The plant-associated Bacillus amyloliquefaciens strains MEP2 18 and ARP2 3 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease.

AIMS: This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP(2) 3 and MEP(2) 18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. METHODS AND RESULTS: The antifungal compounds were isolated by acid precipitation of cell-free supernatants, purified by RP-HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP-HPLC eluted fractions showed the presence of surfactin C(15) , fengycins A (C(16) -C(17)) and B (C(16)) isoforms in supernatants from strain ARP(2) 3 cultures, whereas the major lipopeptide produced by strain MEP(2) 18 was iturin A C(15) . Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides-containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide-producing B. subtilis strain. CONCLUSIONS: Both strains, renamed as B. amyloliquefaciens ARP(2) 3 and MEP(2) 18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide-producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. SIGNIFICANCE AND IMPACT OF THE STUDY: Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP(2) 3 and MEP(2) 18 to control plant diseases caused by S. sclerotiorum.

Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Protein-mediated surface structuring in biomembranes.

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

c-Fos associates with the endoplasmic reticulum and activates phospholipid metabolism.

c-Fos, a transcription factor that constitutes DNA-binding AP-1 complexes, regulates gene expression that promotes long-lasting cellular changes. We show that, in addition to its transcription factor activity, c-Fos regulates the metabolism of phospholipids cytoplasmically by an AP-1-independent activity. Two waves of c-Fos expression that promote subsequent waves of stimulation of 32P-orthophosphate incorporation into phospholipids are evidenced in quiescent cultured fibroblasts induced to re-enter the cell cycle. The first wave of c-Fos expression peaks at 7.5 min and returns to control levels by 15 min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second-messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane-biogenesis-related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c-Fos expression. It is interesting that a peptide that blocks AP-1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c-Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell growth.

c-Fos is surface active and interacts differentially with phospholipid monolayers.

The transcription factor c-Fos forms stable Gibbs and Langmuir monolayers at the air-buffer interface. Its marked surface activity is enhanced by penetration into phospholipid films above the protein's own maximum adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid polar head group and the increases of lateral surface pressure generated are comparable to those of membrane-active proteins. The surface activity of c-Fos is strong enough to thermodynamically drive and retain c-Fos at the membrane interface where it may exert direct or indirect effects.

Pyruvate decarboxylase filaments are associated with the cortical cytoskeleton of asci and spores over the sexual cycle of filamentous ascomycetes.

We show that pyruvate decarboxylase (PDC) 8- to 10-nm-diameter filaments, first described in vegetative cells of Neurospora crassa, are ubiquitously present in filamentous fungi. The cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetes N. crassa, N. tetraesperma, Podospora anserina, and Sordaria macrospora. PDC-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and spindle pole bodies. Nocodazole-induced depolymerization of the cortical microtubules results in the loss of PDC-filaments. Moreover, a S. macrospora mutant defective in cortical MT distribution shows abnormal PDC organization. Neurospora asci generated on various metabolic conditions, which modify the presence and relative abundance of PDC-filaments in vegetative cells, have identical patterns of subcellular distribution of these structures. A N. crassa mutant (snowflake) that accumulates giant bundles of PDC-filaments during vegetative growth, shows normal distribution of the filaments during ascogenesis. Thus, the regulation conditioning the presence and supramolecular assembly of PDC-filaments in Neurospora differs between vegetative and sexual cells. Taken together, these results suggest that PDC in filamentous fungi may perform two functions, intervening as an enzyme in vegetative metabolism and as a structural protein associated with the cytoskeleton during sexual development.

Mapping chromosome landmarks in the centromere I region of Neurospora crassa.

Chromosome translocation breakpoints, RFLP heterozygosity in partial chromosome duplications, and RFLP-marked crossover events have been used as chromosomal landmarks to find the position and orientation of cloned regions flanking centromere I of Neurospora crassa. Determination of physical:genetic ratios in genomic regions flanking the loci mei-3, un-2, and his-2 supports previous evidence indicating that recombinational activity is lower in regions flanking centromere I than in the general N. crassa genome. The homogeneous distribution of crossover events found in these regions suggests that there is not a gradient of crossover inhibition in the vicinity of centromere I. Thus, a largely extended centromeric effect and/or a general crossover inhibitory effect operating on linkage group I (LGI) could constitute the basis of these abnormal physical:genetic ratios. A DNA element containing about 76% A+T was isolated from the centromeric end of a cloned region on LGIR. The fragment includes a previously undescribed DNA sequence, highly repeated in the Neurospora genome, which may correspond to centromeric DNA.

[Basic Life Support: the primary ABC(D) of cardiopulmonary resuscitation]. / Basic Life Support: Das primäre ABC(D) der kardiopulmonalen Reanimation.

Basic Life Support [BLS] includes both prompt recognition and immediate support of ventilation and circulation in case of respiratory or cardiac arrest. The major objective of BLS is to provide oxygen to the heart and the brain and to sustain tissue viability until definitive electrical or medical treatment [Advanced Cardiac Life Support = ACLS] can restore spontaneous circulation. BLS focuses on the primary survey A B C [D]: Airway [to open the airway]-Breath [to assess the presence or absence of spontaneous breathing and to provide ventilation]-Circulation [to assess pulselessness and to perform chest compression-Defibrillation [might perhaps no longer belong solely to ACLS because of the widespread availability of Automade External Defibrillators]. The key for successful ACLS, especially defibrillation, is the prompt initiation of correct rescue breathing, and correct chest compressions in a patient with a cardiac arrest. Promptly and optimally performed BLS is most effective and one major key to save life, thus, both family physicians and specialists must be able to administer optimal BLS-survival will be poor if one link in the chain of survival is inadequate!