Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.     

Modeling of adaptations to physical training by using a recursive least squares algorithm

Busso, Thierry, Christian Denis, Régis Bonnefoy,André Geyssant, and Jean-René Lacour. Modeling ofadaptations to physical training by using a recursive least squaresalgorithm. J. Appl. Physiol. 82(5):1685-1693, 1997.The present study assesses the usefulnessof a systems model with time-varying parameters for describing theresponses of physical performance to training. Data for two subjectswho undertook a 14-wk training on a cycle ergometer were used to testthe proposed model, and the results were compared with a model withtime-invariant parameters. Two 4-wk periods of intensive training wereseparated by a 2-wk period of reduced training and followed by a 4-wkperiod of reduced training. The systems input ascribed to the trainingdoses was made up of interval exercises and computed in arbitraryunits. The systems output was evaluated one to five times per week byusing the endurance time at a constant workload. The time-invariantparameters were fitted from actual performances by using the leastsquares method. The time-varying parameters were fitted by using arecursive least squares algorithm. The coefficients of determinationr² were 0.875 and0.879 for the two subjects using the time-varying model, higher thanthe values of 0.682 and 0.666, respectively, obtained with thetime-invariant model. The variations over time in the model parametersresulting from the expected reduction in the residuals appearedgenerally to account for changes in responses to training. Such a modelwould be useful for investigating the underlying mechanisms ofadaptation and fatigue.

     

Similarity of maize and sorghum genomes as revealed by maize RFLP probes

Summary Densely saturated genetic maps of neutral genetic markers are a prerequisite either for plant breeding programs to improve quantitative traits in crops or for evolutionary studies. cDNA and genomic clones from maize were utilized to initiate the construction of a RFLP linkage map in Sorghum bicolor. To this purpose, an F₂ population was produced from starting parental lines IS 18729 (USA) and IS 24756 (Nigeria) that were differentiated with regard to many morphological and agronomical traits. A total of 159 maize clones were hybridized to the genomic DNA of the two parents in order to detect polymorphism: 154 probes hybridized to sorghum and 58 out of these were polymorphic. In almost all of the cases hybridization patterns were similar between maize and sorghum. The analysis of the segregation of 35 polymorphic clones in an F₂ population of 149 individuals yielded five linkage groups. The three principal ones recall regions of maize chromosomes 1, 3 and 5: in general, colinearity was maintained. A possible inversion, involving a long region of maize chromosome 3, was detected. Simulations were also performed to empirically obtain a value for the lowest number of individuals of the F₂ population needed to obtain the same linkage data.Prof. E. Ottaviano, to whom this paper is dedicated, suddenly died on June 7^th, 1991     

Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes

Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3' untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Crystal structure of a stress inducible protein from Mycoplasma pneumoniae at 2.85 Å resolution

Journal of Structural and Functional Genomics -     

Root proliferation in perennial grasses of low and high palatability

Root proliferation of desirable (Stipa clarazii andS. tenuis) and undesirable (S.ambigua)perennial grasses was studied in semiarid rangelands of Central Argentina(40°39S, 62°54W) in 1998. On 17 September, soil coreswereremoved from the edge of the plant, metal structures lined with screen mesh(hereafter called bags) were buried in the holes, and root-free soil was placedinto these structures. Numbers of green tillers and circumference per plant hadpreviously been determined. Since plants were of unequal size among species,root length and root dry weight data are reported on a per green tiller basis.Half of the plants was defoliated to 5 cm stubble height on 17September and/or 12 October, while the other half remained undefoliated(controls). Bags were destructively harvested either 20 days after the firstdefoliation (first sampling) or 56 days after the second defoliation (secondsampling) by digging out soil very carefully around each bag. Roots were washedfrom soil, root length estimated by the line intercept method, root dry weightdetermined after oven-drying, and root length per unit root dry weightcalculated from the two measured variables. Root length and dry weight weremorethan 96% greater on defoliated and undefoliated plants ofS. clarazii than on those of S.tenuisor S. ambigua for both sampling dates. Root length perunitroot dry weight, however, was more than 43% greater (p < 0.05) inS. tenuis than in S. clarazii andS. ambigua during the second sampling. Defoliated plantshada similar root length and root dry weight than undefoliated plants in all threespecies, although plants of S. tenuis defoliated twiceshowed a greater (p < 0.05) root length than undefoliated controls. Rootlength and root dry weight were similar between sampling periods, except onundefoliated plants of S. tenuis which had a greater (p<0.05) root length and root dry weight at the first than at the second sampling.Although root length per unit root dry weight may be greater inS. tenuis than in S. clarazii andS. ambigua, greater root length and dry weight increasesinS. clarazii after defoliation appear determinant incontributing to explain its greater competitive ability and defoliationtolerance when compared with the other two species.Nomenclature of taxa followed.     

Expression of soluble recombinant proteins in a cell-free system using a 96-well format

For structural and functional genomics programs, new high-throughput methods to obtain well-expressing and highly soluble proteins are essential. Here, we describe a rapid procedure to express recombinant proteins in an Escherichia coli cell-free system using a 96-well format. The identification of soluble proteins is performed by the Dot Blot procedure using an anti-His tag antibody. The applications and the automation of this method are described.