POMC gene-derived peptides activate melanocortin type 3 receptor on murine macrophages, suppress cytokine release, and inhibit neutrophil migration in acute experimental inflammation.

To investigate the relevance of adrenocorticotrophic hormone (ACTH) therapy in human gouty arthritis, we have tested the effect of several ACTH-related peptides in a murine model of experimental gout. Systemic treatment of mice with ACTH4-10 (MEHFRWG) (10-200 microgram s. c.) inhibited neutrophil accumulation without altering peripheral blood cell counts or circulating corticosterone levels. A similar effect was seen with alpha- and beta-melanocyte stimulating hormones (1-30 microgram s.c.). In vivo release of the chemokine KC-(detected in the lavage fluids before maximal influx of neutrophils) was significantly reduced (-50 to -60%) by ACTH4-10. Macrophage activation in vitro, determined as phagocytosis and KC release, was inhibited by ACTH and ACTH4-10 with approximate IC50 values of 30 nM and 100 microM, respectively. The melanocortin receptor type 3/4 antagonist SHU9119 prevented the inhibitory actions of ACTH4-10 both in vitro and in vivo. However, melanocortin type 3, but not type 4, receptor mRNA was detected in mouse peritoneal macrophages by RT-PCR. Therefore, we propose that activation of this receptor type by ACTH4-10 and related amino acid sequences attenuates KC release (and possibly production of other cytokines) from macrophages with consequent inhibition of the host inflammatory response, thus providing a notional anti-inflammatory mechanism for ACTH that is unrelated to stimulation of glucocorticoid release.     

The effects of p-mercuribenzenesulfonate on purified spectrin and actin

The compound p-mercuribenzenefulfonate was found to affect the self-association behavior of both spectrin and actin. The reagent brings about the depolymerization of F-actin, as judged from the decrease in the fluorescence of an attached pyrene label, with a second-order rate constant an order of magnitude less than that for the disruption of isolated erythrocyte cytoskeletons. Therefore, it is unlikely that the depolymerization of actin is the rate-determining step in the mercurial-dependent disruption of the erythrocyte cytoskeleton. Low reagent concentrations caused an initial rapid dissociation of spectrin tetramers at a rate comparable with that of cytoskeleton disruption. Prolonged incubation, or higher reagent concentrations, resulted in subsequent aggregation of spectrin. The reagent also prevented the interaction between spectrin and actin, presumably through its depolymerization of actin and its effects on spectrin. The early event in the disruption of isolated erythrocyte cytoskeletons by p-mercuribenzenesulfonate thus appears to be the dissociation of spectrin oligomers. Subsequent depolymerization of actin brought about by the reagent then results in total disruption of the cytoskeleton.     

Transitions: Homo erectus and the Acheulian: the Ethiopian sites of Gadeb and the Middle Awash

Significant differences in activity-site patterning and artifact composition at Middle Pleistocene localities in Ethiopia's high plains and Afar Rift are indications of both single-episode, small-site residues from small groups and multi-purpose, multiple occupation sites from which larger, temporary groupings might be inferred. Reconstructions of palaeo-environments and geography show that large assemblages relate to relatively stable topography, such as stream channels or lake margins, while small assemblages are more common in deltaic situations of rapid sedimentation and burial in the Rift that preclude possibility of reoccupation. A model for Acheulian socio-economic behaviour within the drier African savanna, based (in part) on chimpanzee behaviour in open savanna habitats, is proposed. This takes the form of organization into relatively extensive territories, each with several more closed-vegetation core areas, of limited extent close to permanent water, surrounded by more extensive areas of drier, open savanna, regularly exploited and patrolled by small groups. Movement from one core area to another was rapid and made together as a group, to minimize danger from large predators and competition from other hominid groups.     

Oxidant membrane injury by the neutrophil myeloperoxidase system. II. Injury by stimulated neutrophils and protection by lipid-soluble antioxidants

The stimulated human neutrophil can damage a variety of target cells, and in some models, a mechanism involving secretion of myeloperoxidase and H2O2 has been demonstrated. We explored the characteristics of this cell-cell interaction by using neutrophils and our recently described liposome model target cell system. Exposure of 51Cr-labeled liposomes to phorbol myristate acetate-stimulated human neutrophils resulted in release of 25 to 30% of the radioactivity. 51Cr release was abrogated by omission of the neutrophils, the phorbol ester or halide (iodide), replacement of the phorbol by an inactive congener, or addition of azide, cyanide, or catalase. Neutrophils from patients with hereditary absence of myeloperoxidase (MPO) or a failure of H2O2 formation (chronic granulomatous disease) did not cause liposome lysis unless purified MPO or a source of H2O2, respectively, was added. These data indicate that 51Cr release from liposomes is a consequence of the secretion of MPO and H2O2, which combine with extracellular halides to form a membrane lytic system. The influence of liposome composition on injury was then examined, with a focus on physiologically relevant lipid soluble antioxidants. Liposomes containing either alpha-tocopherol (0.33 to 1.67% of molar fraction of lipid) or beta-carotene (1.67% of molar fraction of lipid) were markedly resistant to lysis by the cellfree MPO-H2O2-chloride system. When the major structural lipid phosphatidyl choline was replaced by dipalmitoyl phosphatidyl choline, a synthetic phospholipid with no oxidizable double bonds, the resultant liposomes were totally resistant to lysis by the MPO-H2O2-chloride system. The addition of iodide to this system (i.e., both chloride and iodide present) changed the pattern of protection dramatically in that alpha-tocopherol and beta-carotene were no longer protective and the resistance of dipalmitoyl phosphatidyl choline liposomes was partial rather than complete. In contrast to iodide, the addition of bromide or thiocyanate did not have a major effect on the protection by antioxidants. Finally, we demonstrated protection by alpha-tocopherol or dipalmitoyl phosphatidyl choline against liposome lysis by phorbol-activated neutrophils. These studies illustrate the use of model phospholipid membranes in the characterization of oxygen-dependent cell-mediated cytotoxicity. Activated neutrophils lyse liposome targets through a MPO-dependent mechanism. Target properties, especially the content of lipid-soluble antioxidants, have a marked influence on susceptibility to lysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Photoreactions of human lens monomeric crystallins

Human lens beta s- and gamma A-crystallins exhibit very similar tryptophan fluorescence emission maxima (329 nm). gamma A isolated from infant human lenses is photo-oxidized by 300 nm irradiation and forms water-insoluble aggregates; beta s or gamma A from young human lenses form a small amount of water-soluble crosslinked species. At least part of the mechanism of photodamage by 300 nm irradiation is photogeneration of the oxidant H2O2 via the generation of O2- radical, this reaction occurs via photosensitization by the tryptophan photo-oxidation product N-formylkynurenine (N-FK) or related species. These results indicate that even though the tryptophan residues of beta s- and gamma A-crystallins are in hydrophobic (buried) microenvironments as compared to those of the alpha- and beta-crystallins, the photogeneration of N-FK is sufficient to produce O2- and H2O2.     

Transmucosal triglyceride transport rates in proximal and distal rat intestine in vivo.

Transmucosal transport rates for triolein in proximal and distal intestine were compared in unanesthetized rats. Emulsified [1-14-C] triolein together with bile and pancreatic juice from donor rats was infused for 6 hr into either the duodenum or the midpoint of the small intestine at such a rate that absorption was essentially complete in both regions of the intestine. Lymph was collected from the thoracic duct during triolein infusion and for an additional 6-hr period. The decrease in the rate of lymphatic output of labeled fat was found to follow a simple exponential function in all animals. This rate of decrease (decay rate) was used to calculate the half-times of lipid turnover through the intestinal wall and the fractional output rates. Distal intestine transported lipid 40% more slowly than proximal intestine, and the difference was associated with a greater accumulation of triglyceride in the distal intestinal wall. Chylomicron synthesis and/or release is the rate-limiting step for distal lymphatic fat transport in vivo, whereas fat uptake from the lumen is rate limiting for proximal intestine.     

Evidence for multiple lytic pathways used by cytotoxic T lymphocytes

Previous data generated by ourselves and others questioned the role of degranulation as a mechanism to explain CTL-mediated cytotoxicity. In this report we examine this tissue in greater depth. CTL-mediated lysis was probed with three different inhibitors. 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene inhibits degranulation in a wide range of cell types, including CTL. EGTA, through chelation of Ca2+, also inhibits degranulation processes in CTL, and would inhibit other events or processes dependent on extracellular Ca2+. We also used prolonged exposure to PMA to exhaust PKC activity in CTL. Using these inhibitors, we have defined three pathways of lysis used by CTL. One pathway requires Ca2+, is PMA sensitive, but does not depend on degranulation. The second pathway is independent of Ca2+, is not PMA sensitive, and also does not depend on degranulation. All primary CTL and cloned CTL lyse most target cells via pathway I. However, when confronted with certain target cells (which we have referred to previously as Ca2+-independent target cells), pathway II is induced. When pathway II is induced, pathway I apparently shuts down. We show here that pathway II does not depend on protein synthesis, and that it also leads to DNA solubilization in target cells. A limited number of cloned CTL use pathways I and II as just described, but use in addition, and simultaneously, a third pathway that appears to involve degranulation. This pathway is seen irregularly in most CTL clones, and may be influenced by levels of IL-2 in the culture medium.     

Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.