Development of antigens ABH and Ii in children between 0 to 16 years old]

The ABH and I, i erythrocyte antigens of 926 children from birth to sixteen years were tested in a semi-quantitative assay in order to follow their development. Results show that total activities of A, B and H erythrocyte antigens are reached within 13 and 24 months, the main modifications being observed within the first three months of life. However, these activities depend on the agglutinins used in the test; notably the results with anti-H lectins from Ulex europaeus and Laburnum alpinum are not totally identical. The decrease of i reactivity and the increase of I reactivity are simultaneous, but at birth the I reactivity of cord red cells is widely distributed, while that of i is more homogeneous. The increase in H activity tested with Laburnum alpinum is directly related to the decrease of foetal hemoglobin in the children's red cells.     

Novel functional profiling approach combining reverse phase protein microarrays and human 3‐D ex vivo tissue cultures: Expression of apoptosis‐related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3‐D ex vivo culture technique in combination with reverse‐phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3‐D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.     

A phenotype-driven ENU mutagenesis screen for the identification of dominant mutations involved in alcohol consumption

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of dominant mutations involved in the regulation and modulation of alcohol-drinking behavior. The chemical mutagen ENU was utilized in the generation of 131 male ENU-mutant C57BL/6J mice (G0). These ENU-treated mice were paired with wild-type C57BL/6J mice to generate G1 and subsequent generations. In total, 3327 mice were generated. Starting with G1, mice were screened for voluntary oral self-administration of 10% (v/v) alcohol vs. water in a two-bottle paradigm. From these mice, after a total period of 5 weeks of drinking, 43 mutants fulfilled the criteria of an “alcohol phenotype,” that is, high or low ethanol intake. They were then selected for breeding and tested in a “confirmation cross” (G2–G4) for inheritance. Although we did not establish stable high or low drinking lines, several results were obtained in the context of alcohol consumption. First, female mice drank more alcohol than their male counterparts. Second, the former demonstrated greater infertility. Third, all animals displayed relatively stable alcohol intake, although significantly different in two different laboratories. Finally, seasonal and monthly variability was observed, with the highest alcohol consumption occurring in spring and the lowest in autumn. In conclusion, it seems difficult to identify dominant mutations involved in the modulation or regulation of voluntary alcohol consumption via a phenotype-driven ENU mutagenesis screen. In accordance with the findings from knockout studies, we suggest that mainly recessive mutations contribute to an alcohol-drinking or alcohol-avoiding phenotype.     

Highly potent side-chain to side-chain cyclized enkephalin analogues containing a carbonyl bridge: synthesis, biology and conformation.

Six novel cyclic enkephalin analogues have been synthesized. Cyclization of the linear peptides containing basic amino acid residues in position 2 and 5 was achieved by treatment with bis(4-nitrophenyl)carbonate. It was found that some of the compounds exibit unusually high mu-opioid activity in the guinea pig ileum (GPI) assay. The 18-membered analogue cyclo(N(epsilon),N(beta)-carbonyl-D-Lys2,Dap5)-enkephalinamide turned out to be one of the most potent mu-agonists reported so far. NMR spectra of the peptides were recorded and structural parameters were determined. The conformational space was exhaustively examined for each of them using the electrostatically driven Monte Carlo method. Each peptide was finally described as an ensemble of conformations. A model of the bioactive conformation of this class of opioid peptides was proposed.