Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones.

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.

Corpuscles of Stannius and stanniocalcin-like immunoreactivity in the white sucker (Catostomus commersoni): evidence for a new cell-type.

The distribution of stanniocalcin immunoreactivity was examined in the corpuscles of Stannius of the white sucker (Catostomus commersoni) by using a chum salmon stanniocalcin antiserum, Western blotting, and light and electron microscopy. The white sucker possesses at least two stanniocalcin-immunoreactive corpuscles in the most posterior portions of the kidneys. Immunocytochemistry and ultrastructure revealed two cell-types in the corpuscle parenchyma, only one of which was immunoreactive. The nonimmunoreactive cells contained dense-cored vesicles and long processes that extended between the immunoreactive cells and terminated at perivascular spaces. When corpuscle extracts were subjected to electrophoresis and Western blotting, three nonreduced stanniocalcin-like immunoreactive bands (approximately 56, 61, and 64 kDa) were observed. However, in the presence of a reductant, a diffuse band migrating in the range of 28 to 32 kDa was noted. The results of this study on the white sucker demonstrate the presence of a dimeric stanniocalcin-like molecule and present evidence of a previously uncharacterized cell-type in the corpuscles of Stannius.

Deciphering posttranslational processing events in the pituitary of a neopterygian fish: cloning of a gar proopiomelanocortin cDNA.

A cDNA that codes for the polypeptide hormone precursor proopiomelanocortin (POMC) was cloned and sequenced from a gar (Lepisosteus osseus) pituitary cDNA library. The gar POMC cDNA is 1237 bp and contains a 780-bp open reading frame. The deduced amino acid sequence for gar POMC is 259 amino acids in length. The general organization of gar POMC is very similar to that of other gnathostome POMC sequences. The beta-endorphin sequence had 91% sequence identity with sockeye A beta-endorphin and 71% sequence identity with Xenopus laevis beta-endorphin. Three melanocyte-stimulating hormone (MSH) core sequences [HFR(W)] were detected. The gar alpha-MSH sequence was identical to the alpha-MSH sequence in rat POMC. The gar beta-MSH sequence had 77% sequence identity with salmonid forms of beta-MSH and 53% sequence identity with tetrapod forms of beta-MSH. The gamma-MSH region of gar POMC only had 26% primary sequence identity with tetrapod gamma-MSH sequences. Gar gamma-MSH had an incomplete MSH core sequence (HRF), an apparent internal deletion of five amino acids, and lacked flanking paired basic amino acids essential for proteolytic cleavage. The apparent degenerate nature of gar gamma-MSH is discussed in light of the absence of this sequence in salmonid fish.

Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure.

A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3' untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3' untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes-gar and bowfin; one chondrostean fish-the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.

The corpuscles of Stannius in arawana (Osteoglossum bicirrhosum), an ancient teleost.

The corpuscles of Stannius of arawana (Osteoglossum bicirrhosum), an ancient teleost, were examined by routine light and electron microscopy and following their immunoreactivity to salmon and trout stanniocalcin antisera. Periodic acid-Schiff positive cells of the corpuscles of Stannius had a follicular arrangement and demonstrated a strong immunohistochemical reaction with both stanniocalcin antisera. Fine structural analysis of the paired, posteriorly located, and perirenal ovoid glands revealed two morphologically distinct cell types the basal laminae of which were ramified by nerve terminals. Immunocytochemistry demonstrated that osmiophilic secretory granules in both cell types were immunoreactive to the stanniocalcin antisera. When extracts of arawana corpuscles of Stannius were subjected to sodium dodecyl sulphate electrophoresis and Western blot analysis a diffuse molecular weight band was evident ( approximately 68 kDa) in the non-reduced condition. In all cases, immunoreactivity was abolished by preabsorption of the antisera with salmon stanniocalcin or with a crude extract of arawana corpuscles of Stannius. The corpuscles of Stannius of arawana are similar to those in more recent teleosts with respect to cell structure and their anatomical distribution but their stanniocalcin is more similar in molecular weight to that present in at least one other non-teleost actinopterygian (the gar) which has an ancient lineage.

Isolation and biological activity of [Trp5]bradykinin from the plasma of the phylogenetically ancient fish, the bowfin and the longnosed gar.

The holostean fish occupy an important position in vertebrate phylogeny as extant representatives of a ancient group of ray-finned fish with evolutionary connections to present-day teleosts. Incubation of heat-denatured plasma from the bowfin Amia calva with trypsin generated bradykinin-like immunoreactivity. The primary structure of bowfin bradykinin was established as Ala-Pro-Pro-Gly-Trp-Ser-Pro-Phe-Arg. This amino acid sequence contains one amino acid substitution (Phe5 --> Trp) compared with mammalian bradykinin. The same peptide was generated in heat-denatured plasma from the longnosed gar Lepisosteus osseus. Treatment of plasma from either the bowfin or gar with glass beads under conditions previously shown to activate Factor XII in the plasma of mammals and reptiles did not generate bradykinin. Bolus injections of synthetic bowfin bradykinin (0.1, 0.3, and 1.0 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in an immediate fall in arterial blood pressure of 5-10 min duration that was followed by a dose-dependent rise in pressure that was sustained for 30-60 min. There was no change in heart rate following bradykinin administration. The data suggest that the kallikrein-kinin system may predate the appearance of teleosts and may play a role in cardiovascular regulation in holosteans.

Detection of N-acetylated forms of alpha-MSH and beta-endorphin in the intermediate pituitary of the holostean fishes, Lepisosteus spatula, Lepisosteus osseus, and Amia calva.

Acid extracts of the intermediate pituitaries of the gars, L. spatula and L. osseus, were fractionated by Sephadex G-50 column chromatography and analyzed by radioimmunoassay. This procedure revealed that immunoreactive forms of N-acetylated beta-endorphin- and alpha-MSH-sized material were present in equimolar amounts and represented the major end products of the POMC biosynthetic pathway in these species. Cation-exchange chromatography indicated that multiple N-acetylated forms of beta-endorphin were present in the intermediate pituitaries of the two species of gar, and that these forms differed in their net positive charge and in their apparent molecular weight. Reversed-phase HPLC analysis of the alpha-MSH-related material indicated that up to 90% of the total MSH in the pituitary of the gar was N-acetylated. Furthermore, the predominant form of alpha-MSH in both species of gar was N,O-diacetyl-ACTH(1-13)-NH2. Nearly identical results were obtained following the analysis of alpha-MSH-related peptides in the intermediate pituitary of the bowfin, A. calva. The pattern of posttranslational processing of POMC observed in the intermediate pituitaries of holostean fishes is very similar to the processing events observed in lungfishes, turtles, and mammals; hence, the processing of POMC has been remarkably conserved during vertebrate evolution.