Optimizing purebred selection for crossbred performance using QTL with different degrees of dominance

A method was developed to optimize simultaneous selection for a quantitative trait with a known QTL within a male and a female line to maximize crossbred performance from a two-way cross. Strategies to maximize cumulative discounted response in crossbred performance over ten generations were derived by optimizing weights in an index of a QTL and phenotype. Strategies were compared to selection on purebred phenotype. Extra responses were limited for QTL with additive and partial dominance effects, but substantial for QTL with over-dominance, for which optimal QTL selection resulted in differential selection in male and female lines to increase the frequency of heterozygotes and polygenic responses. For over-dominant QTL, maximization of crossbred performance one generation at a time resulted in similar responses as optimization across all generations and simultaneous optimal selection in a male and female line resulted in greater response than optimal selection within a single line without crossbreeding. Results show that strategic use of information on over-dominant QTL can enhance crossbred performance without crossbred testing.     

Adipose Tissue-Derived Stem Cells Reduce Acute and Chronic Kidney Damage in Mice

Acute and chronic kidney injuries (AKI and CKI) constitute syndromes responsible for a large part of renal failures, and are today still associated with high mortality rates. Given the lack of more effective therapies, there has been intense focus on the use stem cells for organ protective and regenerative effects. Mesenchymal stem cells (MSCs) have shown great potential in the treatment of various diseases of immune character, although there is still debate on its mechanism of action. Thus, for a greater understanding of the role of MSCs, we evaluated the effect of adipose tissue-derived stem cells (AdSCs) in an experimental model of nephrotoxicity induced by folic acid (FA) in FVB mice. AdSC-treated animals displayed kidney functional improvement 24h after therapy, represented by reduced serum urea after FA. These data correlated with cell cycle regulation and immune response modulation via reduced chemokine expression and reduced neutrophil infiltrate. Long-term analyses, 4 weeks after FA, indicated that AdSC treatment reduced kidney fibrosis and chronic inflammation. These were demonstrated by reduced interstitial collagen deposition and tissue chemokine and cytokine expression. Thus, we concluded that AdSC treatment played a protective role in the framework of nephrotoxic injury via modulation of inflammation and cell cycle regulation, resulting in reduced kidney damage and functional improvement, inhibiting organ fibrosis and providing long-term immune regulation.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.     

Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.     

Fibril assembly and carotenoid overaccumulation in chromoplasts: a model for supramolecular lipoprotein structures.

Chromoplast development in ripening bell pepper fruits is characterized by a massive synthesis of carotenoid pigments, resulting in their distinctive red color. We have shown that 95% of these pigments accumulate in chromoplasts in specific lipoprotein fibrils. In addition to carotenoids, purified fibrils contain galactolipids, phospholipids, and a single, 32-kD protein, designated fibrillin, which has antigenically related counterparts in other species. Fibrils were reconstituted in vitro when purified fibrillin was combined with carotenoids and polar lipids in the same stoichiometric ratio found in fibrils in vivo. Antibodies directed against fibrillin were used to isolate a fibrillin cDNA clone and, in immunological studies, to follow its accumulation during the chloroplast-to-chromoplast transition under different conditions. A model for fibril architecture is proposed wherein carotenoids accumulate in the center of the fibrils and are surrounded by a layer of polar lipids, which in turn are surrounded by an outer layer of fibrillin. Topological analysis of purified fibrils verified this structure. Collectively, these results suggest that the process of fibril self-assembly in chromoplasts is an example of a general phenomenon shared among cells that target excess membrane lipids into deposit structures to avoid their destabilizing or toxic effects. In addition, we have shown that abscisic acid stimulates this phenomenon in chromoplasts, whereas gibberellic acid and auxin delay it.     

Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in Capsicum chromoplasts.

In plants, zeta-carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two-step desaturation reaction: phytoene-->phytofluene--> zeta-carotene. Using Capsicum annuum chromoplast membranes and the reconstitution system previously described [Camara, B., Bardat, F. & Monéger, R. (1982) Eur. J. Biochem. 127, 255-258], we have attempted to purify the desaturase(s) catalyzing these reactions. The two activities were coincidental during all the purification procedures. Only a single polypeptide with 56 +/- 2 kDa was detected by SDS/PAGE of all active fractions. The enzyme contained protein-bound FAD. Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein. The antibodies were used to isolate a full-length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide-binding site. Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase. The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening. However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C. annuum fruits.     

Purification and characterization of farnesyl pyrophosphate synthase from Capsicum annuum

Farnesyl pyrophosphate synthase (FPP) displaying dimethylallyl transferase activity (EC 2.5.1.1) and geranyl transferase activity (EC 2.5.1.10) was purified from Capsicum fruits. This prenyltransferase has a molecular mass of 89,000 +/- 5000 Da resulting from the association of two apparently identical subunits having a molecular mass of 43,000 +/- 2000 Da. Antibodies raised against Capsicum FPP synthase selectively blocked the transferase activity. Analysis of the immunological relationships between FPP synthase and geranylgeranyl pyrophosphate synthase (EC 2.5.1.1, EC 2.5.1.10 and EC 2.5.1.30) revealed that these two enzymes though performing the same mechanism of catalysis and accepting identical substrates have different antigenic determinants. Thus, in connection to previous work, this immunological study suggests that Capsicum FPP is strictly located in the extraplastidial compartment.