Collection of rice phloem sap from stylets of homopterous insects severed by YAG laser

Pure phloem sap from rice plant was collected by a method similarto the aphid technique using leafhoppers and planthoppers instead.A Yttrium-Aluminium Garnet laser was used to sever the insectstylets. Sap exuded from the plant through the stylet for upto 3 hr at a rate of 0.2 µl/hr. Analysis of the sap forsugars revealed that the only carbohydrate present was sucrose;its content was estimated to be 17% and remained at this levelfor 3 hr. (Received April 16, 1980; )     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

Synergic effects of kallikrein-kinin and prostaglandins on renin release on infusion of isolated hog kidney with aldosterone

The effects of infusion of a large amount of aldosterone into the renal artery of isolated perfused hog kidney on the release of renin, prostaglandins (PG) and kinin and the excretion of urinary kallikrein were investigated. Infusion of aldosterone at a rate of 100 ng/min (100 to 800 ng/ml of perfusate) resulted in significant releases of renin, PG (PGE₂, 6-0-PGF_1α), and kinin and increase in urinary kallikrein. Infusion of aldosterone and an inhibitor of kallikrein, aprotinin, decreased the releases of renin, PG and kinin and infusion of aldosterone with indomethacin decreased the release of PG but increased that of kinin and urinary kallikrein without significant change in renin releases. These findings suggest that the release of renin by aldosterone may result from synergic effects of renal PG and the kallkrein-kinin system.     

Two Rab7 isotypes, EhRab7A and EhRab7B, play distinct roles in biogenesis of lysosomes and phagosomes in the enteric protozoan parasite Entamoeba histolytica

Rab7 small GTPase plays a crucial role in the regulation of trafficking to late endosomes, lysosomes and phagosomes. While most eukaryotes encode a single Rab7, the parasitic protist Entamoeba histolytica possesses nine Rab7. In this study, to understand the significance of the presence of multiple Rab7 isotypes, a role of two representative Rab7 isotypes, EhRab7A and EhRab7B, was investigated. EhRab7B was exclusively localized to acidic vacuoles containing lysosomal proteins, e.g. amoebapore-A and cysteine protease. This lysosome localization of EhRab7B was in good contrast to EhRab7A, localized to a non-acidic compartment in steady state, and only partially colocalized with lysosomal proteins. Overexpression of EhRab7B resulted in augmentation of late endosome/lysosome acidification, similar to the EhRab7A overexpression. Expression of EhRab7B-GTP mutant caused dominant-negative phenotypes including decrease in late endosome/lysosome acidification and missecretion of lysosomal proteins, while EhRab7A-GTP enhanced acidification but did not affect either intracellular or secreted cysteine protease activity. Expression of either EhRab7B or EhRab7B-GTP mutant caused defect in phagocytosis, concomitant with the disturbed formation and disassembly of prephagosomal vacuoles, the compartment previously shown to be linked to efficient ingestion. Altogether, these data indicate that the two Rab7 isotypes play distinct but co-ordinated roles in lysosome and phagosome biogenesis.     

Small GTPase Rab21 Mediates Fibronectin Induced Actin Reorganization in Entamoeba histolytica: Implications in Pathogen Invasion

The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as ‘invadosomes’, promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots.     

Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65?kcal?mol(-1) was observed between WT-protein and W101A mutant.     

Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with gamma-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.     

Two cysteine protease inhibitors, EhICP1 and 2, localized in distinct compartments, negatively regulate secretion in Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica uniquely possesses two isotypes of ICPs, a novel class of inhibitors for cysteine proteases. These two EhICPs showed a remarkable difference in the ability to inhibit cysteine protease (CP) 5, a well-established virulence determinant, whereas they equally inhibited CP1 and CP2. Immunofluorescence imaging and cellular fractionation showed that EhICP1 and EhICP2 are localized to distinct compartments. While EhICP1 is localized to the soluble cytosolic fraction, EhICP2 is targeted from lysosomes to phagosomes upon erythrocyte engulfment. Overexpression of either EhICP1 or EhICP2 caused reduction of intracellular CP activity, but not the amount of CP, and decrease in the secretion of all major CPs, suggesting that both EhICPs are involved in the trafficking and/or interference with the major CP activity. These data indicate that the two EhICPs, present in distinct subcellular compartments, negatively regulate CP secretion, and, thus, the virulence of this parasite.     

Evaluation of metabolic alteration in transgenic rice overexpressing dihydroflavonol-4-reductase

BACKGROUND AND AIMS: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars. METHODS: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified. KEY RESULTS: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased. CONCLUSIONS: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells.