Quantitative trait loci for agronomic traits in an elite barley population for Mediterranean conditions

Advances in plant breeding through marker-assisted selection (MAS) are only possible when genes or quantitative trait loci (QTLs) can contribute to the improvement of elite germplasm. A population of recombinant inbred lines (RILs) was developed for one of the best crosses of the Spanish National Barley Breeding Program, between two six-row winter barley cultivars Orria and Plaisant. The objective of this study was to identify favourable QTLs for agronomic traits in this population, which may help to optimise breeding strategies for these and other elite materials for the Mediterranean region. A genetic linkage map was developed for 217 RILs, using 382 single nucleotide polymorphism markers, selected from the barley oligonucleotide pool assay BOPA1 and two genes. A subset of 112 RILs was evaluated for several agronomic traits over a period of 2 years at three locations, Lleida and Zaragoza (Spain) and Fiorenzuola d’Arda (Italy), for a total of five field trials. An important segregation distortion occurred during population development in the region surrounding the VrnH1 locus. A QTL for grain yield and length of growth cycle was also found at this locus, apparently linked to a differential response of the VrnH1 alleles to temperature. A total of 33 QTLs was detected, most of them for important breeding targets such as plant height and thousand-grain weight. QTL × environment interactions were prevalent for most of the QTLs detected, although most interactions were of a quantitative nature. Therefore, QTLs suitable for MAS for most traits were identified.     

Elevated plasma viscosity in extreme hemodilution increases perivascular nitric oxide concentration and microvascular perfusion

We tested the hypothesis that high-viscosity (HV) plasma in extreme hemodilution causes wall shear stress to be greater than low-viscosity (LV) plasma, leading to enhanced production of nitric oxide (NO). The perivascular concentration of NO was measured in arterioles and venules and the tissue of the hamster chamber window model, subjected to acute extreme hemodilution, with a hematocrit (Hct) of 11% using Dextran 500 (n = 6) or Dextran 70 (n = 5) with final plasma viscosities of 1.99 +/- 0.11 and 1.33 +/- 0.04 cp, respectively. HV plasma significantly increased the periarteriolar, perivenular, and tissue NO concentration by 2.0, 1.9, and 1.4 times the control (n = 7). The NO concentration with LV plasma was not statistically different from control. Arteriolar shear stress was significantly increased in HV plasma relative to LV plasma in arterioles but not in venules. Aortic endothelial NO synthase (eNOS) protein expression was increased with HV plasma but not with LV plasma. There was a weak correlation between perivascular NO concentration and the locally calculated shear stress induced by the procedures, when blood viscosity was corrected according to Hct values previously determined in studies of microvascular Hct distribution. The finding that the periarteriolar and venular NO concentration in HV plasma was the same although arteriolar shear stress was significantly greater than venular shear stress maybe be due to differences in vessel wall metabolism between arterioles and venules and the presence of NO transport through the blood stream in the microcirculation. Results support the concept that in extreme hemodilution HV plasma maintains functional capillary density through a NO-mediated vasodilatation.     

In vivo and in vitro evaluation of cell-mediated immunity in patients with paracoccidioidomycosis

The cellular immune response to specific and nonspecific agents was investigated. both in vivo and in vitro, in 19 patients with paracoceidioidomycosis. In addition, the immunologic study of an investigator aceidentally inoculated with P. brasiliensis was included in this study. Nearly half of the patients showed depressed cell-mediated immune responses, as evaluated by intradermal tests with an antigenic preparation from P. brasiliensis (P.b.Ag.), ubiquitous antigens, and by the ability to develop sensitization to 2,4-dinitrochlorobenzene. A similar proportion of impaired responses was observed when the patients' lymphocytes were cultured with phytohemagglutinin (PHA). C'. albicans antigen and P.h.Ag. A factor was detected in the plasma of some patients which reduced the ability of patients' and normal lymphocytes to undergo blastic transformation. A positive correlation was found between the ability to develop delayed cutaneous hypersensitivity reactions to P.b.Ag. and other ubiquitous antigens, normal in vitro responsiveness to PHA and the absence of humoral blastogenic inhibitory factor. The inhibition of leukocyte migration, but not lymphocyte transformation, correlated positively with delayed hypersensitivity. The percentage of T lymphocytes was significantly reduced in the group of patients, being the absolute number and percentage of B cells bearing receptors tor complement normal. Two polar immunological patterns emerged. One characterized by positiveness in the skin test to P.b.Ag. and lack of significant abnormalities in cellular immunity, and another anergic to P.b.Ag., with cell mediated immunity severely depressed. Between the two polar groups, there were patients with intermediary patterns of immune response. This paper also includes the results obtained with the administration of transfer factor and levamisole to some of the patients.     

Rabies virus glycoprotein expression in Drosophila S2 cells: Influence of re-selection on protein expression

The aim of this study was to achieve expression of recombinant rabies virus glycoprotein (rRVGP) in Drosophila S2 cells. For this, a cDNA coding for the selection hygromycin antibiotic and the cDNA encoding the RVGP protein under the control of the constitutive actin promoter (Ac) were cloned in an expression plasmid, which was transfected into S2 cells. S2 cell populations (S2AcRVGPHy) showed rRVGP expression in cell lysates, attaining concentrations up to 1.5 μg/10⁷ cells (705 μg/L). Of the transfected cells, 20% were shown to express the rRVGP. Cell subpopulations selected by limiting dilution expressed higher rRVGP yields and 90% of the cells were shown to express the rRVGP. Cell populations re-selected by addition of hygromycin were shown to express 10 times higher rRVGP yields. The data presented here show that Drosophila S2 cells can be efficiently transfected with an expression/selection plasmid for rRVGP expression, allowing its synthesis with a high degree of physical and biological integrity. The importance of subpopulation selection was indicated by the increasing rRVGP yields during these procedures.     

Statistical mechanical deconvolution of thermal transitions in macromolecules. III. Application to double-stranded to single-stranded transitions of nucleic acids

The statistical mechanical deconvolution theory for macromolecular conformational transitions is extended to the case of nucleic acids transitions involving strand separation. It is demonstrated that the partition function, Q, as well as all the relevant thermodynamic quantities of the system, can be calculated from experimental scanning calorimetric data. In particular, it is shown that important thermodynamic parameters such as the size of the average cooperative unit during strand separation, the mean helical segment length, and the mean coil-segment length can be calculated from the average excess enthalpy function 〈ΔH〉. The theory is applied to the double-stranded to single-stranded transition of the system poly(A)·poly(U) at different salt concentrations. It is shown that the mean helical segment length is a monotonically decreasing function of the temperature well before strand separation occurs. On the other hand, the mean coil segment length remains practically constant until temperatures very close to T_m. Both experimental findings clearly indicate that the unfolding of poly(A)·poly(U) proceeds through the formation of many short helical sequences. The cooperative unit for the strand separation is calculated to be about 120 base pairs and essentially independent of the salt concentration. The existence of a minimum helical segment length of 10 ± 2 base pairs within the double-stranded form is calculated.     

Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage

The development of an appropriate technique for the identification of autolysin-defective mutants of pneumococcus has been a fundamental step to carry out studies on the molecular characteristics of the lytic enzymes of Streptococcus pneumoniae and its bacteriophage. Our results show that the principal pneumococcal autolysin (an amidase) is responsible for the separation of the daughter cells at the end of the cell division. On the other hand, this system provides a reliable experimental model to support the extended idea concerning the modular organization of most proteins. The comparative analyses of the deduced amino acid sequences of these enzymes, as well as the construction of functional chimeric phage-bacterial enzymes, demonstrate that the C-terminal domain, which contains a large number of repeated amino acid motifs, is the substrate-binding domain, whereas the N-terminal domain provides enzymatic specificity. We propose that the pneumococcal lytic enzymes have evolved by modular exchange providing examples of the types of novel genes that the bacteria or the phage might create to allow them to become adapted to new environmental situations.