits are determined by bacterial community metabolic networks, it is important to gain insights into the principles that govern bacteria-bacteria interactions. Here, we applied an integrative framework linking individual strain-level traits to community structuring in a simplified synthetic bacterial community (SSC8) that promotes the growth of ungrafted watermelon. By combining mono- and coculture assays with genome-scale metabolic modeling and metabolomic profiling of spent media, we characterized directional interactions and resource dependencies among community members. Our findings show that positive interactions dominated the community network, accounting for 55% of all pairwise combinations, indicating a high prevalence of growth-promoting effects among strains. Genome-scale metabolic modeling showed that functional divergence among strains enhanced the potential for metabolic complementarity as phylogenetic distance increased. Integrating metabolic modeling with metabolomics further suggested that Pseudomonas azotifigens Q6 not only benefited from all other community members, but also exhibited mutualistic interactions with the other three strains, with metabolite exchange involving compounds such as L-lysine and L-cysteine. Pseudomonas azotifigens Q6 acted as an important driver of community composition by affecting the abundance of several other consortium members in vitro. These findings highlight the role of metabolic complementarity in driving community structuring by promoting selective persistence of specific strains. Our work provides mechanistic insights into microbial interaction networks in vitro and offers a conceptual foundation for the rational design of functionally robust and plant-beneficial microbiomes.

Bacteria↗

Proteome analysis of sugar beet leaves under drought stress.

Drought is one of the major factors limiting the yield of sugar beet (Beta vulgaris L.). The identification of candidate genes for marker-assisted selection (MAS) could greatly improve the efficiency of breeding for increased drought tolerance. Drought-induced changes in the proteome could highlight important genes. Two genotypes of sugar beet (7112 and 7219-P.69) differing in genetic background were cultivated in the field. A line-source sprinkler irrigation system was used to apply irrigated and water deficit treatments beginning at the four-leaf stage. At 157 days after sowing, leaf samples were collected from well-watered and drought-stressed plants for protein extraction and to measure shoot biomass and leaf relative water content. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and quantitatively analyzed using image analysis software. Out of more than 500 protein spots reproducibly detected and analyzed, 79 spots showed significant changes under drought. Some proteins showed genotype-specific patterns of up- or downregulation in response to drought. Twenty protein spots were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), leading to identification of Rubisco and 11 other proteins involved in redox regulation, oxidative stress, signal transduction, and chaperone activities. Some of these proteins could contribute a physiological advantage under drought, making them potential targets for MAS.

Algorithms↗