cDNA sequence and chromosome localization of pig α1,3 galactosyltransferase

Human serum contains natural antibodies (NAb), which can bind to endothelial cell surface antigens of other mammals. This is believed to be the major initiating event in the process of hyperacute rejection of pig to primate xenografts. Recent work has implicated galoctosyl 1,3 galactosyl 1,4 N-acetyl-glucosaminyl carbohydrate epitopes, on the surface of pig endothelial cells as a major target of human natural antibodies. This epitope is made by a specific galactosyltransferase (1,3 GT) present in pigs but not in higher primates. We have now cloned and sequenced a full-length pig 1,3 GT cDNA. The predicted 371 amino acid protein sequence shares 85% and 76% identity with previously characterized cattle and mouse 1,3 GT protein sequences, respectively. By using fluorescence and isotopic in situ hybridization, the GGTA1 gene was mapped to the region q2.10–q2.11 of pig chromosome 1, providing further evidence of homology between the subterminal region of pig chromosome 1q and human chromosome 9q, which harbors the locus encoding the AB0 blood group system, as well as a human pseudogene homologous to the pig GGTA1 gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L36152     

Concerted evolution in a segment of the first domain exon of polymorphic MHC class II β loci

Genetic exchange of sequence information between members of a gene family, generally denoted gene conversion, causes a phenomenon called concerted evolution meaning that non-allelic genes do not evolve independently. The possible significance of this phenomenon in the evolution of major histocompatibility complex (MHC) class II genes has been investigated in the present study. The results of a phylogenetic analysis of human, mouse, bovine, and chicken class II sequences were consistent with the occurrence of gene conversion between polymorphic class II genes (i. e. DPB, DQB, and DRB) but not between these genes and the monomorphic DOB gene or between class II genes. Gene conversion between polymorphic genes appears to be restricted to a gene segment between approximately nucleotide positions 94–286 in the first domain exon. Due to this genetic exchange, there is a greater interlocus similarity both at the DNA and protein level in this region than in the rest of the sequence. The region encodes a functionally important part of the class II molecule including more than half of the -chain residues of the antigen binding site and the residues in the helix assumed to form contact with the T-cell receptor. The observed similarity in the -helical region of class II molecules may be functionally significant for the utilization of the T-cell repertoire for antigen recognition in the immune system.     

Structural basis for feedback inhibition of the deoxyribonucleoside salvage pathway: studies of the Drosophila deoxyribonucleoside kinase

Deoxyribonucleoside kinases are feedback inhibited by the final products of the salvage pathway, the deoxyribonucleoside triphosphates. In the present study, the mechanism of feedback inhibition is presented based on the crystal structure of a complex between the fruit fly deoxyribonucleoside kinase and its feedback inhibitor deoxythymidine triphosphate. The inhibitor was found to be bound as a bisubstrate inhibitor with its nucleoside part in the nucleoside binding site and with its phosphate groups partially occupying the phosphate donor site. The overall structure of the enzyme--inhibitor complex is very similar to the enzyme--substrate complexes with deoxythymidine and deoxycytidine, except for a conformational change within a region otherwise directly involved in catalysis. This conformational change involves a magnesium ion, which is coordinated in the inhibitor complex to the phosphates and to the primary base, Glu52, that normally is positioned close to the 5'-OH of the substrate deoxyribose.     

Class II genes of miniature swine

Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.     

Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard,bicarbonate-rich freshwater

Summary Carbonic anhydrase (CA) activities in gills and venous blood, acid-base balance, and haematological variables were studied during environmental hypercapnia in rainbow trout (Salmo gairdneri). Batches of 8–10 fish were exposed to about 3 or 13 mmHg in flow-through tests of various duration from 4 h to 80 days.After initial acidosis, blood pH rose above pre-experimental values. At 3 mmHg it became normal again within 21 days, while at 13 mmHg the overshoot lasted for 80 days. In fish acclimated for 3 weeks or more to 13 mmHg , blood HCO ₃ ^– increased four to five times while plasma Cl^– levels were lower and K⁺ higher. Na⁺ levels did not show any consistent trend associated with exposure to hypercapnia. After an initial acidaemia, Hct, Hb, and RBC remained relatively constant.Patterns of change in CA activity differed between gills and erythrocytes. Initially, blood CA decreased at both levels. It then began rising after about 3 weeks and tended to reach pre-experimental values by 80 day's hypercapnia. At 13 mmHg , gill CA increased to twice the pre-experimental level. Compared with blood CA, gill CA appeared to be more specifically involved in fish acclimation to hypercapnia, which demands an increase in blood bicarbonate to provide a sufficient buffering capacity. Increased CA indicates that the gill enzyme may play a more important role than blood CA in acid-base regulation in fish during hypercapnia.Abbreviations CA carbonic anhydrase - Hb haemoglobin - Hct haematocrit value - RBC red blood cells     

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.     

Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.     

Stage-specific apoptosis of male germ cells in the rat: mechanisms of cell death studied by supravital squash preparations

Apoptosis has been proposed as a mechanism by which testis germ cells are removed during normal and various pathological conditions. To establish a new rapid way to detect stage-specific apoptosis in male rat germ cells, their supravital morphology was examined from carefully squashed monolayers of living cells, after several established toxic treatments, using a phase contrast microscope. The results were compared with early detection of apoptosis using annexin V and propidium iodide (PI) stainings. The apoptosis of type-A spermatogonia and round spermatids proceeded in a similar way to somatic cells, while intermediate and type-B spermatogonia, and particularly the dividing spermatocytes, possessed characteristics not entirely typical for apoptosis. Death of elongated spermatids was difficult to assess owing to their compacted chromatin. As the first phases of degeneration seemed different in various germ cell classes, the final stage (karyopycnosis) was similar for most cells. Degenerating cells also showed positive reactions for annexin V and PI. The 'living cell method' provides rapid and accurate possibilities for analysis of stage-specific apoptosis during spermatogenesis. This method is not influenced by artefacts induced by fixation, embedding and sectioning. It may be developed further for routine analyses of the accurate stage-specific effects of various physical and chemical effects on mammalian and human spermatogenesis.