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.     

Kinetics of synthesis and/or conformational changes of biological macromolecules

The analogy, in both the thermodynamics and the kinetics, of reversible polymerizations on templates (in the case wherein these are catalyzed by exoenzymes) to helix–coil transitions (in the case where these proceed only from one end of each macromolecular chain) is presented. A suggestion, based on this analogy, is made concerning the possible nature of biological control of synthesis of macromolecules (enzyme induction and repression). The equations governing the kinetics of these one-dimensional cooperative processes are presented and their solutions discussed.     

Torsional constant of 27-mer DNA oligomers of different sequences.

We have studied the torsional elastic constant (alpha) of short DNA (27mer) oligomers of various sequence by fluorescence polarization anysotropy (FPA) measurements. The lowest alpha values were found in samples with sequence rich in AA dinucleotides or containing the alternating d(A-T) x d(A-T) motif. The torsional rigidity of our DNA samples was compared to that calculated according to the current values of twist angle fluctuations derived for ten dinucleotide steps by recent analyses of DNA crystal structure database. The values of torsional rigidity derived from crystals are higher than our experimental ones, obtained by FPA analysis, suggesting that packing force in crystals may notably hinder the dinucleotide twist angle fluctuations that occur in solution. This behaviour is more evident for samples containing AA, TA and AT steps. In all the samples there is about a twofold change of the alpha value in the 10-40 degrees C range. An activation enthalpy (Delta H (#)) of about 17.4 kJ mol(-1), on average, was obtained for the temperature dependence of eight of the ten samples studied. A correlation with the stacking energy is discussed.     

Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum

The distribution of phosphoribulose kinase (PRK) in the cyanelles of Cyanophora paradoxa Korschikoff and Glaucocystis nostochinearum Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum against ribulose 1,5-bisphosphate carboxylase/oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extracts of C. paradoxa showed that 93% of the enzyme was in the soluble fraction. Quantitative immunoelectron microscopy showed that more than 99% of the PRK in the cyanelle of C. paradoxa was localized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algal chloroplasts do not contain phosphoribulose kinase.     

Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF)

Summary Corticotropin releasing factor (CRF) synthesizing neurons, located in the hypothalamic paraventricular nucleus (PVN), are the main central regulators of the pituitary-adrenal cortex endocrine axis. The hormone production and release of CRF-synthesizing neurons is regulated by neuronal messages and feedback action(s) of glucocorticoids secreted by the adrenal gland. In order to characterize the latter mechanism, glucocorticoid receptor (GR)-immunoreactive (IR) sites were studied in hypothalamic paraventricular neurons of intact, long-term adrenalectomized, and adrenalectomized plus glucocorticoid treated animals, by means of ultrastructural immunocytochemical labelling. In intact animals, glucocorticoid receptor immunoreactivity was found predominantly in the nuclei of parvocellular neurons. Following adrenalectomy GR-immunoreactivity was localized in the cytoplasm of the cells, and there was a concomitant disappearance of the label from the nuclei. After corticosterone administration to adrenalectomized animals, GR-IR sites were again concentrated within the cell nuclei. Immunocytochemical double labelling studies performed on adrenalectomized plus corticosterone-replaced animals demonstrated glucocorticoid receptor-IR sites in the cell nuclei of parvocellular paraventricular neurons that expressed CRF-immunoreactivity in their cytoplasm.These ultrastructural data indicate that the intracellular location of glucocorticoid receptor is dependent on the availability of glucocorticoids by the neurons. The simultaneous expression of GR- and CRF-immunoreactivity in parvocellular paraventricular neurons supports the concept of a direct feedback action of glucocorticoids upon CRF-synthesizing neurons.Supported by NIH Research Grants NS19266 (W.K.P. and Zs.L.), NS20832 (M.C.B.) and a joint grant (INT-8703030) awarded by the National Science Foundation and the Hungarian Academy of Sciences (Zs.L. and W.K.P.). R.M.U. is a recipient of NIMH Pre-doctoral Fellowship and M.C.B. an NIH Research Carcer Development Award     

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K_m for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H₂ adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).     

Detection and partial purification of two lipases fromCandida rugosa

Summary Two distinct lipases produced byCanadida rugosa were identified and separated by a high resolution anion-exchange column (Mono Q) after an ethanol extraction of the crude lipase. From this Mono Q column, lipase I eluted at 0.05 M NaCl whereas lipase II eluted at 0.15 M NaCl. The less anionic nature of lipase I was also confirmed by native polyacrylamide gel electrophoresis as well as isoelectrophoresis. Both proteins have an apparent molecular weight of 58,000 by SDS-PAGE. The isoelectric points of lipase I and II are 5.6 and 5.8 respectively.