Exercise attenuates the effects of hypercholesterolemia on endothelium-dependent relaxation in coronary arteries from adult female pigs.

We tested the hypothesis that exercise training (Ex) attenuates the effects of hypercholesterolemia on endothelium-dependent relaxation in left anterior descending coronary arteries. Adult female pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were trained or remained sedentary (Sed) for 16 wk, yielding four groups of pigs: 1) NF-Sed, 2) NF-Ex, 3) HF-Sed, and 4) HF-Ex. Sensitivity (EC(50)) to bradykinin (BK) was impaired in HF-Sed arteries. Ex improved BK-induced relaxation such that the EC(50) and maximal response to BK in HF-Ex arteries was not different from that in NF-Sed and NF-Ex. ACh-induced constriction was less in HF-Ex arteries than in HF-Sed, NF-Sed, and NF-Ex. To determine the mechanism(s) by which HF and Ex affected responses to BK and ACh, vasoactive responses were assessed in the presence of N(G)-nitro-L-arginine methyl ester [L-NAME; to inhibit nitric oxide (NO) synthase], indomethacin (Indo; to inhibit cyclooxygenase), and L-NAME + Indo. L-NAME inhibited BK-induced relaxation in NF (not HF) arteries. Indo did not significantly alter relaxation to BK in NF arteries; however, relaxation was enhanced in HF-Sed arteries. Double blockade with L-NAME + Indo attenuated BK-induced relaxation in NF arteries and eliminated relaxation in HF arteries. Neither L-NAME nor Indo altered constrictor responses to ACh in NF or HF arteries; however, double blockade with L-NAME + Indo attenuated constriction to ACh in NF-Ex arteries. Endothelium-independent relaxation to sodium nitroprusside was enhanced in HF-Sed and HF-Ex arteries. Collectively, these results indicate that HF impaired endothelial function in coronary arteries by impairing production of NO and by enhancing production of a constrictor that was inhibited by Indo. Ex attenuated the effects of hypercholesterolemia by improving NO-mediated, endothelium-dependent relaxation and by reducing the influence of the Indo-sensitive constrictor.     

Re-orientation and integration of the classical and interspecific linkage maps of the long arm of tomato chromosome 7

To obtain reliable classical and integrated interspecies maps of the long arm of chromosome 7 of tomato, detailed mapping work was undertaken and several phenotypic and molecular markers were assigned loci on both maps to provide reliable cross-reference points. To maximise the value of the new maps, pair-wise segregation data for classical genetic markers from the literature were included, based on large segregating populations with readily scorable phenotypes. In addition, to increase confidence in these maps, introgression lines were used to confirm important map locations. The revised classical map is based on two- and three-point test-cross data from a number of F₂ and BC₁ mapping populations. The integrated interspecies map is based on F₂ mapping populations derived from crosses of Lycopersicon esculentum with Lycopersicon pennellii (LA716). The genetic analyses for both maps were performed using the computer package JoinMap. The revised composite classical map indicates that some of the map positions reported in the literature are incorrect. The linear order of the classical markers common to both the revised classical and integrated interspecies maps are in complete agreement. Production of the integrated interspecies map resulted in re-orientation of the existing molecular map. Received: 13 September 2000 / Accepted: 20 December 2000     

Amplification of the secretory IgA response to Toxoplasma gondii using cholera toxin

Our study demonstrates that cholera toxin (CT) markedly enhances the intestinal anti-T. gondii antibody response following oral immunisation of mice with a T. gondii sonicate (TSo) and CT. The antibodies induced were mostly IgA and secretory IgA but a small quantity of IgG was also produced. In contrast, no intestinal anti-T. gondii IgM antibodies were detected. Anti-CT IgA antibodies were also present in intestinal secretions but in much lower quantities than the T. gondii-specific IgA. No anti-CT IgG nor IgM antibodies were detected. Western blot analysis showed that CT induced not only an increase of the intensity of the intestinal IgA antibody response to the 30-kDa band but also induced intestinal IgA antibodies against other major T. gondii proteins (p22, and the 28-kDa antigen) as recognised by specific monoclonal antibodies. The amplification of the anti-T. gondii secretory IgA response by means of an appropriate adjuvant may be one major step leading towards an orally induced immune protection against toxoplasmosis.     

Genetic diversity for restriction fragment length polymorphisms and heterosis for two diallel sets of maize inbreds

Summary Changes that may have occurred over the past 50 years of hybrid breeding in maize (Zea maize L.) with respect to heterosis for yield and heterozygosity at the molecular level are of interest to both maize breeders and quantitative geneticists. The objectives of this study were twofold: The first, to compare two diallels produced from six older maize inbreds released in the 1950's and earlier and six newer inbreds released during the 1970's with respect to (a) genetic variation for restriction fragment length polymorphisms (RFLPs) and (b) the size of heterosis and epistatic effects, and the second, to evaluate the usefulness of RFLP-based genetic distance measures in predicting heterosis and performance of single-cross hybrids. Five generations (parents, F₁; F₂, and backcrosses) from the 15 crosses in each diallel were evaluated for grain yield and yield components in four Iowa environments. Genetic effects were estimated from generation means by ordinary diallel analyses and by the Eberhart-Gardner model. Newer lines showed significantly greater yield for inbred generations than did older lines but smaller heterosis estimates. In most cases, estimates of additive x additive epistatic effects for yield and yield components were significantly positive for both groups of lines. RFLP analyses of inbred lines included two restriction enzymes and 82 genomic DNA clones distributed over the maize genome. Eighty-one clones revealed polymorphisms with at least one enzyme. In each set, about three different RFLP variants were typically found per RFLP locus. Genetic distances between inbred lines were estimated from RFLP data as Rogers' distance (RD), which was subdivided into general (GRD) and specific (SRD) Rogers' distances within each diallel. The mean and range of RDs were similar for the older and newer lines, suggesting that the level of heterozygosity at the molecular level had not changed. GRD explained about 50% of the variation among RD values in both sets. Cluster analyses, based on modified Rogers' distances, revealed associations among lines that were generally consistent with expectations based on known pedigree and on previous research. Correlations of RD and SRD with f₁ performance, specific combining ability, and heterosis for yield and yield components, were generally positive, but too small to be of predictive value. In agreement with previous studies, our results suggest that RFLPs can be used to investigate relationships among maize inbreds, but that they are of limited usefulness for predicting the heterotic performance of single crosses between unrelated lines.Joint contribution from Cereal and Soybean Research Unit, USDA, Agricultural Research Service and Journal Paper no. J-13929 of the Iowa Agric and Home Economics Exp Stn, Ames, IA 50011. Projects no. 2818 and 2778A.E.M. is presently at the Iowa State University on leave from University of Hohenheim, D-7000 Stuttgart 70, Federal Republic of Germany     

Design and validation of a neutral protein scaffold for the presentation of peptide aptamers

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.     

Cleavage of rabaptin-5 blocks endosome fusion during apoptosis.

Cells undergoing apoptosis exhibit striking changes in membrane organization, including plasma membrane blebbing and invagination, vacuolation and fragmentation of organelles, and alterations in the surface expression of receptors. The underlying mechanisms for these changes are unknown, though alterations in vesicular fusion are likely to play a role. Using a cell-free system based on Xenopus laevis egg extracts we have found that endosome fusion is blocked during apoptosis. Inhibition of fusion is prevented by Bcl-2 or Bcl-xL, two negative regulators of apoptosis, or by specific inhibitors of members of the caspase family of apoptotic proteases. Selective cleavage of Rabaptin-5, an essential and rate-limiting component of endosome fusion, is responsible for the loss of fusion activity. Cleavage of Rabaptin-5 also occurs in cellular models for apoptosis. These results suggest that inactivation of Rabaptin-5 and inhibition of vesicle transport lead to fragmentation of endosomes and inhibition of the endocytic pathway during the execution phase of apoptosis. We propose that parallel changes to other membrane transport pathways would give rise to general membrane fragmentation in apoptotic cells. These changes are likely to play an important role in the generation of apoptotic bodies and their recognition by phagocytosing cells.     

Association of the fusion protein NSF with clathrin-coated vesicle membranes.

N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion.     

A simple method for measurement of the haemoglobin binding capacity of canine haptoglobin

The potential of a series of related compounds to induce haemolytic anaemia in dogs highlighted the need for a reliable and sensitive technique to identify changes in plasma haptoglobin concentration. An indirect method established for human samples was adapted for use with canine plasma. This method measures the haemoglobin binding capacity of plasma, which is directly proportional to the functional haptoglobin concentration. The efficacy of this technique was investigated on samples taken from Beagle dogs which had previously received small quantities of water intravenously. A substantial reduction in haemoglobin binding capacity was recorded before other haematological parameters were significantly affected. It was concluded that measurement of haemoglobin binding capacity by this method provides a valid reflection of haptoglobin status in the dog.     

Molecular marker-facilitated studies in an elite maize population: I. Linkage analysis and determination of QTL for morphological traits

Restriction fragment length polymorphisms (RFLPs) and one morphological marker were used to investigate quantitative trait loci (QTL) for morphological and physiological traits evaluated on 150 F₂₃ maize (Zea mays L.) lines derived from the cross of elite U.S. Corn Belt inbreds Mo17 and H99. F₂₃ lines were grown in a replicated experiment and evaluated for plant and ear heights and flowering traits. QTL were identified for each trait, and genetic effects were determined. Estimated gene action for the flowering traits was predominantly overdominance. Both parents contributed toward increased values for anthesis and silk emergence. QTL for increased plant and ear heights were usually contributed by the taller parent, Mo17. Estimated gene action for these traits was mainly partial to overdominance. QTL for plant height were located in the vicinity of loci defined by alleles with qualitative effects on plant height.