Characterisation of troponin-T from salmonid fish

Five major troponin-T isoforms were isolated from the myotomal muscles of Atlantic salmon: three from fast muscle (Tn-T1F, Tn-T2F and Tn-T3F) and two from slow muscle (Tn-T1S and Tn-T2S). In addition to their presence in troponin preparations, these proteins were also recognised to be Tn-T on the basis of immunoreaction with anti-troponin-T antibodies and partial amino acid sequence. The electrophoretic mobility in the presence of SDS of the various Tn-Ts increases in the order: 1S < 1F < 2S < 2F 3F. Compositional analysis shows that the higher M_r forms (1F and 1S) contain considerably more proline, glutamic acid and alanine than the lower-M_r forms (2F, 3F and 2S). Every isoform lacks cysteine and phosphoserine is present only in isoforms 2F and 3F. All of the Tn-Ts, with the exception of isoform 1F, are N-terminally blocked. CNBr fragments from same cell type Tn-Ts yield identical sequences over at least fifteen Edman cycles. Two full-length cDNA sequences, presumed to represent 1S and 3F, or isoforms that are highly similar, are reported. As documented for higher vertebrate Tn-Ts, the predicted primary structures display a non-uniform distribution of charged amino acids and greater divergence at each end than in the central section. The most striking difference between the two salmonid proteins is the presence of a N-terminal (proline-, glutamic acid- and alanine-rich) extension of about fifty amino acids in Tn-T1S (278 amino acids) that is missing from the fast muscle Tn-T (223 amino acids). The sequences also differ in that 1S lacks the known phosphorylation site while the fast-type isoform contains serine next to the initiating methionine. Of the two, the slow isoform has accumulated the greater number of substitutions.     

Characterisation of cathelicidin gene family members in divergent fish species

Cathelicidins are antimicrobial peptides, well studied in mammals and found to be multifunctional proteins, important in the fight against bacterial invasion. Cathelicidins in fish have only recently been identified and little is known about their function and importance in the immune system of fish. In this study we have identified several novel cathelicidin proteins in far related fish species such as Atlantic cod (Gadus morhua) and Arctic charr (Salvelinus alpinus). Atlantic cod was found to have at least three cathelicidin genes of which two are nearly identical except for a nine-amino acid duplication in the antimicrobial peptide region. The predicted mature peptides of cod were found to be unusual peptides, made mainly of arginine, glycine and serine (RGS) residues and form a novel class of antimicrobial peptides. Cathelicidin in Arctic charr and brook trout (Salvelinus fontinalis) were found to have an exon deletion in the cathelin region of the protein, which would lead to the deletion of the predicted loop 2 of cathelin and its adjacent beta-strands. This is the first report of a deletion of a whole exon in the family of the cathelicidins. Infection of fish with pathogenic bacteria caused an upregulation of the expression of the cathelicidins in Arctic charr and Atlantic cod and indicates a role of these proteins in fish innate immunity.     

Patterns in the biosynthesis and action of human gamma-interferon

The highest yields of gamma-interferon activity were obtained by using a fraction of mononuclears recovered from freshly collected donor blood in ficoll-verografin density gradient without using hemolysis. Unification of mononuclears from individual donors into a common pool stimulated interferon production. Staphylococcal enterotoxins A and B, concanavalin A, and lentyl-lectin were found to be the most effective inducers. Immobilization of inducers on neutral carriers reduced their effectiveness. Upon induction with lectin the synthesis was complete within 24 hours, and with enterotoxin in 3 days. In the latter instance the synthesis dynamics was of a two-phase nature. Gamma-interferon produced the antiviral condition later (in 10 hours) than alpha-interferon.     

A macrophage migration-inhibiting factor in preparations of alpha- and gamma-interferon

Native preparations of human alpha- and gamma-interferon synthesized in stationary, suspension or roller leukocyte cultures regularly showed high levels of migration inhibition factor (MIF) activity a significant portion of which was lost in the course of interferon purification and concentration. Minimal losses of MIF activity were achieved when medicinal preparations of interferon were made by the method of negative immunoabsorption on immobilized antibodies to inductor antigens and chromatography on Sephadex G-25.     

Melatonin induced increase in gamma-interferon production by murine splenocytes.

Previously, we demonstrated that production of gamma-interferon (gamma-IFN) by the mouse splenocytes isolated at night was higher than from those isolated in the morning. In this paper we show that melatonin increased gamma-IFN production by murine splenocytes. Moreover, this stimulating effect was significantly higher (10 times) in the cells isolated at night than in those isolated in the morning (2 times).     

Imbalance of alpha- and gamma-interferon levels in patients with herpetic keratitis

The paper presents data on elevated serum and lacrimal fluid alpha- and gamma-interferon (INF) levels in patients with herpetic keratitis (HK) in the course of the disease depending on its clinical form and stage. A more significant increase in the levels of alpha- and gamma-INF was noted in the lacrimal fluid, which was indicative of the important role of local antiherpetic corneal and conjunctival protection. The determination of serum and lacrimal fluid IFN levels in patients with HK may be used to predict the clinical course of the disease.     

Mechanisms of macrophage activation in rheumatoid arthritis: the role of gamma-interferon

Gamma-interferon (gamma-IFN) is a potent inducer of surface expression of class II MHC molecules in vitro. Enhanced HLA-DR expression is a characteristic immuno-histological feature of rheumatoid joints. To assess the possible relevance of gamma-IFN to macrophage (M phi) activation in rheumatoid arthritis (RA) we investigated the spontaneous and mitogen-induced production of gamma-IFN by RA lymphocytes using a sensitive radioimmunoassay. Synovial fluids (SF) from a variety of inflammatory and non-inflammatory rheumatic diseases did not contain measurable amounts of IFN. RA lymphocytes from peripheral blood (PBL) and joints failed to show spontaneous gamma-IFN production. RA and control PBL were equally responsive to both mitogen stimulation and to the addition of exogenous interleukin 2 (IL-2) as control PBL. SF lymphocytes from RA patients showed a significantly decreased PHA-stimulated gamma-IFN production and this was in contrast to the SF lymphocytes from patients with other inflammatory joint diseases who showed significantly increased gamma-IFN production compared with matched PB lymphocytes. These results show that gamma-IFN production by peripheral blood and joint cells from patients with RA is normal and it remains to be established whether gamma-IFN is the factor responsible for the macrophage activation seen in the disease.     

Kinetics of mucosal ileal gamma-interferon response during cryptosporidiosis in immunocompetent neonatal mice

 The kinetics of serum and ileal interferon-gamma (IFN-γ) content were determined during recovery from cryptosporidiosis in NMRI suckling mice. A total of 60 mice aged 4 days were inoculated by intragastric gavage with 10⁴ cryptosporidia (n = 30) or phosphate-buffered saline (n = 30). Six animals per group were killed on days 0, 3, 6, 9 and 13 postinoculation. Blood samples and ileum were collected. Experimental infection was followed by a rise in parasite load in the ileum starting on day 3 postinfection, which peaked at day 6 postinoculation. Ileal IFN-γ levels increased rapidly in parasitized mice from day 3 to day 6, then fell rapidly. These levels were significantly higher than the control values (day 3 P<0.05, days 6 and 9 P<0.001). IFN-γ secretion began before parasite excretion, but the curves of these two parameters correlated positively. Recovery from cryptosporidiosis in immunocompetent neonatal mice is thus associated with an early and marked increase in ileal IFN-γ content. Received: 22 February 1996 / Accepted:15May 1996     

Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).     

Candidacidal mechanisms of peritoneal macrophages activated with lymphokines or gamma-interferon

The mechanisms by which resident peritoneal macrophages, activated in vitro by lymphokines (LK) or recombinant gamma-interferon (IFN), kill Candida parapsilosis or C. albicans were studied. Resident non-activated peritoneal macrophages killed C. parapsilosis (55.5% SD 6.8%), but not C. albicans. This killing was completely inhibited by superoxide dismutase (SOD), partially by dimethyl sulphoxide (DMSO), but not by catalase or azide. Killing correlated with a brisk lucigenin-dependent chemiluminescence (CL) response by macrophages interacting with C. parapsilosis. No enhanced luminol-dependent CL response was observed in this system. This suggests that C. parapsilosis is killed by resident macrophages via a mechanism dependent on the presence of superoxide anion. By contrast, killing of C. parapsilosis by activated macrophages (49.0% SD 5.9%) was not inhibited by SOD or DMSO, suggesting the induction of a non-oxidative candidacidal mechanism. C. albicans was killed only by macrophages activated with IFN (52.0% SD 3.7%) or LK (55.7% SD 2.8%). Inhibition of killing by SOD was greater in IFN- than in LK-activated macrophages. Conversely, killing by LK-, but not IFN-, activated macrophages was significantly inhibited by catalase, DMSO or azide. The killing by LK-activated macrophages, and its inhibition by scavengers, correlated with the luminol-dependent CL response. The non-killing resident macrophages interacting with C. albicans made lucigenin-dependent CL responses similar to those of activated macrophages. The mechanisms enabling killing of C. albicans induced by activation appear to be different for LK and IFN, and appear to depend upon the myeloperoxidase systems and superoxide respectively.     

The synergism of gamma-interferon and tumor necrosis factor in whole body hyperthermia

It is suggested that gamma-interferon (gamma-IFN) can be added to tumor necrosis factor (TNF) to improve therapy in whole body hyperthermia (WBH). The amount of r-HTNF is about 3.5 x 10(6) units per week with about 3.5 x 10(4) units of rH gamma-INF intravenously. The body temperature should be 40 degrees C for one half hour. An i.p. injection of secobarbital (24 mg/kg) should be given as well.     

原癌基因启动子中G-四链体的研究进展

G-四链体是富含G的核酸序列形成的一种非典型的DNA二级结构,在多个原癌基因启动子中能够负调控基因表达,进而抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡及减缓肿瘤细胞迁移等,为肿瘤靶向治疗提供了新靶点。本文阐述了多个原癌基因启动子G-四链体结构特点、小分子配体对G-四链体的稳定作用以及启动子中的G-四链体的基因表达调控作用。     

Serum values of interleukin-10, gamma-interferon and vitamin A in female adolescents

Many studies have demonstrated that vitamin A deficiency (VAD) affects the immunomodulated response mediated by cytokines. However, these studies are controversial. The purpose of the present study was to analyze Interleukin-10, gamma-Interferon and vitamin A serum concentrations in adolescents. Seventy three female, not pregnant adolescents (15.95 +/- 1.10 years old), of a low socioeconomic condition were studied. Serum retinol was determined by HPLC using the Bieri method. International reference standards were considered to define VAD (serum retinol < 20 microg/dL), risk of VAD (20-30 microg/dL) and vitamin A sufficiency (>30 microg/dL). Serum concentrations of Interleukine-10 (IL-10) and gamma-Interferon (gamma-IFN) were detected by an ELISA method (pg/mL). The data were analyzed using the SAS/STAT statistical program; the results were presented as mean +/- Standard deviation and the differences between mean values were analyzed by the ANOVA test. The prevalence of VAD in adolescents was 6.85% (serum retinol <20 microg/dL) and 41.10% adolescents had VAD risk (20-30 microg/dL). Adolescents with VAD showed a significant increase of gamma-IFN serum concentration (p = 0.01). Correlation between serum retinol and gamma-IFN was r = -0.29 (p = 0.01). Adolescents represent a VAD risk group. Low serum levels of retinol were correlated with high levels of gamma-IFN, this cytokine has been associated with chronics inflammatory processes and it can contribute to increase the morbidity and mortality in this population.     

Steroid responsive prolonged thrombocytopenia in dengue

Prolonged thrombocytopenia in a usual case of dengue virus infection is uncommon. Dengue-related thrombocytopenia is self-limiting and responds within 3-5 days. An underlying immunological disorder may be responsible for delayed return of platelet count to a normal level. We present a case of prolonged thrombocytopenia in a case of dengue hemorrhagic fever. The response to steroids suggests a possible immunological dysfunction.     

Characterisation of chicken viperin

The identification of immune pathways that protect against pathogens may lead to novel molecular therapies for both livestock and human health. Interferon (IFN) is a major response pathway that stimulates multiple genes targeted towards reducing virus. Viperin is one such interferon stimulated gene (ISG) that helps protect mammals from virus and may be critical to protecting chickens in the same way. In chickens, ISGs are not generally well characterised and viperin, in concert with other ISGs, may be important in protecting against virus. Here we identify chicken viperin (ch-viperin) and show that ch-viperin is upregulated in response to viral signature molecules. We further show that viperin is upregulated in response to virus infection in vivo. This data will benefit investigators targeting the antiviral pathways in the chicken.