Trichomonas vaginalis: identification of soluble and membrane-associated phospholipase A1 and A2 activities with direct and indirect hemolytic effects

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.     

Monoamine transporter inhibitors and norepinephrine reduce dopamine-dependent iron toxicity in cells derived from the substantia nigra

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean +/- SD uptake of 100 microm 59FeCl3 in RCSN-3 cells was 25 +/- 4 pmol per min per mg, which increased to 28 +/- 8 pmol per min per mg when complexed with dopamine (Fe(III)-dopamine). This uptake was inhibited by 2 microm nomifensine (43%p < 0.05), 100 microm imipramine (62%p < 0.01), 30 microm reboxetine (71%p < 0.01) and 2 mm dopamine (84%p < 0.01). The uptake of 59Fe-dopamine complex was Na+, Cl- and temperature dependent. No toxic effects in RCSN-3 cells were observed when the cells were incubated with 100 microm FeCl3 alone or complexed with dopamine. However, 100 microm Fe(III)-dopamine in the presence of 100 microm dicoumarol, an inhibitor of DT-diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 microm nomifensine, 30 microm reboxetine and 2 mm norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe3+, (2) the uptake of Fe-norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe-norepinephrine complex even when DT-diaphorase is inhibited. These results support the proposed neuroprotective role of DT-diaphorase and norepinephrine.     

The institute for genomic research Osa1 rice genome annotation database

We have developed a rice (Oryza sativa) genome annotation database (Osa1) that provides structural and functional annotation for this emerging model species. Using the sequence of O. sativa subsp. japonica cv Nipponbare from the International Rice Genome Sequencing Project, pseudomolecules, or virtual contigs, of the 12 rice chromosomes were constructed. Our most recent release, version 3, represents our third build of the pseudomolecules and is composed of 98% finished sequence. Genes were identified using a series of computational methods developed for Arabidopsis (Arabidopsis thaliana) that were modified for use with the rice genome. In release 3 of our annotation, we identified 57,915 genes, of which 14,196 are related to transposable elements. Of these 43,719 non-transposable element-related genes, 18,545 (42.4%) were annotated with a putative function, 5,777 (13.2%) were annotated as encoding an expressed protein with no known function, and the remaining 19,397 (44.4%) were annotated as encoding a hypothetical protein. Multiple splice forms (5,873) were detected for 2,538 genes, resulting in a total of 61,250 gene models in the rice genome. We incorporated experimental evidence into 18,252 gene models to improve the quality of the structural annotation. A series of functional data types has been annotated for the rice genome that includes alignment with genetic markers, assignment of gene ontologies, identification of flanking sequence tags, alignment with homologs from related species, and syntenic mapping with other cereal species. All structural and functional annotation data are available through interactive search and display windows as well as through download of flat files. To integrate the data with other genome projects, the annotation data are available through a Distributed Annotation System and a Genome Browser. All data can be obtained through the project Web pages at http://rice.tigr.org.     

Anuran fauna from Salta city, Argentina

The loss and transformation of habitat by anthropogenic activities is one of the main causes of biodiversity decrease. Amphibians are declining all over the world and one of the causes of this decline is thought to be habitat reduction. However, there are anuran populations in highly disturbed habitats, like cities. This study presents the ecological baseline needed to understand anuran communities in urban environments, for Salta City. Argentina (ca. 502,000 inhabitants). The sample was stratified by environments defined by infrastructure and geographic location. The species were identified by visual encounter techniques and by auditory transect during the 2002-2003 reproductive period. Three families, seven genera and twelve species were recorded and the species richness decreased from city border to center. Similarity index values among geographic areas was higher East-West (94.7) than South-West (33.3). Bufo arenarum was the only species recorded in all environments. Hyla andina and Odontophrynus americanus had a restricted distribution and are here considered vulnerable. The border effect can explain some distribution pattern in these populations. A map of the potential distribution of recorded species for Salta is provided.     

Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

Two new gorgonian genera (Octocorallia: Primnoidae) from Southern Ocean waters

Tauroprimnoa austasensis gen. nov., sp. nov. and Digitogorgia kuekenthali gen. nov., sp. nov. are described and illustrated from Southern Ocean waters. The most distinctive characters of the newly proposed genera are, in Tauroprimnoa, the existence of four marginal scales, two abaxials with a strong thorn, and the presence of a single abaxial longitudinal row of body scales. In the case of Digitogorgia, the colony branching pattern, the structure of the opercular scales, and the presence of a complete cycle of accessory opercular scales are the distinct features to distinguish it from previously known genera. Tauroprimnoa austasensis sp. nov. is reported from the Eastern Weddell Sea, Antarctica, while Digitogorgia kuekenthali sp. nov. has been found in the SubAntarctic waters off Burdwood Bank and, in the south east of Isla Nueva in Chilean Patagonia.     

Quinazolinone linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates: Design,synthesis and biological evaluation as potential anticancer agents

A series of novel quinazolinone linked pyrrolobenzodiazepine (PBD) conjugates were synthesized. These compounds 4a–f and 5a–f were prepared in good yields by linking C-8 of DC-81 with quinazolinone moiety through different alkane spacers. These conjugates were tested for anticancer activity against 11 human cancer cell lines and found to be very potent anticancer agents with GI₅₀ values in the range of <0.1–26.2 μM. Among all the PBD conjugates, one of the conjugate 5c was tested against a panel of 60 human cancer cells. This compound showed activity for individual cancer cell lines with GI₅₀ values of <0.1 μM. The thermal denaturation studies exhibited effective DNA binding ability compared to DC-81 and these results are further supported by molecular modeling studies. The detailed biological aspects of these conjugates on A375 cell line were studied. It was observed that compounds 4b and 5c induced the release of cytochrome c, activation of caspase-3, cleavage of PARP and subsequent cell death. Further, these compounds when treated with A375 cells showed the characteristic features of apoptosis like enhancement in the levels of p53, p21 and p27 inhibition of cyclin dependent kinase-2 (CDK2) and suppression of NF-κB. Moreover, these two compounds 4b and 5c control the cell proliferation by regulating anti-apoptotic genes like (B-cell lymphoma 2) Bcl-2. Therefore, the data generated suggests that these PBD conjugates activate p53 and inhibit NF-κB and thereby these compounds could be promising anticancer agents with better therapeutic potential for the suppression of tumours.     

In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: relevance to Alzheimer's disease and other oxidative stress-related neurodegenerative disorders

Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH) and has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Abeta(1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro- and antiapoptotic factor proteins. The present study was undertaken to test the hypothesis that intraperitoneal injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitoneally (ip) with D609 and subsequently treated in vitro with the oxidants Fe(2+)/H(2)O(2) (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals), and AD-relevant amyloid beta-peptide 1-42 [Abeta(1-42)]. Brain mitochondria isolated from the gerbils previously injected ip with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal [a lipid peroxidation product], 3-nitrotyrosine, and cytochrome c release compared to oxidant-treated brain mitochondria isolated from saline-injected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These antiapoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.     

Purification of a moderate thermotolerant Bacillus coagulans BTS1 lipase and its properties in a hydro-gel system

An alkaline thermotolerant lipase of Bacillus coagulans BTS1 was successively purified by ammonium sulfate precipitation and DEAE anion exchange chromatography. The purified lipase immobilized in alginate beads showed an optimal activity at pH 7.5 and 55 degrees C. A pH of 5.0 or 10.0 completely quenched the activity of immobilized lipase. The alginate-bound lipase retained its activity following exposure to most of the organic solvents including amines, alkanes and alcohols. Chloride salt of Al3+, Co2+, Mg2+ and NH4+ modulated the lipase activity of alginate-immobilized enzyme. The alginate entrapped lipase showed a preferentially high activity towards p-nitrophenyl palmitate (C: 16) and activity of matrix increased following exposure to SDS. Moreover, the immobilized lipase retained more than 50% of its activity after 3rd cycle of reuse.     

Preventive effect of tannic acid on 2-acetylaminofluorene induced antioxidant level, tumor promotion and hepatotoxicity: a chemopreventive study

Tannic acid, present in almost every food derived from plants, has been widely investigated as a chemopreventive agent because, apart from its use as a food additive, pharmacological studies have demonstrated its many health-promoting properties. In this study, we show the modulatory effect of tannic acid on 2-acetylaminofluorene (2-AAF)-mediated hepatic oxidative stress and cell proliferation in rats. 2-AAF (50 mg/kg body weight) caused reduction in hepatic glutathione content and the activities of hepatic anti-oxidant enzymes and phase-II metabolizing enzymes with an enhancement of xanthine oxidase activity, lipid peroxidation and hydrogen peroxide content. 2-AAF treatment also induced serum oxaloacetate and pyruvate transaminase, lactate dehydrogenase and gamma-glutamyl transpeptidase. Treatment of rats orally with tannic acid (125 and 250 mg/kg body weight) resulted in significant recovery of hepatic glutathione content, antioxidant and phase-II metabolizing enzymes. Also, significant decreases in lipid peroxidation, xanthine oxidase, hydrogen peroxide generation and liver damage marker enzymes were observed. The antiproliferative efficacy of the tannic acid was also evaluated. The promotion parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in the diet with partial hepatectomy (PH) were also significantly suppressed, dose dependently, by tannic acid. Hence, we propose that tannic acid might suppress the promotion stage via inhibition of oxidative stress and polyamine biosynthetic pathway.