Ribosomal DNA as a convenient probe to follow segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process

Summary Restriction fragment length polymorphism of the wheat nuclear ribosomal DNA has been studied in several steps of a breeding scheme, including parental genotypes, F1 hybrid, F9 generation, and anther-derived doubled haploid lines obtained from F9. Ribosomal DNA represents a suitable molecular marker in following segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process. It has been shown to undergo variations among the first cycle-doubled haploid lines in the relative amount of two different sizes of ribosomal DNA repeat units. The specificity and peculiar properties of the plant system used allowed us to assign an intrachromosomal location (short arm of the chromosomes 1B, 1R or 6B) to several ribosomal DNA repeat units that differ by the length of their nontranscribed spacer region.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Oxidative stress induced by corticosterone administration in broiler chickens (Gallus gallus domesticus) 1. Chronic exposure

Two trials were conducted to evaluate the effects of short-term administration of corticosterone (CORT) on the induction of oxidative injury in broiler chickens (Gallus gallus domesticus). Twelve broiler chickens of 30 and of 40 days of age were respectively employed in Trial 1 and 2. Half of the chickens were administered subcutaneously with CORT (4 mg/kg body weight [BW] in corn oil), while another half served as controls (corn oil) in each trail. In Trial 1, a blood sample was obtained from each chicken immediately before administration and at 1 and 3 h after injection. In Trial 2, the liver and heart were obtained after 3 h of CORT exposure. Short-term administration of CORT resulted in enhanced proteolysis and gluconeogenesis. There were no obvious changes in lipid peroxidation status of the heart and liver, whereas a decrease in lipid peroxidation in the plasma was observed after acute CORT exposure. The significantly increased plasma nonenzymatic antioxidants (uric acid [UA] and total antioxidant capacity) in concert with the enhanced enzymatic antioxidant activity (SOD in heart) during short-term CORT administration indicate preventive changes to counteract the oxidative injury, and these may be tissue specific.     

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.     

Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.     

Thyroid status,but not insulin status,affects expression of avian uncoupling protein mRNA in chicken

The aim of this study was to investigate the hormonal regulation of the avian homolog of mammalian uncoupling protein (avUCP) by studying the impact of thyroid hormones and insulin on avUCP mRNA expression in chickens (Gallus gallus). For 3 wk, chicks received either a standard diet (control group), or a standard diet supplemented with triiodothyronine (T(3); T3 group) or with the thyroid gland inhibitor methimazole (MMI group). A fourth group received injections of the deiodinase inhibitor iopanoic acid (IOP group). During the 4th wk of age, all animals received two daily injections of either human insulin or saline solution. The results indicate a twofold overexpression of avUCP mRNA in gastrocnemius muscle of T3 birds and a clear downregulation (-74%) in MMI chickens compared with control chickens. Insulin injections had no significant effect on avUCP mRNA expression in chickens. This study describes for the first time induction of avUCP mRNA expression by the thermogenic hormone T(3) in chickens and supports a possible involvement of avUCP in avian thermogenesis.     

Assessment of age-related glucose oxidation rates of broiler chickens by using stable isotopes

During their relatively short commercial lifespan of six weeks, broiler chickens undergo very pronounced age- or body weight-related changes in metabolic rate and body composition. The present study was aimed to assess the age-related changes in glucose oxidation rate of broiler chickens by using 13C-labeled glucose. The methodology for this breath test needed to be established first. Broiler chickens aged from two to six weeks were placed in open-circuit respiration cells and received a single intubation of U-13C6-glucose, followed by breath sampling for 4 hours and mass spectrometric analysis of 13C: 12C ratio in the exhaled air. Simultaneously, CO2 concentration in the respiration cell air was continuously monitored in order to calculate the cumulative percentage dose recovery (CPDR). With respect to the methodology, an oral dose of 2 mg U-13C6-glucose per kg body weight while maintaining a CO2 in the concentration of 0.4 to 0.5% was considered to be optimal. The three-parameter Gompertz curve fitted the CPDR values very well. Pronounced age-related changes in exogenous glucose oxidation rates in rapidly growing meat-type chickens were assessed. Young broiler chickens spend only a relatively low percentage of ingested glucose for immediate oxidation. In contrast, broiler chickens approaching the age of maximal absolute growth rate oxidize a greater proportion of the recently ingested glucose relative to the non-oxidative disposal pathways. This shift in the exogenous partitioning is discussed in relation to age-dependent changes in glucose turnover, lipid oxidation and deposition and metabolic heat production.     

Impaired development of broiler chickens by stress mimicked by corticosterone exposure

The effects of corticosterone (CORT) administration on the development of muscular tissues of broiler chickens (Gallus gallus domesticus) fed with diets differing in lipid content were investigated. The experimental chickens were given one of two experimental diets: high lipid diet (9.9% crude fat) or control diet, from 21 d of age. At 28 d of age, half of the chickens in each dietary treatment were exposed to CORT treatment, supplemented with 30 mg CORT/kg diet for 12 days, while the other half continued to consume the former diet. The zootechnical parameters were recorded at 21, 28, 35 and 39 d, and a blood sample was obtained from 8 birds of each group, respectively. The growth performance of broiler chickens was significantly depressed by CORT administration, but not by dietary treatment. Corticosterone treatment resulted in enhanced energy expenditure. The results indicate that the development of breast muscle was more susceptible to stress mimicked by CORT administration. The results suggest that corticosterone administration enhanced hepatic fatty acid synthesis and resulted in the redistribution of energy to abdominal store from peripheral tissues. Diet rich in lipid content was favorable to the central fat deposit in stressed broiler chickens.