Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 2. Quantitative trait loci (QTL) for grain yield, plant height, ear height and grain moisture

A previous genetic map containing 117 microsatellite loci and 400 F(2) plants was used for quantitative trait loci (QTL) mapping in tropical maize. QTL were characterized in a population of 400 F(2:3) lines, derived from selfing the F(2) plants, and were evaluated with two replications in five environments. QTL determinations were made from the mean of these five environments. Grain yield (GY), plant height (PH), ear height (EH) and grain moisture (GM) were measured. Variance components for genotypes (G), environments (E) and GxE interaction were highly significant for all traits. Heritability was 0.69 for GY, 0.66 for PH, 0.67 for EH and 0.23 for GM. Using composite interval mapping (CIM), a total of 13 distinct QTLs were identified: four for GY, four for PH and five for EH. No QTL was detected for GM. The QTL explained 32.73 % of the phenotypic variance of GY, 24.76 % of PH and 20.91 % of EH. The 13 QTLs displayed mostly partial dominance or overdominance gene action and mapped to chromosomes 1, 2, 7, 8 and 9. Most QTL alleles conferring high values for the traits came from line L-14-4B. Mapping analysis identified genomic regions associated with two or more traits in a manner that was consistent with correlation among traits, supporting either pleiotropy or tight linkage among QTL. The low number of QTLs found, can be due to the great variation that exists among tropical environments.     

The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic,manganese-containing enzyme

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition

Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 90% protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs from Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues of Caenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies.     

Effect of genetic modifications by selection for immunological tolerance on fungus infection in mice

Two strains of mice genetically selected for extreme phenotypes of immunological tolerance to ovalbumin, susceptible (TS) and resistant (TR), were experimentally infected with Sporothrix schenckii. The objective was to observe whether the genetic modifications produced by the selection might be associated with interstrain differences in adaptive immune and innate responses to infection. Therefore, we evaluated the LD(50), CFU, phagocytic index, fungicidal activity, pro-inflammatory cytokines, specific antibody titres, and the delayed-type hypersensitivity reactivity. TR mice were tenfold more susceptible to infection than TS mice, as shown by LD(50) (5 x 10(6) conidia i.v.). In TS mice, the resistance was a consequence of the tissue fungal load reduction, consistent specific T-cell-mediated immunity, and tumour necrosis factor (TNF)-alpha activity at onset of infection. In TR mice, these responses were not precociously detected. Therefore, the absence of CD4(+) T-cell response in the first week of infection might explain the non-clearance of pathogen in TR mice. However, TR mice did show an increase in TNF level and delayed-type hypersensitivity response after the first week post-infection; there was also expansion and increase in granulomatous foci and CFU in the spleen. The expansion of granulomatous foci and the increase in TNF-alpha and tissue fungal load to damaging levels induced severe tissue destruction, general failure of the organs, cachexy and death in TR mice. The results show that genetic selection for extreme phenotypes of immunological tolerance also modified the responses to S. schenckii infection.     

Distinction among the puparia of three blowfly species (Diptera: Calliphoridae) frequently found on unburied corpses

Calliphorid larvae are important in the decomposition of carrion. Since these larvae are present in the primary stages of succession on carcasses, they may be important indicators of death time and the movement of corpses in homicide investigations. In this study we examined the morphological differences among puparia of Chrysomya megacephala, C. putoria and Cochliomyia macellaria. Puparia of the three species (N=30, each) were obtained from the F2 generation bred in culture medium at 25 degrees C, and 60% relative humidity on a 12 h photoperiod. The interspecific differences found were related to the conspicuousness of six tubercles located in the region near the posterior spiracles and to the distance between the two peritrema involving the spiracles. The latter were (mean +/- SD) 15.2 +/- 3.1 microm for C. megacephala, 18.8 +/- 2.8 microm for C. putoria and 16.5 +/- 3.5 microm for C. macellaria. The results of the present study may be useful in forensic entomology.     

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.     

Superovulatory response, embryo quality and fertility after treatment with different gonadotrophins in native cattle

We studied native Mertolengo cattle to evaluate superovulatory (SOV) treatments, subsequent fertility of donors and pregnancy rate of recovered embryos. In Experiment 1 we compared superovulatory response (SR), embryo quality and plasma progesterone (P4) levels between donors treated with eCG (10 cows and 5 heifers) vs. FSH (pure, FSH-1, n=10 cows and crude, FSH-2, n=10 cows), during progestagenic impregnation. We also compared fertilization rates and embryo quality of bred and inseminated eCG and FSH-1 donors. Significantly more viable embryos were yielded by FSH than by eCG treated donors. Less FSH-1 than FSH-2-treated donors showed SR, but the response was identical in responder donors of both groups. Fertilization rates were significantly higher in bred than in inseminated donors. Plasma P4 levels were only significantly different (higher) between responder and non-responder donors on the day of embryo recovery. Experiment 2 compared FSH treatments (FSH-2, crude, n=11 cows and FSH-3, pure, n=10 cows) started at the midluteal phase. The mean number of viable embryos was significantly higher in FSH-3 than in FSH-2 treated donors. Both FSH treatments exerted a similar luteotrophic effect upon injection. The FSH-2 donors treated during the midluteal phase yielded more ova and showed significantly higher plasma P4 levels at all sampling days than those treated during progestagenic impregnation. The pregnancy rates of recipient cows were 67% and 46% for fresh and frozen-thawed embryos respectively. In Experiment 3, the fertility of donors (n=20) after SOV treatments was compared with that of untreated cows (n=40). Time to conception of donors, after mating with a bull 14 days after embryo recovery, was identical to that of control cows. There was some delay to conception in eCG-treated cows, but the difference was not significant. These preliminary results suggest that response to SOV treatments in Mertolengo cattle might be affected by the type of gonadotrophin and by the treatment protocol. The fertility of a traditional breeding season after SOV treatments was not impaired. Cryopreserved embryo banking can be used to preserve the breed.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.