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.     

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.     

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.     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

Toxigenic Potential of Some Fusarium Isolates from Southeast Asia

The isolation and characterization of 10 isolates of six Fusarium spp. from plant and soil samples collected in Southeast Asia is reported. The ability of these isolates to produce trichothecenes both in liquid cultures (CZ, GYEP, and MYRO) and on rice was assessed, and their toxigenic potential was examined by skin assay and gavage studies with culture filtrates. Although culture filtrates of all the isolates caused minor damage to test animals, only that of F. equiseti DAOM 189762 produced trichothecenes.     

Amidation of C3 at the thiolester site: stimulation of chemiluminescence and phagocytosis by a new inflammatory mediator

We studied the ability of particle-bound and fluid-phase C3b monomers and monomeric amidated C3 (prepared by treatment of purified human C3 with ammonium chloride or methylamine) to stimulate chemiluminescence of human polymorphonuclear leukocytes (PMN) and monocytes and to promote phagocytosis in the absence of antibody. Particle-bound C3b evoked chemiluminescence from both PMN and monocytes, and fluid-phase C3b (0.5 mg/ml) elicited significant chemiluminescence from PMN but not from monocytes. Amidated forms of C3, both particle bound and fluid phase, were potent stimulators of chemiluminescence from phagocytic cells and caused a significantly greater response than did C3b. The phagocytosis of 1-micron microspheres by PMN and monocytes was enhanced by coating them with purified C3b in an antibody-free system. Microspheres coated with amidated C3 were avidly phagocytized, and to a greater degree than were C3b-coated microspheres. In a direct binding assay with tritiated monomeric C3b and amidated C3, the affinity of the PMN complement receptor type 1 for C3b (Ka = 4.9 X 10(7) L/M) was similar to that for amidated C3 (Ka = 5.7 X 10(7) L/M). However, there was a fourfold increase in the number of apparent binding sites for amidated C3. This increase did not reflect binding of amidated C3 to the complement receptor type 3, because blocking of CR3 with the monoclonal antibody OKM 10 failed to decrease binding of amidated C3. In sites of increased ammoniagenesis, such as the kidney in chronic renal failure, amidated C3 may play a role as an inflammatory mediator by stimulating oxidative metabolism in phagocytic cells.