VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.     

Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase

Particulate methane monooxygenase (pMMO) is a three-subunit integral membrane enzyme that catalyzes the oxidation of methane to methanol. Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and most questions regarding its molecular structure, active site composition, chemical mechanism, and genetic regulation remain unanswered. Copper ions are believed to play a key role in both pMMO regulation and catalysis, and there is some evidence that the enzyme contains iron as well. A number of research groups have solubilized and purified or partially purified pMMO. These preparations have been characterized by biochemical and biophysical methods. In addition, aspects of methane monooxygenase gene regulation and copper accumulation in methanotrophs have been studied. This review summarizes for the first time the often controversial pMMO literature, focusing on recent progress and highlighting unresolved issues.     

Antimicrobial activity in vitro of plumbagin isolated from Plumbago species

Plumbagin is a naturally occurring naphthoquinone isolated from roots of Plumbago scandens. The plant was collected at the Campus of Funda??o Oswaldo Cruz, Rio de Janeiro, Brazil. P. scandens is used as a traditional medicine for the treatment of several diseases. The antimicrobial activity of plumbagin was evaluated using the macrodilution method. The compound exhibited relatively specific activity against bacteria and yeast. The minimum inhibitory concentration test showed the growth inhibiton of Staphylococcus aureus at a concentration of 1.56 g/ml and of Candida albicans at a concentration of 0.78 g/ml. These results suggest the naphthoquinone plumbagin as a promising antimicrobial agent.     

Genetic detoxification of an aroA Salmonella enterica serovar Typhimurium vaccine strain does not compromise protection against virulent Salmonella and enhances the immune responses towards a protective malarial antigen

Live Salmonella vaccines are limited in use by the inherent toxicity of the lipopolysaccharide. The waaN gene encodes a myristyl transferase required for the secondary acylation of lipid A in lipopolysaccharide. A waaN mutant exhibits reduced induction of the inflammatory cytokines associated with lipopolysaccharide toxicity. Here the characteristics of a Salmonella enterica serovar Typhimurium aroA waaN mutant (SK100) in vitro and in vivo compared with its parent aroA strain (SL3261) were described. Phenotypic analysis of purified lipopolysaccharide obtained from SK100 confirmed that the physical and biological activities of the lipopolysaccharide had been altered. Nevertheless both strains had similar patterns of colonization and persistence in mice and significantly the aroA waaN mutant was equally as effective as the parent at protecting against challenge with wild-type S. Typhimurium. Furthermore, a SK100 strain was constructed expressing both tetanus toxin fragment C and the circumsporozoite protein of a malaria parasite. In marked contrast to its isogenic parent, the new attenuated strain induces significantly enhanced immune responses against the circumsporozoite protein. The waaN mutation enhances the ability of this strain to elicit immune responses towards guest antigens. This study provides important insights into the development of safe and effective multivalent Salmonella vaccines.     

Intercropped Silviculture Systems,a Key to Achieving Soil Fungal Community Management in Eucalyptus Plantations

Fungi are ubiquitous and important contributors to soil nutrient cycling, playing a vital role in C, N and P turnover, with many fungi having direct beneficial relationships with plants. However, the factors that modulate the soil fungal community are poorly understood. We studied the degree to which the composition of tree species affected the soil fungal community structure and diversity by pyrosequencing the 28S rRNA gene in soil DNA. We were also interested in whether intercropping (mixed plantation of two plant species) could be used to select fungal species. More than 50,000 high quality sequences were analyzed from three treatments: monoculture of Eucalyptus; monoculture of Acacia mangium; and a mixed plantation with both species sampled 2 and 3 years after planting. We found that the plant type had a major effect on the soil fungal community structure, with 75% of the sequences from the Eucalyptus soil belonging to Basidiomycota and 19% to Ascomycota, and the Acacia soil having a sequence distribution of 28% and 62%, respectively. The intercropping of Acacia mangium in a Eucalyptus plantation significantly increased the number of fungal genera and the diversity indices and introduced or increased the frequency of several genera that were not found in the monoculture cultivation samples. Our results suggest that management of soil fungi is possible by manipulating the composition of the plant community, and intercropped systems can be a means to achieve that.     

Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment

Mutations in the mitochondrial DNA are one of the most important causes of sensorineural hearing loss, especially in the 12S ribosomal RNA (rRNA) gene. We have analyzed the mtDNA 12S rRNA gene in a cohort of 443 families with hearing impairment, and have identified the A1555G mutation in 69 unrelated cases. A1555G is not a fully penetrant change, since only 63% of subjects with this change have developed hearing impairment. In addition, only 22% of the 183 A1555G deaf subjects were treated with aminoglycosides. Two novel nucleotide changes (T1291C and T1243C) were identified. T1243C was found in five deafness cases and one control sample. Mutation T1291C was detected in all maternally related individuals of a pedigree and in none of 95 control samples. Conservation analysis and comparison of the 12S rRNA structure with the 16S rRNA of Escherichia coli showed that the T at nucleotide 1243 and A at nucleotide 1555 are conserved positions. Prediction of RNA secondary structure showed changes in all 12S rRNA variants, the most severe being for T1291C. The reported data confirm the high prevalence of mutation A1555G in deafness cases and the major role of the 12S rRNA gene in hearing. The two novel changes reported here might have different contributions as deafness-related variants. T1291C fulfills the criteria of a disease-causing change. As in the case of mutation A1555G, the underlying phenotype of T1291C is not homogeneous for all family members, providing evidence for the implication of environmental and/or additional genetic factors.     

Proteomic Analysis of Liquid Biopsy from Tumor-Draining Vein Indicates that High Expression of Exosomal ECM1 Is Associated with Relapse in Stage I-III Colon Cancer

BACKGROUND: The analysis of exosomes in blood obtained from the tumor-draining mesenteric vein (MV) can identify tumor biomarkers before they reach target organs and form the premetastatic niche where circulating tumor cells can anchor. Our group has recently shown that microRNAs in plasma from the MV—but not the peripheral vein (PV)—have been related to liver metastases in colon cancer (CC) patients. Here we examine the exosomal protein cargo in plasma from the MV and paired PV in 31 CC patients. PATIENTS AND METHODS: The study included patients who were initially diagnosed with stage I-III CC and 10 healthy controls. Exosomes from the MV and PV of all patients and controls were isolated by ultracentrifugation and confirmed by cryogenic transmission electron microscopy. High-throughput proteomic analysis by mass spectrometry was used to identify expression levels of exosomal proteins. Findings were confirmed by Western blot. RESULTS: Exosomal ECM1 protein was more highly expressed in patients than in controls and was 13.55 times higher in MV from relapsed than relapse-free patients. High exosomal ECM1 expression was associated with liver metastases. Patients with high exosomal ECM1 expression in MV—but not PV—plasma had shorter time to relapse than those with low ECM1 expression (P = .04). CONCLUSION: High levels of exosomal ECM1 protein can identify CC patients with a higher risk of relapse. The analysis of exosomes isolated from the tumor-draining MV is a promising method for the identification of biomarkers before they reach the target organ.     

Decolorization of textile dyes by enzymatic extract and submerged cultures of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus sajor-caju PS2001 was screened in Petri dish plates to assess the dye-decolorizing ability of industrial textile dyes. P. sajor-caju PS2001 was also cultivated in solid-state fermentation containing sawdust of Pinus sp. and wheat bran to obtain the enzymatic extract, showing laccase and manganese-peroxidase activity, which was used to test the capacity to degrade the textile dyes. Additional tests of decolorization were performed in liquid cultures. Anthraquinone-type textile dyes proved to be substrates for the enzymatic system of P. sajor-caju PS2001. Cultures in Petri dish plates showed that the anthraquinone dye Reactive Blue 220 can act as a redox mediator for the enzymatic reactions involved in the decolorization process, and enables the azo dye degradation. Reactive Blue 220 and Acid Blue 280 were completely decolorized in 30 min and 60 min, respectively, during the tests with precipitated enzymatic extract, while the azo dyes showed resistance to degradation. Additionally, in submerged cultures with dyes, veratryl alcohol oxidases and lignin peroxidase activities were observed. These results suggest that the strain P. sajor-caju PS2001 has great potential for use in the bioremediation technology of recalcitrant pollutant such as textile effluents.