Transposition, amplification, and divergence in the origin of the DNF15 loci, a polymorphic repetitive sequence family on chromosomes 1 and 3

The loci DNF15S1 and DNF15S2 are members of a small repetitive sequence family at discrete chromosomal locations, namely, 1p36 and 3p21, respectively. Studies of the structure, arrangement, and interrelations of the family suggest that the single copy on chromosome 3 is the original member and that this gave rise to the several members on chromosome 1 by transposition, partial duplication, and amplification. Several restriction fragment length polymorphisms have been discovered at the DNF15S1 locus and these have been assigned to the different subfamilies of the repeat at this locus. The existence of these RFLPs, and the nonallelic restriction site variation also found in this sequence family, suggests that transposition and amplification occurred as discrete events. We sequenced across the ancient junction between chromosomes 1 and 3 and noted features which might explain the mechanics of the transposition and amplification events.     

Isolation of Glutamyltaurine from Bovine Brains and Proof of Its γ-Linkage by the B/E Linked Scan SIMS Technique

We isolated a glutamyltaurine from bovine brains and determined its structure as gamma-glutamyltaurine (gamma-Glu-Tau; glutaurine) by use of a new mass spectrometric technique [B/E linked scan sputtered ion mass spectrometry (SIMS)], which we have recently shown to be useful for distinguishing the gamma- from the alpha-isomer of glutamyl-dipeptides. Neither the alpha-isomer of glutamyltaurine nor any aspartyltaurines could be detected in bovine brain.     

Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.     

The effect of dehydration on the functional activity of the interrenal gland in adenohypophysectomized Rana catesbeiana frogs. A preliminary report

A significant increase of the content of corticosterone in the blood collected from intravenous cannula or by intracardiac punction has been detected using radioimmunoassay in non-operated and adenohypophysectomized frogs Rana catesbeiana subjected to dehydration in 6.2% mannitol solution during 24 hours. The osmolality of the blood plasma of these animals also increases although less significantly than the growth of plasma corticosterone content. There is a tendency to substantial increase of plasma arginine-vasotocin level prior to the growth of corticosterone level, already after 6 hours of dehydration. Based on the present results and literature data, it is suggested that in adenohypophysectomized frogs lacking endogenous ACTH just the increase of blood arginine-vasotocin level results in a substantial activation of corticosteroid-producing cells of the interrenal gland and in the growth of plasma content of corticosterone.     

The effects of xylazine and alpha-adrenoreceptor antagonists on bovine uterine contractility in vitro

The effects of alpha-adrenoreceptor antagonists prazosin (alpha-1), yohimbine (alpha-2), and idazoxan (alpha-2) on xylazine-induced bovine uterine contractility were tested in vitro. Uterine strips from proestrous/estrous and diestrous cows were mounted in tissue baths containing Tyrode's solution. Changes in uterine contractility were measured by strain gauge. The following results were observed: 1) Xylazine increased uterine contractility in a dose dependent manner (cumulative concentrations: 10(-8), 3x10(-8), 10(-7), 3x10(-7) and 10(-6)M). 2) Idazoxan (10(-8), 10(-7) and 10(-6)M) and yohimbine (10(-6), 10(-5) and 10(-4)M) antagonized uterine contractility induced by xylazine in a dose-dependent manner. Idazoxan was approximately 50 to 100 times more potent than yohimbine. 3) Prazosin (10(-5)M) did not alter the effect of xylazine on uterine contractility. These results suggested that xylazine-induced uterine contractility in the cyclic cow is directly mediated by myometrial alpha-2 adrenoreceptors.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.     

Long-Term Acceptance of Major Histocompatibility Complex-Mismatched Cardiac Allograft Induced by a Low Dose of CTLA4IgM Plus FK506

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2^b) neonatal hearts were transplanted to CBA/J (H-2^k) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.     

Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.