The effect of substance P on the regional lymph node antibody response to antigenic stimulation in capsaicin-pretreated rats

The undecapeptide substance P (SP) contained in primary afferent nerves is thought to mediate that part of the neurogenic inflammatory response consisting of vasodilation and plasma extravasation. This response is diminished in rats pretreated as neonates with the neurotoxin capsaicin. It is not known whether primary afferent nerves influence cellular responses of the immune response to antigenic stimulation. Using 6- to 12-wk-old Sprague-Dawley rats pretreated as neonates with capsaicin, we examined the regional lymph node response to a s.c. antigenic stimulus of sheep red blood cells. The number of cells secreting antigen-specific antibody in these animals was reduced by more than 80% using direct and indirect plaque assay methods. The reduced antibody response in capsaicin-pretreated animals was reversed by a s.c. infusion of SP given over a 4-hr period at the injection site immediately after antigen stimulation. This response had a threshold at approximately 1.0 X 10(-5) M SP. SP1-7 (1.0 X 10(-5) M) was without effect but an infusion of SP5-11 (1.0 X 10(-5) M) reversed the effects of capsaicin treatment indicating a carboxyl-terminal effect of SP. The results suggest that the reduced response of capsaicin-treated animals to an antigenic stimulus is due to an effect of capsaicin on the SP-containing primary afferent nerves rather than a toxic effect of capsaicin on the immune system.     

High-resolution 1H NMR study of the solution structure of alamethicin

A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.     

Nature of the thymocytes associated with dendritic cells and macrophages in thymic rosettes

Thymic rosettes, structures consisting of 3-30 thymic lymphoid cells attached to a central macrophage or dendritic cell, were released from mouse thymus tissue by collagenase digestion. They were shown to be preexistent structures within the thymus, but to be subject to extensive exchange with free thymocytes under certain conditions. An isolation procedure was developed, using a new technique of zonal unit-gravity elutriation, which minimized exchange and produced a completely pure sample of the larger rosettes. The rosette-associated thymocytes were analyzed by two- and three-color immunofluorescent staining and flow cytometry. The dominant cell type was a small, CD4+CD8+, cortical-type thymocyte. However, all of the established thymus subpopulations defined by CD4 and CD8, including CD4-CD8+ and CD4+CD8- mature thymocytes and CD4-CD8- early thymocytes, were also present in rosettes. Very few of the cells present were of an intermediate or transitional phenotype. Rosette-associated thymocytes were somewhat enriched in large dividing thymocytes, in CD4-CD8- thymocytes, and in mature thymocytes expressing the T-cell antigen receptor-CD3 complex. Their most striking characteristic was a marked depletion in small thymocytes lacking surface H-2K expression, a major population among free thymocytes. The physiological role of the rosette structure is discussed, and it is suggested that the heterogeneity of the associated thymocytes in part reflects the existence of different types of rosettes in different areas of the thymus.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

RESPONSE OF ANTARCTIC FUR SEALS TO IMMOBILIZATION WITH KETAMINE, A KETAMINE-DIAZEPAM OR KETAMINE-XYLAZINE MIXTURE, AND ZOLETIL®

Adult Antarctic fur seals (Arctocephalus gazella) were immobilized with Zoletil^® ( n = 172), ketamine ( n = 30), ketamine mixed with diazepam ( n = 23) and with ketamine mixed with xylazine ( n = 45). Response to all drugs was highly variable. There was a relationship between dose rate and level of immobilization in females given Zoletil^®. Males were slightly more sensitive to Zoletil^® than females but this could have been due to the greater body mass and lower mass-specific metabolic rate of males. The dose required to achieve a level of immobilization declined with greater body mass for Zoletil^® and ketamine but not for ketamine-diatepam. Ketamine and ketamine-sedative mixtures commonly caused mild tremoring and occasionally caused convulsions. Neither reaction was seen with Zoletil^®. Mean doses were, Zoletil^® 1.5 mg/ kg, ketamine 6.9 mg/kg, ketamine-diazepam 6.3 mg/kg ketamine and 6.3 μg/kg diazepam, and ketamine-xylazine 7.3 mg/kg ketamine and 0.62 mg/ kg xylazine. Zoletil^® performs at least as well on Antarctic fur seals as ketamine but it may cause respiratory depression. The dose of ketamine required for Antarctic fur seals was greater than for most other species of seals.     

Siblings with chromosome mosaicism,microcephaly, and growth retardation: the phenotypic expression of a human mitotic mutant?

Summary We report male and female siblings with extreme microcephaly and mental retardation, growth retardation, and multiple chromosome mosaicism. Mental retardation associated with chromosome mosaicism does not always carry a low recurrence risk.     

Characterization of the sulfonylurea receptor on beta cell membranes

Specific, high affinity sulfonylurea receptors were characterized on membranes of an insulin-secreting hamster beta cell line (HIT cells). Saturable binding of the sulfonylurea, [3H]glyburide, was linear up to 0.8 mg/ml membrane protein. Scatchard analysis of equilibrium binding data at room temperature indicated the presence of a single class of saturable, high affinity binding sites with a Kd of 0.76 +/- 0.04 nM and a Bmax of 1.09 +/- 0.13 pmol/mg protein, n = 9. The insulin secretory potency of glyburide, glipizide, tolbutamide, tolazamide, and carboxytolbutamide was compared to the ability of these ligands to displace [3H]glyburide from the sulfonylurea receptor. Tolbutamide, tolazamide, and glipizide demonstrated reasonable agreement with ED50 values of 15 microM, 3 microM, and 30 nM and Ki values of 25.3 microM, 7.2 microM, and 45 nM, respectively. The inactive tolbutamide metabolite, carboxytolbutamide, at the highest concentration tested, only partially displaced [3H]glyburide from the receptor and was a very poor secretagogue. At 37 degrees C the affinity of [3H]glyburide binding, Kd = 2.0 nM, was similar to the ED50 of 5.5 nM when the free glyburide concentrations were corrected for binding of the drug to albumin. These studies suggest that sulfonylureas initiate their biologic effect through a high affinity, specific interaction with sulfonylurea receptors on the beta cell membrane.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

P transposition in Drosophila provides a new tool for analyzing postreplication repair and double-strand break repair.

A genetic screen has been developed in Drosophila for identifying host-repair genes responsible for processing DNA lesions formed during mobilization of P transposable elements. Application of that approach to repair deficient mutants has revealed that the mei-41 and mus302 genes are necessary for recovery of P-bearing chromosomes undergoing transposition. Both of these genes are required for normal postreplication repair. Mutants deficient in excision repair, on the other hand, have no detected effect on the repair of transposition-induced lesions. These observations suggest that P element-induced lesions are repaired by a postreplication pathway of DNA repair. The data further support recent studies implicating double-strand DNA breaks as intermediates in P transposition, because the mei-41 gene has been genetically and cytologically associated with the repair of interrupted chromosomes. Analysis of this system has also revealed a striking stimulation of site-specific gene conversion and recombination by P transposition. This result strongly suggests that postreplication repair in this model eukaryote operates through a conversion/recombination mechanism. Our results also support a recently developed model for a conversion-like mechanism of P transposition (Engels et al., 1990). Involvement of the mei-41 and mus302 genes in the repair of P element-induced double-strand breaks and postreplication repair points to a commonality in the mechanisms of these processes.