Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z-disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z-discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha-actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A-bands of leg myofibrils. p700 is in Z-discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z-discs. p400 is in M-lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z-disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.     

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE⁺ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a sup-Estrain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel -strands organized into two orthogonally situated -sheets. The overall conformation of the protein resembles that of a clam — hence the term -clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg¹⁰⁶ and Gln¹¹⁵. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the -clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations pf several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism. Comparison of the C coordinates of apo- and holo-I-FABP with those of other proteins indicates it is a member of a superfamily that currently includes (i) 10 mammalian intracellular lipid binding proteins, (ii) the photoactive yellow protein from the purple photoautotrophic bacterium Ectothiorhodospira halophila and (iii) a group of extracellular lipid binding proteins from a diverse number of phyla that have a common barrel consisting of 8 anti-parallel -strands stacked in two nearly orthogonal sheets. In summary, E. coli-derived I-FABP not only represents a useful model for assessing the atomic details of fatty acid-protein interactions and the mechanisms which regulate acquisition and release of this type of ligand, but also structure/function relationships in other superfamily members.Abbreviations I-FABP Intestinal Fatty Acid Binding Protein - r.m.s root mean square     

Correlation of changes in pineal gland cAMP metabolism with light-mediated decrease in N-acetyltransferase activity

The present study investigated the correlation between the light-mediated decrease in rat pineal N-acetyltransferase (NAT) observed in vivo and changes in cAMP metabolism. While exposing dark-adapted rats to light for short time periods (0–10 min) resulted in a rapid decrease in pineal NAT activity, cAMP exhibited a biphasic response. Following light exposure for 30 s there was a 50% decrease in cAMP levels. However, after 6 min of light exposure the cyclic nucleotide levels had increased 2–3 times above control values. These responses were prevented by phenoxybenzamine pretreatment and the initial decrease was mimicked by i.v. propranolol administration. Examination of cAMP metabolic enzymes, adenylate cyclase and phosphodiesterase revealed an increase in adenylate cyclase activity following 6 min of exposure to light. We discussed how the results observed in vivo compare with those observed using cultured pinealocytes.     

Ascorbic acid accumulation by cultured rat adrenocortical cells

Summary The influence of adrenocorticotrophin (ACTH) on radiolabeled ascorbic acid (AA) accumulation by adrenocortical cells was examined in primary cultures of collagenase dissociated glands from adult male rats. The cells were ACTH responsive by morphological and steroidogenic criteria. After 5 d in AA-free medium, cells pretreated with 100 mU/ml ACTH for 3 d took up two to three times more AA over a 2 h period than did untreated controls (4.0 to 10.0 nmol versus 1.7 to 3.4 nmol AA/μg DNA). In contrast, ACTH administered on Day 6 concurrently with AA inhibited AA accumulation compared to cultures exposed to AA alone. This acute inhibitory effect of ACTH was in the order of 30% in cultures pretreated with ACTH for 3 d but was not significant (7%) without ACTH pretreatment. The results show that ACTH has distinct long term stimulatory and acute inhibitory effects on AA accumulation by adrenocortical cells and suggest that both maximal AA accumulation and the responsiveness to acute inhibition of AA accumulation by ACTH may depend on the maintenance of the differentiated state of the adrenal cortex. This work was supported by a grant and research associateship to N. A. from the National Cancer Institute of Canada.     

Properties of a partially-purified preparation of the prostaglandin-forming oxygenase from sheep vesicular gland

The fatty acid oxygenase of sheep vesicular glands was solubilized with Tween-40 and purified 60-fold using ammonium sulfate precipitation and DEAE-cellulose chromatography. Glycerol (50%) stabilized the activity at all stages of purification and allowed long-term storage at −60°. The partially purified enzyme contained less than 0.7 nmoles of iron per mg of protein and less than 0.1 nmole of copper per mg of protein. Although the K_I values for aspirin, BL-2338, flurbiprofen and ibuprofen remained relatively unchanged during purification, the apparent K_I value for inhibition by indomethacin decreased from 120 to 2.7 μ$\text{M}$.     

Susceptibility of Freshly Picked Ear Corn to Invasion by Fungi

High-moisture ear corn held 9 days at 23 C remained free of visible mold or evidence of Aspergillus flavus invasion provided there was no mechanical damage of the kernels.     

Sexual segregation and its ontogeny in squirrel monkey social structure

Four groups of squirrel monkeys were observed to determine the primary basis of adult group structure. These studies included observations on (1) intact and gonadectomized males and females during and after breeding; (2) intact adults only; (3) subadults only; and, (4) a mixed group of adults and subadults. The spatial distribution of subjects in each group was used as a basic measure of social organization. It was found that for adults, regardless of hormonal status, between-sex distances were consistently greater than within-sex distances. This sexually segregated adult structure was largely attributable to the females' attaction to one another and overt rejection of the males. Subadults by themselves did not show any clear sexual segregation. However, in the presence of an adult structure, the subadults gradually manifested the segregated pattern of the adults by gravitating toward same-sex adults. These results indicate that the socialization process, rather than endogenous hormonal status, is the major determinant of adult social structure in squirrel monkeys.