Genetic Diversity of a Natural Population of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis> Nodulating Plants of <Emphasis Type="Italic">Vicia faba</Emphasis> in the Vesuvian Area

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H′ decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.     

Bioenergetics shapes cellular death pathways in Leber's hereditary optic neuropathy: a model of mitochondrial neurodegeneration

Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.     

A preliminary report on the use of transfer factor for treating stage D3 hormone-unresponsive metastatic prostate cancer

As conventional treatments are unsuccessful, the survival rate of stage D3 prostate cancer patients is poor. Reports have suggested the existence of humoral and cell-mediated immunity (CMI) against prostate cancer tumour-associated antigens (TAA). These observations prompted us to treat stage D3 prostate cancer patients with an in vitro produced transfer factor (TF) able to transfer, in vitro and in vivo, CMI against bladder and prostate TAA. Fifty patients entered this study and received one intramuscular injection of 2–5 units of specific TF monthly. Follow-up, ranging from 1 to 9 years, showed that complete remission was achieved in 2 patients, partial remission in 6, and no progression of metastatic disease in 14. The median survival was 126 weeks, higher than the survival rates reported in the literature for patients of the same stage.     

Integration of mRNA Expression Profile,Copy Number Alterations,and microRNA Expression Levels in Breast Cancer to Improve Grade Definition

Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number.     

Technological and molecular diversity of Lactobacillus plantarum strains isolated from naturally fermented sourdoughs

Thirty Lactobacillus (L.) plantarum strains, isolated from sourdough, were identified by biochemical tests as well as 16S rDNA sequencing and differentiated on the basis of technological properties, such as amylase, protease, phytase and antirope activities. These properties were shown to be widely differing among the strains, indicating a significant technological diversity. Genetic differentiation was achieved by restriction endonuclease analysis-pulsed field gel electrophoresis (REA-PFGE) that allowed the L. plantarum strains to be divided into 10 different genomic groups. Moreover, 32 different starters were employed in dough making experiments; each starter consisted of a single strain of L. plantarum associated with a maltose positive or a maltose negative yeast. The technological properties of the doughs were greatly influenced by the type of strain included in the starter. The time of leavening and the acidification activities detected in the dough were enhanced by the presence of L. plantarum strains. The bacterial and yeast contents and fermentation properties were statistically treated by principal component analysis (PCA), which allowed the discrimination of different typologies of dough. The study of the peculiar characteristics of different strains of L. plantarum is fundamental for a better understanding of their potential in affecting the nutritional value, quality and stability of the baked goods. L. plantarum strains are able to differentially influence the dough quality when employed as starters.     

Protein carbonylation, cellular dysfunction, and disease progression

Carbonylation of proteins is an irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. Whereas moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high-molecular-weight aggregates that are resistant to degradation and accumulate as damaged or unfolded proteins. Such aggregates of carbonylated proteins can inhibit proteasome activity. Alarge number of neurodegenerative diseases are directly associated with the accumulation of proteolysis-resistant aggregates of carbonylated proteins in tissues. Identification of specific carbonylated protein(s) functionally impaired and development of selective carbonyl blockers should lead to the definitive assessment of the causative, correlative or consequential role of protein carbonylation in disease onset and/or progression, possibly providing new therapeutic approaches.     

A novel factor XI missense mutation (Val371Ile) in the activation loop is responsible for a case of mild type II factor XI deficiency

Coagulation factor XI (FXI) is the zymogen of a serine protease that, when converted to its active form, contributes to blood coagulation through proteolytic activation of factor IX. FXI deficiency is typically an autosomal recessive disorder, characterized by bleeding symptoms mainly associated with injury or surgery. Of the more than 100 FXI gene mutations reported in FXI-deficient patients, most are associated with a proportional decrease in FXI functional and immunologic levels (type I defects), whereas only a few mutations leading to the presence of dysfunctional molecules in plasma have been molecularly analyzed to date (type II deficiencies). We report the functional and molecular characterization of a missense mutation (Val371Ile) identified, in the heterozygous state, in a 25-year-old Italian male with mild FXI deficiency. Laboratory analysis revealed reduced functional FXI levels (34%), but normal antigen levels (102%), distinctive of a type II defect. Given the proximity of Val371 to the FXI activation site, a possible interference with zymogen activation was postulated. Expression experiments of the FXI-Val371Ile recombinant protein, followed by activation assays, showed both a different time course in FXI activation and a slight delay in factor IX activation by thrombin-activated FXI.