Changing the substrate specificity of a chitooligosaccharide oxidase from Fusarium graminearum by model-inspired site-directed mutagenesis

Chitooligosaccharide oxidase (ChitO) catalyzes the oxidation of C1 hydroxyl moieties on chitooligosaccharides and in this way displays a different substrate preference as compared to other known oligosaccharide oxidases. ChitO was identified in the genome of Fusarium graminearum and a structural model revealed that one active site residue (Q268) was likely to be involved in the recognition of the N-acetyl moiety on the chitooligosaccharide substrates. The substrate specificity of wild type ChitO and the Q268R mutant were examined and confirmed that Q268 is indeed involved in N-acetyl recognition.     

Ventilation-perfusion relationships following experimental pulmonary contusion.

Ventilation-perfusion changes after right-sided pulmonary contusion (PC) in swine were investigated by means of the multiple inert gas elimination technique (MIGET). Anesthetized swine (injury, n = 8; control, n = 6) sustained a right-chest PC by a captive-bolt apparatus. This was followed by a 12-ml/kg hemorrhage, resuscitation, and reinfusion of shed blood. MIGET and thoracic computed tomography (CT) were performed before and 6 h after injury. Three-dimensional CT scan reconstruction enabled determination of the combined fractional volume of poorly aerated and non-aerated lung tissue (VOL), and the mean gray-scale density (MGSD). Six hours after PC in injured animals, Pa(O(2)) decreased from 234.9 +/- 5.1 to 113.9 +/- 13.0 mmHg. Shunt (Q(S)) increased (2.7 +/- 0.4 to 12.3 +/- 2.2%) at the expense of blood flow to normal ventilation/perfusion compartments (97.1 +/- 0.4 to 87.4 +/- 2.2%). Dead space ventilation (V(D)/V(T)) increased (58.7 +/- 1.7% to 67.2 +/- 1.2%). MGSD increased (-696.7 +/- 6.1 to -565.0 +/- 24.3 Hounsfield units), as did VOL (4.3 +/- 0.5 to 33.5 +/- 3.2%). Multivariate linear regression of MGSD, VOL, V(D)/V(T), and Q(S) vs. Pa(O(2)) retained VOL and Q(S) (r(2) = .835) as independent covariates of Pa(O(2)). An increase in Q(S) characterizes lung failure 6 h after pulmonary contusion; Q(S) and VOL correlate independently with Pa(O(2)).     

Enhanced endothelin-1-mediated leg vascular tone in healthy older subjects.

Advanced age is associated with a decreased leg blood flow and reduced physical activity. Endothelin (ET-1), a powerful vasoconstrictor, may play a role in the increased leg vascular tone in older men. objectives: to assess the ET-1-mediated vascular tone in the legs of healthy sedentary older men, both before and after 8 wk of exercise training. methods: in 8 younger subjects (19-50 yr) and 8 older men (67-76 yr), bilateral leg blood flow was measured using venous occlusion plethysmography before and after antagonizing ET-1 (using selective ET(A/B)-receptor antagonists). In older men, reversibility of the observations was assessed after 8 wk of cycling. results: ET-receptor inhibition increased leg blood flow significantly more in older men compared with younger individuals (29 +/- 9% and 10 +/- 4%, respectively, P < 0.05). Eight-week cycling training increased baseline blood flow in older men. The blood flow response to ET-receptor inhibition in older men was not affected by the training program (25 +/- 8%, P > 0.05 for comparison with pretraining). The flow ratio (blood flows infused leg/noninfused leg) decreased significantly by training from 26 +/- 8% to 7+3% (P < 0.05). CONCLUSION: the increased baseline vascular tone in aging is at least in part mediated by the endothelin. Eight-weeks cycling training in older sedentary men decreased leg vascular tone and seems to partly decrease the ET-1-mediated vascular tone.     

Genome annotation and antimicrobial properties of Bacillus toyonensis VU‐DES13, isolated from the Folsomia candida gut

Antibiotic resistance necessitates the search for new bioactive compounds with novel mechanisms of action. Natural products derived from bacteria and fungi are widely used in the field of medicine and new environments can be explored as sources of antimicrobials. Bacteria associated with springtails have shown high inhibitory activity against pathogens. Here, we characterized a bacterial strain with high potential for antimicrobial activity, isolated from the gut of the springtail Folsomia candida Willem (Collembola: Isotomidae). The strain was characterized using the ‘analytical profile index’ and the ‘minimal inhibitory concentration’ assay to test for antibiotic resistance. Agar overlay and agar disk diffusion assays were used to test the inhibitory activity of the strain and its extract against a variety of pathogens, and reporter assays were used to investigate the mode of action. High‐performance liquid chromatography was used to analyze and fractionate the extract of bacterial culture, followed by additional assays on the fractions. The genome of the strain was screened for presence of antibiotic resistance genes and secondary metabolite gene clusters. The isolate was identified as Bacillus toyonensis Jiménez et al., but it displayed differences in metabolic profile when compared to the type species. The isolate was highly resistant to penicillin and inhibited the growth of a variety of pathogenic microorganisms. Genome analysis revealed an enrichment of resistance genes for β‐lactam antibiotics compared to the type isolate. Also, secondary metabolite clusters involved in the production of siderophores, bacteriocins, and nonribosomal peptide synthetases were identified. In conclusion, a unique Bacillus strain was isolated from the gut of F. candida, for which we provide evidence of inhibitory activity against an array of pathogens. This, coupled with high resistance to penicillin as substantiated by the presence of resistance genes, points to the potential of B. toyonensis VU‐DES13 to provide a new source of antimicrobial compounds.     

Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model

Oxygen starvation triggers the shiftdown of the obligate aerobe Mycobacterium bovis BCG to a state of dormancy. Two-dimensional electrophoresis showed a drastic up-regulation of the alpha-crystallin homolog, the putative response regulator Rv3133c, and the two conserved hypothetical proteins Rv2623 and Rv2626c in dormant bacilli.     

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.     

The Cf-ECP2 gene is linked to, but not part of, the Cf-4/Cf-9 cluster on the short arm of chromosome 1 in tomato

A gene has been identified in tomato, which confers resistance to Cladosporium fulvum through recognition of the pathogenicity factor ECP2. Segregation analysis of F₂ and F₃ populations showed monogenic dominant inheritance, as for previously reported Cf resistances. The gene has been designated Cf-ECP2. Using several mapping populations, Cf-ECP2 was accurately mapped on chromosome 1, 7.7 cM proximal to TG236 and 6.0 cM distal to TG184. Although Cf-ECP2 is linked to Cf-4, it is not located in the Hcr9 cluster “Milky Way”. Therefore, Cf-ECP2 is the first functional Cf homologue on chromosome 1 that does not belong to this Hcr9 cluster. No recombination events between Cf-ECP2 and CT116 have been observed in three populations tested, representing 282 individuals. The low value for the physical distance per cM around CT116 reported previously and the high probability that Cf-ECP2 is also a member of a Hcr9 cluster will facilitate cloning of the locus. Received: 15 June 1999 / Accepted: 24 August 1999     

Hematopoietic cell fate and the initiation of leukemic properties in primitive primary human cells are influenced by Ras activity and farnesyltransferase inhibition

The Ras pathway transduces divergent signals determining normal cell fate and is frequently activated in hematopoietic malignancies, but the manner in which activation contributes to human leukemia is poorly understood. We report that a high level of activated H-Ras signaling in transduced primary human hematopoietic progenitors reduced their proliferation and enhanced monocyte/macrophage differentiation. However, the exposure of these cells to a farnesyltransferase inhibitor and establishment of a moderate level of Ras activity showed increased proliferation, an elevated frequency of primitive blast-like cells, and progenitors with enhanced self-renewal capacity. These results suggest that the amplitude of Ras pathway signaling is a determinant of myeloid cell fate and that moderate Ras activation in primitive hematopoietic cells can be an early event in leukemogenesis.     

Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.     

Lipoprotein kinetics in the metabolic syndrome: pathophysiological and therapeutic lessons from stable isotope studies

Dyslipoproteinaemia is a cardinal feature of the metabolic syndrome that accelerates atherosclerosis. It is usually characterised by high plasma concentrations of triglyceride-rich and apolipoprotein (apo) B-containing lipoproteins, with depressed concentrations of high-density lipoprotein (HDL). Dysregulation of lipoprotein metabolism in these subjects may be due to a combination of overproduction of very-low-density lipoprotein (VLDL) apoB-100, decreased catabolism of apoB-containing particles, and increased catabolism of HDL apoA-I particles. These abnormalities may be consequent on a global metabolic effect of insulin resistance that increases the flux of fatty acids from adipose tissue to the liver, the accumulation of fat in the liver, the increased hepatic secretion of VLDL-triglycerides and the remodelling of both low-density lipoprotein (LDL) and HDL particles in the circulation; perturbations in lipolytic enzymes and lipid transfer proteins contribute to the dyslipidaemia. Our in vivo understanding of the kinetic defects in lipoprotein metabolism in the metabolic syndrome has been chiefly achieved by ongoing developments in the use of stable isotope tracers and mathematical modelling. Knowledge of the pathophysiology of lipoprotein metabolism in the metabolic syndrome is well complemented by extensive cell biological data. Nutritional modifications and increased physical exercise may favourably alter lipoprotein transport in the metabolic syndrome by collectively decreasing the hepatic secretion of VLDL-apoB and the catabolism of HDL apoA-I, as well as by increasing the clearance of LDL-apoB. Pharmacological treatments, such as statins, fibrates or fish oils, can also correct the dyslipidaemia by several mechanisms of action including decreased secretion and increased catabolism of apoB, as well as increased secretion and decreased catabolism of apoA-I. The complementary mechanisms of action of lifestyle and drug therapies support the use of combination regimens to treat dyslipidaemia in the metabolic syndrome.