The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Interaction between the Ectomycorrhizal Fungus Paxillus involutus, Damping-off Fungi and Pinus resinosa Seedlings

Paxillus involutus, an ectomycorrhizal fungus, had an inhibitory effect on the root pathogenic fungus Fusarium moniliforme and two isolates of F. oxysporum when grown in paired cultures on modified Melin Norkrans’ medium. In contrast, one isolate of F. oxysporum was not inhibited and another damping-off fungus, Cylindrocarpon destructans inhibited growth of Pax. involutus in similar paried cultures. Survival of Pinus resinosa (red pine) seedlings was increased significantly when they were grown in vitro concomitantly with either Pax. involutus and F. moniliforme or Pax. involutus and the three isolates of F. oxysporum, compared with seedlings inoculated with either F. moniliforme or F. oxysporum isolates alone. pax. involutus showed no protective effect against C. destructans. The number of colony forming units of Fusarium spp. was reduced significantly in the root extract and rhizosphere substrate of P. resinosa seedlings inoculated with Pax. involutus. Spore germination of Fusarium spp. was reduced significantly when treated with culture filtrate of Pax. involutus and root extract of P. resinosa seedlings inoculated with Pax. involutus. Neither colony forming units nor spore germination of C. destructans was affected either by culture filtrate of Pax. involutus or root extract of P. resinosa seedlings inoculated with Pax. involutus.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Protein synthesis in rabbit reticulocytes. A study of the mechanism of Co-eIF-2 action

The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.     

Protein synthesis in rabbit reticulocytes. Purification and properties of an Mr 80,000 polypeptide (Co-eIF-2A80) with Co-eIF-2A activity

The high molecular weight protein complex, Co-eIF-2, contains both Co-eIF-2A and Co-eIF-2C activities (Bagchi, M. K., Banerjee, A. C., Roy, R., Chakravarty, I., and Gupta, N. K. (1982) Nucleic Acids Res. 10, 6501-6510). Co-eIF-2A stimulated Met-tRNAf binding to eukaryotic initiation factor-2 (eIF-2) both in the presence and absence of Mg2+. Co-eIF-2C stimulates Met-tRNAf binding to eIF-2 in the presence of Mg2+ by relieving Mg2+ inhibition of ternary complex formation from eIF-2. Co-eIF-2 protein complex contains several polypeptides including Mr 80,000 and 50,000 polypeptides. Three polypeptides (Mr 80,000, 50,000 and 25,000) are present in 0.5 M KCl ribosomal salt wash and each possesses Co-eIF-2A activity. Mr 80,000 polypeptide (Co-eIF-2A80) has been purified to homogeneity and its properties studied. 1) Co-eIF-2A80 stimulated Met-tRNAf binding to eIF-2 and the complex formed was resistant to aurintricarboxylic acid. 2) Co-eIF-2A80 activity was N-ethylmaleimide-resistant and heat-labile; it was destroyed by heating at 55 degrees C for 4 min. 3) Antibodies prepared against homogeneous Co-eIF-2A80 strongly inhibited protein synthesis in reticulocyte lysates and, also, eIF-2 and Co-eIF-2 promoted Met-tRNAf binding to 40 S ribosomes. Inhibition of protein synthesis in reticulocyte lysates was overcome by preincubation of anti-Co-eIF-2A80 with homogeneous Co-eIF-2A80 and was partially overcome by similar preincubation with Co-eIF-2. 4) Upon limited digestion with Staphylococcus aureus V8 protease, the homogeneous Co-eIF-2A80 gave two major polypeptide fragments (Mr 50,000 and 25,000). Upon similar treatment, an Mr 80,000 polypeptide band isolated from the sodium dodecyl sulfate-gel of the Co-eIF-2 protein complex gave four major polypeptide fragments, and two of these fragments (Mr 50,000 and 25,000) were similar to those given by Co-eIF-2A80, indicating that this Mr 80,000 polypeptide band contains the Co-eIF-2A80 component. We suggest that Co-eIF-2A80 is a component of Co-eIF-2 and is also essential for Co-eIF-2 activity and overall peptide chain initiation.     

Comparison of buprenorphine and pethidine given intravenously on demand to relieve postoperative pain.

In a double-blind study of on-demand intravenous analgesia buprenorphine was found to be about 600 times as potent as pethidine. The incidence of side effects was similar with both drugs. The quality of analgesia, subjectively assessed, was good with both drugs using this method of administration. Provided that its low potential for abuse is substantiated, buprenorphine appears to be a powerful analgesic that may successfully be given intravenously on demand.     

Effect of charge transfer bands on the photo-induced DNA cleavage activity of [1-(2-thiazolylazo)-2-naphtholato]copper(II) complexes

Ternary copper(II) complex [Cu(TAN)(O2CMe)] (1), where H-TAN is 1-(2-thiazolylazo)-2-naphthol, is prepared and structurally characterized by X-ray crystallography. The complex has a distorted square pyramidal (4+1) CuN2O3 coordination geometry with the acetate showing chelating axial-equatorial binding mode and TAN as a tridentate ligand bonded to the metal in the basal plane. Complex 1 is one-electron paramagnetic and displays ligand-to-metal charge transfer bands at 575 and 398 nm in dimethylformamide. The reactions of 1 with bases (B) like 1,10-phenanthroline (phen) and kanamycin-A (kan-A) afford ternary complexes of formulation [Cu(TAN)B]+ (B=phen, 2; kan-A, 3) under in situ reaction conditions. Complexes 2 and 3, prepared to explore their DNA binding and photo-induced DNA cleavage activity, display good binding propensity to calf thymus (CT) DNA giving a relative order: 2-3>1. The apparent binding constant (Kapp) for 1 is determined as 9.8 x 10(5)M(-1) from fluorescence quenching experiments using ethidium bromide. The quenching constants (K) values of 1-3, obtained from the Stern-Volmer plots, are 0.28, 0.52 and 0.49, respectively. All the complexes show photo-induced DNA cleavage activity when irradiated with a monochromatic UV light of 365 nm wavelength. A 200 microM complex 1 cleaves approximately 75% supercoiled (SC) DNA on 2h exposure time at 365 nm. A 50 microM solution of 1 in presence of 100 microM phen and kanamycin-A cleaves approximately 99% and approximately 60% SC DNA to its nicked circular form, respectively, for an exposure of 30 min. The complexes also exhibit significant cleavage of SC DNA on irradiation with visible light of wavelengths 532, 575 and 632.8 nm. Control experiments reveal the minor groove binding nature of the complexes. The cleavage reactions involve the formation reactive hydroxyl species as significant inhibition in the presence of dimethyl sulfoxide (DMSO) and catalase is observed. There is no apparent inhibition in cleavage in the presence of singlet oxygen quenchers like sodium azide. The cleavage activity has been found to be higher at the CT band position of 575 nm in comparison to those at 532 and 632.8 nm. The results indicate the involvement of the CT band in the photo-excitation process.     

Rescue of ligand binding of a mutant IGF-I receptor by complementation

The IGF-I receptor binds IGF-I with complex kinetics characterized by a curvilinear Scatchard plot, suggesting receptor heterogeneity and apparent negative cooperativity. To explore the molecular mechanisms underlying these properties, we have characterized the binding of a hybrid receptor formed from a wild-type receptor monomer and a mutant receptor monomer devoid of binding activity. Receptor hybrids were generated by transient co-transfection of cDNAs encoding wild-type and mutant receptors with unique epitope tags. Hybrid receptors were purified from transfected cells by sequential immuno-affinity chromatography and their ligand-binding properties were determined. Complementation produced a hybrid with near wild-type affinity. Dissociation studies demonstrated that the hybrid did not exhibit negative cooperativity.     

Metal-assisted light-induced DNA cleavage activity of 2-(methylthio)phenylsalicylaldimine Schiff base copper(II) complexes having planar heterocyclic bases

Ternary copper(II) complexes [CuLL'](ClO(4)), where HL is NSO-donor Schiff base (2-(methylthio)phenyl)salicylaldimine and L' is NN-donor phenanthroline bases like 1,10-phenanthroline (phen), dipyridoquinoxaline (dpq) and 2,9-dimethyl-1,10-phenanthroline (dmp), are prepared and structurally characterized by X-ray crystallography. The complexes have a distorted square-pyramidal (4+1) CuN(3)OS coordination geometry. While [CuL(phen)](ClO(4)) and [CuL(dpq)](ClO(4)) show axial sulfur ligation, [CuL(dmp)](ClO(4)) has the sulfur bonded at the equatorial site. The one-electron paramagnetic complexes exhibit axial electron paramagnetic resonance (EPR) spectra in dimethylformamide glass at 77 K. The complexes are redox active and a quasireversible electron transfer process near 0.0 V vs saturated calomel electrode (SCE) in DMF-Tris buffer (1:4 v/v at pH 7.2) involving Cu(II)/Cu(I) couple is observed for the phen and dpq complexes. The dmp complex exhibits an irreversible reduction process forming bis(dmp)copper(I) species. A profound effect of the substituents of the phenanthroline bases is observed on the binding of the complexes to the calf thymus (CT) and in the cleavage of supercoiled (SC) pUC19 DNA. The phen and dpq complexes show DNA cleavage activity in presence of mercaptopropionic acid (MPA). The dmp complex is cleavage inactive in presence of MPA. All the complexes show photocleavage activity when irradiated with a monochromatic UV light of 312 nm. The dpq complex also cleaves SC DNA on visible light irradiation at 436, 532 and 632.8 nm but with a longer exposure time and higher complex concentration. The cleavage reactions in presence of MPA are found to involve hydroxyl radical. The photocleavage reactions are found to occur under aerobic conditions showing an enhancement of cleavage in D(2)O and inhibition with azide addition suggesting formation of singlet oxygen as a reactive species. The roles of sulfur of the Schiff base as photosensitizer and the phenanthroline bases as minor groove binder, and their influence on the photocleavage activity are discussed. The quinoxaline ligand exhibits significant photosensitizing effect assisted by the copper(II) center.     

Thermoregulatory responses of rats to conventional preparations of lipopolysaccharide are caused by lipopolysaccharide per se-- not by lipoprotein contaminants

LPS preparations cause a variety of body temperature (T(b)) responses: monophasic fever, different phases of polyphasic fever, and hypothermia. Conventional (c) LPS preparations contain highly active lipoprotein contaminants (endotoxin proteins). Whereas LPS signals predominantly via the Toll-like receptor (TLR) 4, endotoxin proteins signal via TLR2. Several TLR2-dependent responses of immunocytes to cLPS in vitro are triggered by endotoxin proteins and not by LPS itself. We tested whether any T(b) response to cLPS from Escherichia coli 055:B5 is triggered by non-TLR4-signaling contaminants. A decontaminated (d) LPS preparation (free of endotoxin proteins) was produced by subjecting cLPS to phenol-water reextraction. The presence of non-TLR4-signaling contaminants in cLPS (and their absence in dLPS) was confirmed by showing that cLPS (but not dLPS) induced IL-1beta expression in the spleen and increased serum levels of TNF-alpha and IL-1beta of C3H/HeJ mice; these mice bear a nonfunctional TLR4. Yet, both cLPS and dLPS caused cytokine responses in C3H/HeOuJ mice; these mice bear a fully functional TLR4. We then studied the T(b) responses to cLPS and dLPS in Wistar rats preimplanted with jugular catheters. At a neutral ambient temperature (30 degrees C), a low (0.1 microg/kg iv) dose of cLPS caused a monophasic fever, whereas a moderate (10 microg/kg iv) dose produced a polyphasic fever. In the cold (20 degrees C), a high (500 microg/kg iv) dose of cLPS caused hypothermia. All T(b) responses to dLPS were identical to those of cLPS. We conclude that all known T(b) responses to LPS preparations are triggered by LPS per se and not by non-TLR4-signaling contaminants of such preparations.