Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

Characteristic of cruciferous plants is the synthesis of nitrogen- and sulfur-rich compounds, such as glucosinolates and indole alkaloids. The intact glucosinolates have limited biological activity, but give rise to an array of bio-active breakdown products when hydrolysed by endogenous β-thioglucosidases (myrosinases) upon tissue disruption. Both glucosinolates and indole alkaloids constitute an important part of the defence of plants against herbivores and pathogens, with the difference that a basal level of glucosinolates is ever-present in the plant whereas indole alkaloids are true phytoalexins that are de novo synthesised upon pathogen attack. With the completion of the genome sequence of the model plant, Arabidopsis thaliana, which is a crucifer, many genes involved in the biosynthesis of glucosinolates and indole alkaloids have been identified and cytochromes P450 are key players in these pathways. In the present review, we will focus on the cytochromes P450 in the biosynthesis of both groups of compounds. Their functional roles and regulation will be discussed.     

Initial characterization of an autoclaved Toxoplasma vaccine in mice

Toxoplasmosis is a zoonotic protozoal disease that has a major significance from the perspectives of public health and veterinary medicine. Therefore, an obvious long-term goal of many scientists would be the development of an effective vaccine. In this study, autoclaved vaccine was evaluated for its ability to protect mice against Toxoplasma gondii RH challenge as an acute infection model. Results showed that autoclaved Toxoplasma vaccine (ATV) when combined with BCG as an adjuvant was effective in triggering cell mediated immunity as shown by a significant increase in the percentage of splenic CD8+ T-lymphocytes. Following challenge, death of mice vaccinated with ATV was delayed for nine days. There was a significant decrease in parasite density in different organs, and a marked reduction of pathological changes in the liver suggesting that significant immune responses were mounted following vaccination. Future studies are warranted to test the vaccine against challenge with brain cysts as a chronic infection model and to evaluate it with other recent immunization strategies that can further enhance its immunogenicity.     

Origin and hidden diversity within the poorly known Galápagos snake radiation (Serpentes: Dipsadidae)

Galápagos snakes are among the least studied terrestrial vertebrates of the Archipelago. Here, we provide a phylogenetic analysis and a time calibrated tree for the group, based on a sampling of the major populations known to occur in the Archipelago. Our study revealed the presence of two previously unknown species from Santiago and Rábida Islands, and one from Tortuga, Isabela, and Fernandina. We also recognize six additional species of Pseudalsophis in the Galápagos Archipelago (Pseudalsophis biserialis from San Cristobal, Floreana and adjacent islets; Pseudalsophis hoodensis from Española and adjacent islets; Pseudalsophis dorsalis from Santa Cruz, Baltra, Santa Fé, and adjacent islets; Pseudalsophis occidentalis from Fernandina, Isabela, and Tortuga; Pseudalsophis slevini from Pinzon, and Pseudalsophis steindachneri from Baltra, Santa Cruz and adjacent islets). Our time calibrated tree suggests that the genus Pseudalsophis colonized the Galápagos Archipelago through a single event of oceanic dispersion from the coast of South America that occurred at approximately between 6.9?Ma and 4.4?Ma, near the Miocene/Pliocene boundary.www.zoobank.org/urn:lsid:zoobank.org:pub:2679FD19-01E5-48FE-A0DA-A88FF145DE56     

Gender Disparities in the Presentation,Management and Outcomes of Acute Coronary Syndrome Patients: Data from the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE-2)

Background

Gender-related differences in mortality of acute coronary syndrome (ACS) have been reported. The extent and causes of these differences in the Middle-East are poorly understood. We studied to what extent difference in outcome, specifically 1-year mortality are attributable to demographic, baseline clinical differences at presentation, and management differences between female and male patients.

Methodology/Principal Findings

Baseline characteristics, treatment patterns, and 1-year mortality of 7390 ACS patients in 65 hospitals in 6 Arabian Gulf countries were evaluated during 2008–2009, as part of the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE-2). Women were older (61.3±11.8 vs. 55.6±12.4; P<0.001), more overweight (BMI: 28.1±6.6 vs. 26.7±5.1; P<0.001), and more likely to have a history of hypertension, hyperlipidemia or diabetes. Fewer women than men received angiotensin-converting enzyme inhibitors (ACE), aspirin, clopidogrel, beta blockers or statins at discharge. They also underwent fewer invasive procedures including angiography (27.0% vs. 34.0%; P<0.001), percutaneous coronary intervention (PCI) (10.5% vs. 15.6%; P<0.001) and reperfusion therapy (6.9% vs. 20.2%; P<0.001) than men. Women were at higher unadjusted risk for in-hospital death (6.8% vs. 4.0%, P<0.001) and heart failure (HF) (18% vs. 11.8%, P<0.001). Both 1-month and 1-year mortality rates were higher in women than men (11% vs. 7.4% and 17.3% vs. 11.4%, respectively, P<0.001). Both baseline and management differences contributed to a worse outcome in women. Together these variables explained almost all mortality disparities.

Conclusions/Significance

Differences between genders in mortality appeared to be largely explained by differences in prognostic variables and management patterns. However, the origin of the latter differences need further study.     

Short-Term Exposure to Nickel Alters the Adult Rat Brain Antioxidant Status and the Activities of Crucial Membrane-Bound Enzymes: Neuroprotection by L-Cysteine

Nickel (Ni) is an environmental pollutant towards which human exposure can be both occupational (mainly through inhalation) and dietary (through water and food chain-induced bioaccumulation). The aim of this study was to investigate the effects of short-term Ni-administration (as NiCl₂, 13 mg/kg) on the adult rat whole brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), Na⁺,K⁺-ATPase, and Mg²⁺-ATPase; in addition, the potential effect of the co-administration of the antioxidant L-cysteine (Cys, 7 mg/kg) on the above parameters was studied. Twenty-eight male Wistar rats were divided into four groups: A (saline-treated control), B (Ni), C (Cys), and D (Ni and Cys). All rats were treated once daily with intraperitoneal injections of the tested compounds, for 1-week. Rats were sacrificed by decapitation and the above-mentioned parameters were measured spectrophotometrically. Rats treated with Ni exhibited a significant reduction in brain TAS (-47%, p < 0.001, BvsA) that was efficiently limited by the co-administration of Cys (-4%, p > 0.05, DvsA; +83%, p < 0.001, DvsB), while Cys (group C) had no effect on TAS. The rat brain AChE activity was found significantly increased by both Ni (+30%, p < 0.001, BvsA) and Cys (+62%, p < 0.001, CvsA), while it tended to adjust to control levels by the co-administration of Ni and Cys (+13%, p < 0.001, DvsA; −13%, p < 0.001, DvsB). The activity of rat brain Na⁺,K⁺-ATPase was significantly decreased by Ni-administration (−49%, p < 0.001, BvsA), while Cys supplementation could not reverse this decrease (-44%, p < 0.001, DvsA). The activity of Mg²⁺-ATPase was not affected by Ni-administration (−3%, p > 0.05, BvsA), but was significantly reduced when combined with Cys administration (−17%, p < 0.001, DvsA). The above findings suggest that Ni short-term in vivo administration causes a statistically significant decrease in the rat brain TAS and an increase in AChE activity. Both effects can be, partially or totally, reversed to control levels by Cys co-administration; Cys could thus be considered (for future applications) as a potential neuroprotective agent against chronic exposure to Ni. The activity of Na⁺,K⁺-ATPase that was inhibited by Ni, could not be reversed by Cys co-administration. The matter requires further investigation in order to fully elucidate the spectrum of the neurotoxic effects of Ni.     

Microparticles Mediate Hepatic Ischemia-Reperfusion Injury and Are the Targets of Diannexin (ASP8597)

Background & Aims

Ischemia–reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis.

Methods

C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min–24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function.

Results

Microparticles were detected in the circulation 15–30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8⁺ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-κB and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin.

Conclusions

Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.     

Characterization of TioQ, a type II thioesterase from the thiocoraline biosynthetic cluster

An antitumor agent thiocoraline is a thiodepsipeptide marine product derived from two Micromonospora sp. strains that inhibits protein synthesis by binding of its key 3-hydroxyquinaldic acid (3HQA) chromophores to duplex DNA. There are at least two potential pathways via which the 3HQA moiety could be biosynthesized from L-Trp. By biochemical characterization and by preparation of knockouts of an adenylation-thiolation enzyme, TioK, and of two type II thioesterases, TioP and TioQ, found in the thiocoraline biosynthetic gene cluster, we gained valuable insight into the pathway followed for the production of 3HQA.     

Bocavirus episome in infected human tissue contains non-identical termini

Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus [1] in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses. Significance: HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.     

16S rRNA gene sequences analysis of Ficus elastica rubber latex degrading thermophilic Bacillus strain ASU7 isolated from Egypt

A thermophilic Bacillus strain ASU7 was isolated from soil sample collected from Assiut governorate in Upper Egypt on latex rubber-containing medium at 45?°C. Genetically, the 16S bacterial ribosomal RNA gene of the strain ASU7 was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database. Based on phylogenetic analyses, strain ASU7 was identified as Bacillus amyloliquefaciens. The strain was able to utilize Ficus elastica rubber latex as a sole source for carbon and energy. The ability for degradation was determined by measuring the increase in protein content of bacterium (mg/g dry wt), reduction in molecular weight (g/mol), and inherent viscosity (dl/g) of the latex. Moreover, the degradation was also confirmed by observing the growth of bacterium and formation of aldehyde or keto group using scanning electron microscopy (SEM) and shiff's reagent, respectively.