A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum.

The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.     

Temperature dependence of D-glucose transport in reconstituted liposomes

Sodium-dependent D-glucose uptake into proteoliposomes reconstituted from dimyristoylphosphatidylcholine (DMPC) and hog kidney brush border membrane extract is strongly affected by temperature and the physical state of the membranes. This dependence is defined by a nonlinear Arrhenius plot with a break point at 23 degrees C, a temperature not significantly different from the phase transition temperature of the pure lipid (24 degrees C). The transport process is characterized by different activation energies: 35.1 kcal/mol below and 5.5 kcal/mol above the transition temperature. The shift in the break point for the D-glucose transport activity from 15 degrees C, in the brush border membranes, to 23 degrees C in the reconstituted system leads us to conclude that the lipids surrounding the sodium/D-glucose cotransport system can exchange readily with the bulk lipid used for reconstitution. The results thus provide no evidence for the presence of an annulus of specific lipids surrounding the transport system.     

Complete genome sequence of Corynebacterium pseudotuberculosis strain CIP 52.97, isolated from a horse in Kenya

In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.     

Characterization of the new insertion sequence IS91 from an alpha-hemolysin plasmid of Escherichia coli

Summary IS91 is a 1.85 kb insertion sequence originally resident in the -hemolytic plasmid pSU233. The element was transposed sequentially from this plasmid to pA-CYC184, to R388, and to pBR322. Both cointegrates and simple insertions of the element were obtained. A detailed restriction enzyme map of the element is presented. This does not bear any relationship to the maps of previously described insertion sequences. Furthermore, hybridization between these sequences and IS91 could not be demonstrated.Deletion derivatives of IS91 were constructed which are unable to transpose. However, their transposition can be complemented in Trans by wild-type elements. One of these deletion derivatives has been genetically labeled with a kanamycin resistance marker from Tn5. When this new element was complemented for transposition, only about 2% of the transposition products were cointegrates. Thus, the behavior of IS91 is better explained by transposition models that allow direct transposition.Part of this work was carried out by E. Diaz-Aroca as a requirement for her degree in Sciences. The work is published (Esmeralda Diaz-Aroca, Tesina de Licenciatura, Universidad Autónoma de Madrid, 1983) and it contains the complete details of procedures and results of the cloning experiments and the restriction maps of the plasmids shown in this work. It is available from the authors upon request     

Effect of slice thickness on brain magnetic resonance image texture analysis

Background  

The accuracy of texture analysis in clinical evaluation of magnetic resonance images depends considerably on imaging arrangements and various image quality parameters. In this paper, we study the effect of slice thickness on brain tissue texture analysis using a statistical approach and classification of T1-weighted images of clinically confirmed multiple sclerosis patients.     

Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.     

Construction of a physical and transcript map for a 1-Mb genomic region containing the urofacial (Ochoa) syndrome gene on 10q23-q24 and localization of the disease gene within two overlapping BAC clones (<360 kb).

Urofacial (Ochoa) syndrome is an autosomal recessive disease characterized by distorted facial expression and urinary abnormalities. Previously, we mapped the UFS gene to chromosome 10q23-q24 and narrowed the interval to one YAC clone of 1410 kb. Here, we have constructed a BAC/PAC contig of the 1-Mb region using STS content mapping with 42 BAC/PAC-end sequences, 9 previously reported and 16 newly identified microsatellite markers, and 14 EST markers. A total of 26 polymorphic microsatellite markers were genotyped for 31 UFS patients from Colombia and 2 patients from the United States. Haplotype analyses suggest that the UFS gene is located within two overlapping BAC clones, a region of <360 kb of DNA sequence. We tested 42 EST markers previously mapped to the D10S1709-D10S603 interval against the BAC/PAC contig and identified 11 ESTs located in the 1-Mb region. Four of the 11 ESTs mapped to the 360-kb UFS critical region. Shotgun sequencing of the two BAC clones and BLASTN search of the EST databases revealed 3 other ESTs contained in the UFS critical region. These results will facilitate the cloning and identification of the UFS gene.     

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200°C and 5–20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R ² = 0.8861) and for ethanol production (R ² = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190°C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.     

Randia lorenceana (Rubiaceae,Gardenieae), una especie nueva del bosque mesófilo de montaña en el estado de Guerrero,México

Randia lorenceana (Rubiaceae), a new species from the cloud forest in the state of Guerrero, Mexico, is described and illustrated. The new species is related to R. matudae, but it differs from that species in having larger stipules, peduncles and calyx lobes, smaller corolla lobes, anthers, and hypanthium in the staminate flowers, and distinctly pendulous fruits due the of the bending of the peduncles.