N.m.r. studies of metabolism in perfused organs

Several metabolites and intracellular pH in intact organs can be studied in a non-destructive manner by phorphorus nuclear magnetic resonance (31P n.m.r.). This possibility was demonstrated by us nearly five years ago. Since then we have developed the appropriate physiological techniques and improved the n.m.r. method for the study of animal hearts and kidneys. Here we described measurements aimed at clarifying three problesm. (1) Having measured the enzyme-catalysed fluxes between phosphocreatine and ATP by the method of saturation transfer n.m.r., we examine the relations between energy supply and heart rate in the isolated perfused rat heart. (2) We describe experiments to establish the validity of the perfusion model. For the first time, we report 31P n.m.r. measurements of an in vivo rat heart and compare the results with those obtained for the perfused rat heart. (3) Ischaemia and metabolism in rabbit kidneys is investigated to establish the relation between functional and metabolic recovery after a renal transplant operation.     

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.     

Cervical lymphadenitis in guinea pigs: infection via intact ocular and nasal mucosa by Streptococcus zooepidemicus.

The traditional view regarding the pathogenesis of cervical lymphadenitis in guinea pigs is that Lancefield Group C Streptococcus gains access to cervical lymph nodes via an abraded oral mucosa. In this study, it is established that inoculation of intact nasal and conjunctival mucous membranes with Streptococcus zooepidemicus (Lancefield Group C) also can produce the disease. Weanling (SPF) guinea pigs (Cavia porcellus) were divided into two experimental groups of 10 and two control groups of four each. Guinea pigs from each group were individually housed in separate cubicles. Group I was inoculated with 0.05 ml of culture containing 2.8 x 10(7) CFU/ml of S. zooepidemicus into the conjunctiva of the left eye. Group II received a similar inoculum into the left nares. Control groups received 0.05 ml of TSB broth in the same sites. Five of ten guinea pigs in Group II died four to nine days postinoculation. Surviving guinea pigs were euthanatized at intervals between days 4-13 postinoculation. All guinea pigs were necropsied, cultured and examined for evidence of infection. S. zooepidemicus was recovered from 30/50 and 39/46 sites cultured from Groups I and II, respectively. Lymphadenitis was found in cervical lymph nodes from 8/10 guinea pigs in Group I and 10/10 in Group II. The conjunctival and nasal mucosa, therefore, represent potential sites of entry resulting in cervical lymphadenitis in guinea pigs.     

Fate of Surviving Low-birth-weight Infants with Coagulation Deficiencies on the First Day of Life

Fifty-two surviving low-birth-weight infants who had low Thrombotest (Owren) results on the first day, together with the same number of matched controls with higher Thrombotest results, were examined for the integrity of their central nervous system. Gross abnormalities were found in 13·5% of the low Thrombotest group compared with 1·9% in the higher group. Minor brain damage syndromes were more common in the low Thrombotest group. The combined brain damage syndromes were 23·1% in the low Thrombotest group compared with 3·8% in the higher group.It is suggested that the causes of the brain damage in the low Thrombotest group are either non-fatal cerebral haemorrhage or intravascular fibrin deposition associated with disseminated intravascular coagulation.     

Cytotoxic potential of ribonuclease and ribonuclease hybrid proteins

Pancreatic RNase injected into Xenopus oocytes abolishes protein synthesis at concentrations comparable to the toxin ricin yet has no effect on oocyte protein synthesis when added to the extracellular medium. Therefore RNase behaves like a potent toxin when directed into a cell. To explore the cytotoxic potential of RNase toward mammalian cells, bovine pancreatic ribonuclease A was coupled via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M whereas greater than 10(-5) M native RNase was required to inhibit protein synthesis. Cytotoxicity requires both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Compounds that interfere with transferrin receptor cycling and compartmentalization such as ammonium chloride decreased the cytotoxicity of transferrin-RNase. After a dose-dependent lag period inactivation of protein synthesis by transferrin-RNase followed a first-order decay constant. In a clonogenic assay that measures the extent of cell death 1 x 10(-6) M transferrin-RNase killed at least 4 logs or 99.99% of the cells whereas 70 x 10(-6) M RNase was nontoxic. These results show that RNase coupled to a ligand can be cytotoxic. Human ribonucleases coupled to antibodies also may exhibit receptor-mediated toxicities providing a new approach to selective cell killing possibly with less systemic toxicity and importantly less immunogenicity than the currently employed ligand-toxin conjugates.     

An Excess of Deleterious Variants in VEGF-A Pathway Genes in Down-Syndrome-Associated Atrioventricular Septal Defects

About half of people with trisomy 21 have a congenital heart defect (CHD), whereas the remainder have a structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is not causal for abnormal heart development. Atrioventricular septal defects (AVSD) are the most commonly occurring heart defects in Down syndrome (DS), and ∼65% of all AVSD is associated with DS. We used a candidate-gene approach among individuals with DS and complete AVSD (cases = 141) and DS with no CHD (controls = 141) to determine whether rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis contribute to AVSD in this sensitized population. We found a significant excess (p < 0.0001) of variants predicted to be deleterious in cases compared to controls. At the most stringent level of filtering, we found potentially damaging variants in nearly 20% of cases but fewer than 3% of controls. The variants with the highest probability of being damaging in cases only were found in six genes: COL6A1, COL6A2, CRELD1, FBLN2, FRZB, and GATA5. Several of the case-specific variants were recurrent in unrelated individuals, occurring in 10% of cases studied. No variants with an equal probability of being damaging were found in controls, demonstrating a highly specific association with AVSD. Of note, all of these genes are in the VEGF-A pathway, even though the candidate genes analyzed in this study represented numerous biochemical and developmental pathways, suggesting that rare variants in the VEGF-A pathway might contribute to the genetic underpinnings of AVSD in humans.     

A study on the minimum number of loci required for genetic evaluation using a finite locus model

For a finite locus model, Markov chain Monte Carlo (MCMC) methods can be used to estimate the conditional mean of genotypic values given phenotypes, which is also known as the best predictor (BP). When computationally feasible, this type of genetic prediction provides an elegant solution to the problem of genetic evaluation under non-additive inheritance, especially for crossbred data. Successful application of MCMC methods for genetic evaluation using finite locus models depends, among other factors, on the number of loci assumed in the model. The effect of the assumed number of loci on evaluations obtained by BP was investigated using data simulated with about 100 loci. For several small pedigrees, genetic evaluations obtained by best linear prediction (BLP) were compared to genetic evaluations obtained by BP. For BLP evaluation, used here as the standard of comparison, only the first and second moments of the joint distribution of the genotypic and phenotypic values must be known. These moments were calculated from the gene frequencies and genotypic effects used in the simulation model. BP evaluation requires the complete distribution to be known. For each model used for BP evaluation, the gene frequencies and genotypic effects, which completely specify the required distribution, were derived such that the genotypic mean, the additive variance, and the dominance variance were the same as in the simulation model. For lowly heritable traits, evaluations obtained by BP under models with up to three loci closely matched the evaluations obtained by BLP for both purebred and crossbred data. For highly heritable traits, models with up to six loci were needed to match the evaluations obtained by BLP.