Evolution of the apolipoproteins. Structure of the rat apo-A-IV gene and its relationship to the human genes for apo-A-I, C-III, and E

We have determined the nucleotide sequence of the rat apolipoprotein (apo-) A-IV gene and analyzed its structural and evolutionary relationships to the human apolipoprotein A-I, E, and C-III genes. The rat A-IV gene is 2.4 kilobases in size and consists of three exons (142, 126, and 1157 base pairs) interrupted by two introns (277 and 673 base pairs). The 5'-nontranslated region and most of the signal peptide are encoded by the first exon. Thus, the apo-A-IV gene lacks an intron in the 5'-nontranslated region of its mRNA in contrast to all other known apolipoprotein genes. Sequences coding for amphipathic docosapeptides span both the second and third exons of the rat A-IV gene. We demonstrate that this is also true for the human apolipoprotein genes. This gene family seems to have evolved by the duplication of an ancestral minigene that resulted in the formation of two exons. Thereafter, evolution of these sequences was dominated by intraexonic amplification of repeating units coding for amphipathic peptides. Sequence divergence of these repeats resulted in the functional differentiation of the apolipoproteins. However, conservation of the fundamental amphipathic pattern allowed members of this protein family to retain their lipid-binding properties.     

Immunoaffinity isolation of Na+ channels from rat skeletal muscle. Analysis of subunits

Polyclonal antiserum and monoclonal antibodies raised against the sodium channel from rat skeletal muscle sarcolemma have been immobilized on Sepharose and used to immunoaffinity purify this channel directly from skeletal muscle without the intervening purification of surface membranes. These antibodies isolate a approximately 260-kDa protein from whole muscle, although each purifies predominantly a 150-kDa component when isolated sarcolemmal membranes are used as starting material. A 45-kDa band is also found in the material purified from sarcolemma but not that obtained from whole muscle. In addition, these immunoaffinity columns isolate a 38-kDa band from both whole muscle and sarcolemma that copurifies with the 260-kDa protein. In some preparations this component appears as two closely spaced bands of 37 and 39 kDa. These small subunits coelute with the 260-kDa subunit when thiocyanate gradients are used to displace protein bound to the immunoaffinity columns and behave as integral components of the sodium channel. Estimates of stoichiometry were made for the large and small subunits of the muscle channel protein. After correction for labeling efficiency, values consistent with a ratio of one 260-kDa subunit to one 38-kDa subunit were obtained. We conclude that the rat skeletal muscle sodium channel contains a large alpha subunit of approximately 260 kDa that is sensitive to proteolytic nicking during the isolation of sarcolemmal membranes. In addition, at least one 38-kDa beta subunit is associated with each alpha subunit in the native channel.     

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Granulocyte-mediated airway edema in guinea pigs

To determine the role of polymorphonuclear leukocytes (PMNs) in the airway edema that accompanies airway inflammation, we studied the effects of a 1-h exposure to 2 ppm toluene diisocyanate (TDI) on tracheal extravasation of Evans blue dye and on the concentration of PMNs in the tracheal wall. Tracheal Evans blue content was significantly increased by TDI exposure (53.6 +/- 8.0 micrograms/g tracheal tissue (mean +/- SE) for animals exposed to TDI and 16.3 +/- 2.0 for animals exposed to air, P less than 0.0025) as were both the intravascular and extravascular concentration of PMNs in tracheal sections (intravascular PMNs were 28.0 +/- 8.4 X 10(3) cells/mm3 for TDI and 1.5 +/- 1.5 X 10(3) for air, P less than 0.025, extravascular PMNs were 10.9 +/- 4.5 X 10(3) for TDI and 0 for air, P less than 0.05). PMN depletion with vinblastine or with hydroxyurea abolished both the increase in tracheal Evans blue extravasation and the increase in the concentration of intravascular and extravascular PMNs in animals exposed to TDI. PMN depletion with hydroxyurea did not significantly inhibit the increase in tracheal Evans blue extravasation caused by intravenous histamine. Administration of donor PMNs to animals depleted of PMNs with hydroxyurea reconstituted the TDI-induced increase in tracheal Evans blue extravasation (80.4 +/- 17.3 micrograms/g tissue (mean +/- SE) in animals exposed to TDI vs. 21.3 +/- 2.9 in animals exposed to air, P less than 0.025) and in the intravascular concentration of PMNs in tracheal sections [18.5 +/- 3.4 X 10(3) cells/mm3 (mean +/- SE) in animals exposed to TDI vs. 1.3 +/- 1.3 X 10(3) in animals exposed to air, P less than 0.0025].(ABSTRACT TRUNCATED AT 250 WORDS)     

Analyzing the structures, functions and evolution of two abundant gastrointestinal fatty acid binding proteins with recombinant DNA and computational techniques

The structures of intestinal and liver fatty acid binding proteins (FABPs) have been determined from an analysis of the nucleotide sequences of cloned cDNAs. The primary translation product of intestinal FABP mRNA contains 132 residues (Mr = 15 124). Liver FABP mRNA encodes a 127 amino acid polypeptide (Mr = 14 273). In vitro co-translational cleavage and translocation assays showed that neither sequence has a cleavable signal peptide or signal peptide equivalent - suggesting that the FABPs do not enter the secretory apparatus but rather are targeted to the cytoplasm. A variety of computational techniques were used to compare the two FABP sequences. The results indicate that liver and intestinal FABP are paralogous homologues. A superfamily of proteins was defined which includes the FABPs, the cellular retinol and retinoic acid binding proteins, the P2 protein of peripheral nerve myelin, and a polypeptide known as 422 whose synthesis is induced during differentiation of 3T3-L1 cells to adipocytes. No sequence homologies were noted between any of these small molecular weight cytosolic proteins and nonspecific lipid transfer protein (sterol carrier protein 2), phosphatidylcholine transfer protein, serum albumin or apolipoprotein AI. The FABPs may have structural features responsible for lipid-protein interactions that are not present in these non-homologous sequences. The distribution of intestinal and liver FABP mRNAs in adult rat tissues and the changes in FABP gene expression which occur during gastrointestinal development support the notion that these proteins are involved in fatty acid uptake, transport and/or compartmentalization. However, differences in tissue distribution and periods of non-coordinate expression during gastrointestinal ontogeny suggest that the two FABPs have distinct functions. The relationship between intestinal and liver FABPs and similar sized cytosolic FABPs isolated from brain, skeletal and cardiac muscle remains unclear. Recombinant DNA techniques combined with comparative sequence analyses offer a useful approach for defining unique as well as general structure-function relationships in this group of fatty acid binding proteins.     

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.     

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 ml-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and L-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5-120 microM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E2 and prostaglandin F2 alpha to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100 degrees C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.