Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system

The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta. No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.     

Membrane topology of the acyl-lipid desaturase from Bacillus subtilis

The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA). The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.     

ddbRNA: detection of conserved secondary structures in multiple alignments

MOTIVATION: Structured non-coding RNAs (ncRNAs) have a very important functional role in the cell. No distinctive general features common to all ncRNA have yet been discovered. This makes it difficult to design computational tools able to detect novel ncRNAs in the genomic sequence. RESULTS: We devised an algorithm able to detect conserved secondary structures in both pairwise and multiple DNA sequence alignments with computational time proportional to the square of the sequence length. We implemented the algorithm for the case of pairwise and three-way alignments and tested it on ncRNAs obtained from public databases. On the test sets, the pairwise algorithm has a specificity greater than 97% with a sensitivity varying from 22.26% for Blast alignments to 56.35% for structural alignments. The three-way algorithm behaves similarly. Our algorithm is able to efficiently detect a conserved secondary structure in multiple alignments.     

From autopoiesis to neurophenomenology: Francisco Varela's exploration of the biophysics of being

This paper reviews in detail Francisco Varela's work on subjectivity and consciousness in the biological sciences. His original approach to this "hard problem" presents a subjectivity that is radically intertwined with its biological and physical roots. It must be understood within the framework of his theory of a concrete, embodied dynamics, grounded in his general theory of autonomous systems. Through concepts and paradigms such as biological autonomy, embodiment and neurophenomenology, the article explores the multiple levels of circular causality assumed by Varela to play a fundamental role in the emergence of human experience. The concept of biological autonomy provides the necessary and sufficient conditions for characterizing biological life and identity as an emergent and circular self-producing process. Embodiment provides a systemic and dynamical framework for understanding how a cognitive self--a mind--can arise in an organism in the midst of its operational cycles of internal regulation and ongoing sensorimotor coupling. Global subjective properties can emerge at different levels from the interactions of components and can reciprocally constrain local processes through an ongoing, recursive morphodynamics. Neurophenomenology is a supplementary step in the study of consciousness. Through a rigorous method, it advocates the careful examination of experience with first-person methodologies. It attempts to create heuristic mutual constraints between biophysical data and data produced by accounts of subjective experience. The aim is to explicitly ground the active and disciplined insight the subject has about his/her experience in a biophysical emergent process. Finally, we discuss Varela's essential contribution to our understanding of the generation of consciousness in the framework of what we call his "biophysics of being."     

Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR

Strain-specific rRNA-targeted primers were designed for the quantitative detection of Bifidobacterium infantis Y1, B. breve Y8 and B. longum Y10 used in a pharmaceutical probiotic product (VSL-3). PCR and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures. These analysis revealed that B. infantis Y1 was the predominant strain in the probiotic product and that its growth rate was the highest. Since B. infantis Y1, B. breve Y8 and B. longum Y10 are co-cultured during the industrial production of VSL-3, the kinetic characteristics of these strains can explain their different concentrations in the probiotic preparation. A validation of the PCR quantification method was performed by identifying a representative number of isolates from the bacterial mixtures with automated ribotyping. The methodology described represents a useful tool for the specific quantitative detection of bacterial strains and species in complex mixtures such as pharmaceutical preparations, dairy starter cultures, faecal samples and biopsies.     

Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.     

Selection of olive varieties for tolerance to iron chlorosis

Under certain conditions, olive trees grown on calcareous soils suffer from iron chlorosis. In the present study several olive varieties and scion-rootstock combinations were evaluated for their tolerance to iron chlorosis. Plants were grown over several months in pots with a calcareous soil, under two fertilization treatments. These consisted of periodic applications of nutrient solutions containing either, 30 μmol/L FeEDDHA or not Fe. Tolerance was assessed by the chlorosis and growth parameters of plants grown without Fe, compared to those plants grown with Fe. Results show that there are differences in tolerance among olive varieties and that tolerance is mainly determined by the genotype of the rootstock. These results open the way to use tolerant varieties for those conditions where iron chlorosis could become a problem.