Spatial aluminium sensitivity of root apices of two common bean (Phaseolus vulgaris L.) genotypes with contrasting aluminium resistance

The initial response of plants to aluminium (Al) is an inhibition of root elongation. In the present study, short and medium-term effects of Al treatment (20 muM) on root growth and Al accumulation of two common bean (Phaseolus vulgaris L.) genotypes, VAX-1 (Al-sensitive) and Quimbaya (Al-resistant), were studied. Root elongation of both genotypes was severely inhibited during the first 3-4 h of Al treatment. Thereafter, both genotypes showed gradual recovery. However, this recovery continued in genotype Quimbaya until the root elongation rate reached the level of the control (without Al) while the genotype VAX-1 was increasingly damaged by Al after 12 h of Al treatment. Short-term Al treatment (90 microM Al) to different zones of the root apex using agarose blocks corroborated the importance of the transition zone (TZ, 1-2 mm) as a main target of Al. However, Al applied to the elongation zone (EZ) also contributed to the overall inhibition of root elongation. Enhanced inhibition of root elongation during the initial 4 h of Al treatment was related to high Al accumulation in root apices in both genotypes (Quimbaya>VAX-1). Recovery from Al stress was reflected by decreasing Al contents especially in the TZ, but also in the EZ. After 24 h of Al treatment the high Al resistance of Quimbaya was reflected by much lower Al contents in the entire root apex. The results confirmed that genotypic differences in Al resistance in common bean are built up during medium-term exposure of the roots to Al. For this acquisition of Al resistance, the activation and maintenance of an Al exclusion mechanism, especially in the TZ but also in the EZ, appears to be decisive.     

Relevance of the interaction between alphaherpesvirus UL3.5 and UL48 proteins for virion maturation and neuroinvasion

The UL3.5 and UL48 genes, which are conserved in most alphaherpesvirus genomes, are important for maturation of pseudorabies virus (PrV) particles in the cytoplasm of infected cells (W. Fuchs, B. G. Klupp, H. J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996; W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002). In bovine herpesvirus 1 (BoHV-1), the homologous gene products pUL3.5 and pUL48 have been demonstrated to interact physically (N. Lam and G. Letchworth, J. Virol. 74:2876-2884, 2000). Moreover, BoHV-1 pUL3.5 partially complemented a pUL3.5 defect in PrV (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). By using coimmunoprecipitation and yeast two-hybrid studies, we observed a similar interaction between pUL3.5 and pUL48 of PrV, as well as a heterologous interaction between the PrV and BoHV-1 gene products. The relevant domain could be confined to the first 43 amino acids of PrV pUL3.5. Unlike its BoHV-1 homologue, PrV pUL3.5 is processed by proteolytic cleavage, and only an abundant 14-kDa fragment consisting of amino acids 1 to >or=116 could be detected by peptide mass fingerprint analysis of purified wild-type PrV particles, which also contain the pUL48 tegument component. To determine the biological relevance of the protein-protein interaction, pUL3.5-, pUL48-, and double-negative PrV mutants were analyzed in parallel. All deletion mutants were replication competent but exhibited significantly reduced plaque sizes and virus titers in cultured rabbit kidney cells compared to wild-type and rescued viruses, which correlated with a delayed neuroinvasion in intranasally infected mice. Remarkably, the defects of the double-negative mutant were similar to those of pUL48-negative virus. Electron microscopy of cells infected with either deletion mutant revealed the retention of naked nucleocapsids in the cytoplasm and the absence of mature virus particles. In summary, our studies for the first time demonstrate the relevance of the pUL3.5-pUL48 interaction for secondary envelopment of an alphaherpesvirus, give a molecular basis for the observed trans-complementation between the PrV and BHV-1 pUL3.5 homologs, yield conclusive evidence for the incorporation of a proteolytically processed pUL3.5 into PrV virions, and demonstrate the importance of both proteins for neuroinvasion and neurovirulence of PrV.     

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Function of a conserved checkpoint recruitment domain in ATRIP proteins

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.     

Genomic comparison using data mining techniques based on a possibilistic fuzzy sets model

Current copiousness of genomic information stored in biological databases [Mar Albà, M., Lee, M., Pearl, D., Shepherd, F.M.G., Martin, A.J., Orengo, N., Kellam, C.A., 2001. P. VIDA: a virus database system for the organisation of virus genome open reading frames. Nuleic Acids Res. 133-136] makes ultimately feasible the proposal for an application of knowledge management aimed to discover general rules in subcellular phenomena. The goal of this work is primarily to discover relationships between genes by microarray analysis. The tools exploited come from clustering techniques and are mainly based on Knowledge Discovery in Databases (KDD) concepts [Fayyad, U., Piatetsky-Shapiro, G., Smyth, P., 1996. From data mining to knowledge discovery in databases. AI Magazine 17(3), 37-54]. Starting from a data set, each element can be represented by a characteristic matrix, which sums up all data attributes. In this case data mining is oriented to perform a Pattern Recognition of related sequences, hidden in databases [Hand, D.J., Nicholas, A., 2005. Heard finding groups in gene expression data. J. Biomed. Biotechnol. 215-225]. Following a bottom up approach, the next refinement is to compare retrieved data to gather similar features, by dedicated clustering algorithms [Kaufman, L., Rousseeuw, P.J., 1990. Finding groups in data. An Introduction to Cluster Analysis. John Wiley & Sons, New York; Forman, G., Zhang, B., 2000. Distributed Data clustering can be efficient and exact HP. Laboratories Palo Alto HPL-2000, p. 158], driven by fuzzy logic, allowing us to perceive by intuition a common denominator for various genomic families and to anticipate likely future developments.     

Mathematical modeling of laser based potato cutting and peeling

A mathematical model is developed and validated to predict the depth of cut in potato tuber slabs as a function of laser power and travel speed. The model considers laser processing parameters such as input power, spot size and exposure time as well as the properties of the material being cut such as specific heat, thermal conductivity, surface reflectance, etc. The model also considers the phase change of water in potato and the ignition temperature of the solid portion. The composition of the potato tuber is assumed to be of water and solid. The model also assumes that the ablation process is accomplished through ejection of liquid water, debris and water vapour, and combustion of solid. A CO₂ laser operating in c.w. mode was chosen for the experimental work because water absorbs laser energy highly at 10.6 μm, and CO₂ laser units with relatively high output power are available. Slabs of potato tuber were chosen to be laser processed since potato contains high moisture and large amounts of relatively homogeneous tissue. The results of the preliminary calculations and experiments concluded that the model is able to predict the depth of cut in potato tuber parenchyma when subjected to a CO₂ laser beam.     

Molecular phylogeny, biogeography, and systematics of Dicerandra (Lamiaceae), a genus endemic to the southeastern United States

Dicerandra, an endemic mint of the southeastern United States, comprises nine species, all of which are threatened or endangered and restricted to sandhill vegetation and a mosaic of scrub habitats. Molecular phylogenetic analyses of Dicerandra based on data from the nuclear and plastid genomes for all 13 taxa of the genus, identified two strongly supported clades, corresponding to the four annual and to the five perennial species of Dicerandra. However, the nuclear and plastid trees were incongruent in their placement of two perennial taxa, D. cornutissima and D. immaculata var. savannarum, perhaps due to ancient hybridization or to lineage sorting. Based on these analyses, the widespread D. linearifolia is not monophyletic, with populations of D. linearifolia var. linearifolia falling into either western or eastern clades. The western clade, comprising populations of D. linearifolia var. linearifolia and var. robustior, occurs in an area drained by rivers flowing toward the Gulf of Mexico, whereas the eastern clade, comprising populations of D. linearifolia var. linearifolia, D. densiflora, D. odoratissima, and D. radfordiana (i.e., all the annual species), occupies a region drained by rivers flowing to the Atlantic Ocean. Although this pattern of genetic differentiation between populations from these two river drainages has been documented in several animal species, it has not previously been reported for plants. A revised subgeneric classification is presented to reflect the annual and perennial clades.