Activation of human monocyte cytotoxicity by natural and recombinant immune interferon

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.     

Parasites of Pleurobrachia pileus Muller, 1776 (Ctenophora), from Galway Bay, western Ireland

Specimens of Pleurobrachia pileus Müller from Galway Bay,western Ireland, were found to be parasitised by three trematodes,one isopod and one nematode. Among these, the trematode, Opechonabacillaris Molin and didymozoid larvae were the most abundant,infecting over 17% of the Pleurobrachia population. Peak infectionwas during the summer of each year when >40% of P. - pileuswere parasitised. Serious infection was found to be either accompaniedor followed by a sharp decline in the abundance of the ctenophore.A close, but negative, correlation was established between theabundance of P. pileus and the percentage of parasitic infection.The percentage of infection in larger P. pileus was higher thanin small specimens. No parasites were detected from P. pileus1 mm or less in length.     

Insulin receptors are bivalent as demonstrated by photoaffinity labeling.

Insulin receptors in human placental membranes were photoaffinity-labeled with a radioactive human insulin-like growth factor I (hIGF-I) photoprobe N epsilon B28-monoazidobenzoyl 125I-hIGF-I either alone or together with a non-radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl insulin. Precipitation of the solubilized receptors with anti-insulin antibody showed that receptors labeled with the radioactive hIGF-I photoprobe were detected in the immunoprecipitate only when photolabeling was carried out in the presence of the non-radioactive insulin photoprobe. Comparable results were obtained in converse experiments using a radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl 125I-insulin, a non-radioactive hIGF-I photoprobe N epsilon B28-monoazidobenzoyl hIGF-I, and an antibody to hIGF-I. The amount of radioactive receptors precipitated by either the anti-insulin antibody or the anti-hIGHF-I antibody was close to the expected amount. These observations demonstrate that the insulin receptor is bivalent being capable of binding two molecules of ligand.     

Effect of naloxone and prolonged preganglionic stimulation on noncholinergic transmission in the superior cervical ganglion of the cat

In cats anesthetized with sodium pentobarbital, a supramaximal 40-Hz, 30-s train to the cervical sympathetic trunk, during block of ganglionic cholinergic transmission with hexamethonium and scopolamine, produced a delayed, slow, small amplitude contraction of the nictitating membrane that persisted for several minutes after the end of the stimulus train. The post-stimulus component of the response was due to afterdischarge of the ganglion cells, since section of the post-ganglionic axons at the end of the train resulted in elimination of this component. The amplitude of the slow nictitating membrane response was enhanced in a dose-dependent manner by i.v. injection of naloxone. The enhancement was detectable at a dose as low as 1 microgram/kg and was maximal at 10 micrograms/kg. During continuous preganglionic stimulation at 40 Hz, the amplitude of the slow nictitating membrane response reached a peak in 2-4 min and then faded with time until it became undetectable. Time for 90% decay was 82 +/- 5 min (n = 18). The nictitating membrane response to postganglionic nerve stimulation was not modified by prolonged preganglionic stimulation. In three cats, the cervical sympathetic trunk was split into two bundles and one bundle was stimulated continuously at 40 Hz until the slow response disappeared. At this time stimulation of the unconditioned bundle evoked a slow response of normal appearance. This suggests that the process underlying the fade involves only the conditioned axons. Recovery from the fade was slow, the response approaching control by 24 h post-stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin receptor: its subunit structure as determined by photoaffinity labeling

This communication reviews briefly the preparation of two photoreactive arylazido insulins and the use of these insulin derivatives to photolabel the insulin receptor of rat adipocytes. These experiments showed that the receptor contains disulfide linked subunits of 130,000, 90,000, and 40,000 daltons. When not reduced by dithiothreitol, three receptor species of 380,000, 300,000, and 230,000 daltons were detected. Based on the results of photolabeling we propose that the 300,000-dalton species is composed of one 130,000-, one 90,000-, and two 40,000-dalton subunits in disulfide linkages.     

Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings

This study tested the hypothesis that melatonin (Mel) therapy preserved the brain architectural and functional integrity against ischaemic stroke (IS) dependently through suppressing the inflammatory/oxidative stress downstream signalling pathways. Adult male B6 (n = 6 per each B6 group) and TLR4 knockout (ie TLR4^?/?) (n = 6 per each TLR4^?/? group) mice were categorized into sham control (SC^B6), SC^TLR4?/?, IS^B6, IS^TLR4?/?, IS^B6 + Mel (i.p. daily administration) and IS^TLR4?/? + Mel (i.p. daily administration). By day 28 after IS, the protein expressions of inflammatory (HMBG1/TLR2/TLR4/MAL/MyD88/RAM TRIF/TRAF6/IKK‐α/p‐NF‐κB/nuclear‐NF‐κB/nuclear‐IRF‐3&7/IL‐1β/IL‐6/TNF‐α/IFN‐γ) and oxidative stress (NOX‐1/NOX‐2/ASK1/p‐MKK4&7/p‐JNK/p‐c‐JUN) downstream pathways as well as mitochondrial‐damaged markers (cytosolic cytochrome C/cyclophilin D/SRP1/autophagy) were highest in group IS^B6, lowest in groups SC^B6 and SC^TLR4?/?, lower in group IS^TLR4?/? + Mel than in groups IS^TLR4?/? and IS^B6 + Mel and lower in group IS^B6 + Mel than in group IS^TLR4?/? (all P < .0001). The brain infarct volume, brain infarct area and the number of inflammatory cells in brain (CD14/F4‐88) and in circulation (MPO+//Ly6C+/CD11b+//Ly6G+/CD11b+) exhibited an identical pattern, whereas the neurological function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.     

Historical biogeography of the widespread macroalga Sargassum (Fucales,Phaeophyceae)

Sargassum is a cosmopolitan brown algal genus spanning the three ocean basins of the Atlantic, Pacific and Indian Oceans, inhabiting temperate, subtropical and tropical habitats. Sargassum has been postulated to have originated in the Oligocene epoch approximately 30 mya according to a broad phylogenetic analysis of brown macroalgae, but its diversification to become one of the most widespread and speciose macroalgal genera remains unclear. Here, we present a Bayesian molecular clock study, which analyzed data from the order Fucales of the brown algal crown radiation (BACR) group to reconstruct a time-calibrated phylogeny of the Sargassum clade. Our phylogeny included a total of 120 taxa with 99 Sargassum species sampled for three molecular markers – ITS-2, cox3 and rbcLS – calibrated with an unambiguous Sargassaceae fossil from between the lower and middle Miocene. The analysis revealed a much later origin of Sargassum than expected at about 6.7 mya, with the genus diversifying since approximately 4.3 mya. Current geographic distributions of Sargassum species were then analyzed in conjunction with the time-calibrated phylogeny using the dispersal-extinction-cladogenesis (DEC) model to estimate ancestral ranges of clades in the genus. Results strongly support origination of Sargassum in the Central Indo-Pacific (CIP) region with subsequent independent dispersal events into other marine realms. The longer history of diversification in the ancestral CIP range could explain the much greater diversity there relative to other marine areas today. Analyses of these dynamic processes, when fine-tuned to a higher spatial resolution, enable the identification of evolutionary hotspots and provide insights into long-term dispersal patterns.     

β2 adrenoceptor signaling regulates ion transport in 16HBE14o- human airway epithelial cells

We investigated the regulation of Cl⁻ secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective β adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (I_SC), which was inhibited by the β adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the β₂ adrenoceptor subtype, stimulated a similar increase in I_SC, which was inhibited by the β₂ adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl⁻ channel (CaCC), but not K⁺ channel blockers, were able to inhibit the increase in I_SC. “Trimultaneous” recording of I_SC and intracellular cyclic adenosine monophosphate (cAMP) and Ca²⁺ levels in 16HBE14o- epithelia confirmed that the I_SC induced by isoprenaline or procaterol involved both cAMP and Ca²⁺ signaling. Our results demonstrate that β₂ adrenoceptors regulate Cl⁻ secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca²⁺-dependent mechanisms, respectively.