Scavenger receptors may regulate nitric oxide production from macrophages stimulated by LPS

A new crystal form of papain from the latex of Carica papaya, complexed with an inhibitor (Z-Arg-Leu-Val-Gly-CHN2) was obtained by the vapor-diffusion method using a methanol/ethanol mixture as a precipitant. The slat-like crystals are monoclinic, space group P2(1), with unit cell parameters a = 42.6 A, b = 49.8 A, c = 50.5 A, A = 111.9 degrees, and contain one molecule in the asymmetric unit. The crystals are stable in the X-ray beam and diffract beyond 1.8 A. A molecular model has been placed in the unit cell by molecular replacement.     

IFN-gamma-induced TNF-alpha is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN-gamma

Dopamine is formed form L-tyrosine by tyrosine hydroxylase and aromatic L-amino acid decarboxylase. In addition to this pathway, however, the formation of catecholamines, including dopamine, from trace amines such as tyramine by hepatic microsomes has been demonstrated. In this study, we investigated the formation of dopamine from trace amines, using human hepatic microsomes and human cytochrome P450 (CYP) isoforms expressed in yeast. Among the 11 isoforms of human CYP expressed in yeast, CYP2D6 was the only isoform exhibiting strong ability to convert p-tyramine and m-tyramine to dopamine. In studies with human hepatic microsomes, the hydroxylation of tyramine to dopamine was inhibited by bufuralol, a typical substrate for CYP2D isoforms, and anti-CYP2D1 antiserum. This is the first report showing that CYP2D is capable of converting tyramine to dopamine. The Km values of CYP2D6, expressed in yeast, for p-tyramine and m-tyramine were 190.1 +/- 19.5 microM and 58.2 +/- 13.8 microM, respectively. Tyramine is an endogenous compound which exists in the brain as a trace amine but is also an exogenous compound which is found in foods such as cheese and wine. Our results suggest that dopamine is formed from endogenous and/or exogenous tyramine by this CYP2D isoform.     

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.     

Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

Stimulation of nitric oxide production in macrophages by Babesia bovis

Gamma interferon (IFN-gamma)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-gamma, B. bovis merozoites stimulated NO production, as indicated by the presence of increased L-arginine-dependent nitrite (NO2-) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, were also found to induce L-arginine-dependent NO2- production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.     

Stimulation of macrophages by Bacillus firmus: production of nitric oxide and cytokines

Immunostimulatory properties of gram-positive Bacillus firmus were investigated under in vitro conditions using murine peritoneal macrophages. B. firmus stimulated in a concentration and time dependent manner the secretion of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10), but it had no influence upon interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) production. It also substantially augmented production of nitric oxide (NO) induced by exogenous IFN-gamma. Inhibitory experiments using neutralizing antibodies against TNF-alpha and/or IL-10 have demonstrated that these cytokines are responsible for triggering the underlying mechanism(s) leading to enhanced NO production. The cytokine-stimulatory and NO-costimulatory properties could participate in the antiinfectious and anticancer effects of B. firmus, detected previously in the in vivo experiments.     

The role of nitric oxide in the regulation of aldosterone synthesis by adrenal glomerulosa cells

The role of nitric oxide (NO) in the regulation of aldosterone synthesis in the adrenal glomerulosa is not known. In this study, we observed that liberators of NO such as S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (Snp) and spermine nonoate (SNO) could significantly inhibit angiotensin II (AII) and ACTH-induced aldosterone synthesis in isolated rat and cultured human adrenal glomerulosa cells. To evaluate more precisely whether glomerulosa cells express NO synthase, we performed immunoblotting experiments with antibodies specific for the endothelial type ecNO synthase as well as the neuronal NO synthase. This revealed the presence of the ecNO synthase in rat adrenal capsules, in normal and in adenomatous human adrenal glomerulosa tissue, as well as in freshly dispersed rat adrenal glomerulosa cells. Furthermore, on immunohistochemical analysis, rat adrenal glomerulosa cell sections showed strongly positive staining for ecNO synthase. These results suggest that NO may be an important negative modulator of adrenal glomerulosa steroidogenesis.     

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia

This brief history of the dental diamond bur is intended to provide both a historical perspective and an evaluation of the current state of bur technology. An understanding of the origins of dental diamonds and the issues facing manufacturers transforms the dentist from a simple user into an informed consumer. The author contends that this can improve dental care and enable the dentist to collaborate with manufacturers in developing improved dental burs.     

Regulation of nitric oxide production by rat alveolar macrophages in response to silica exposure

Research conducted primarily over the past 5-8 years on the psychosocial effects of pediatric chronic physical disorders on children and their families is reviewed. A large body of studies show that both children and their mothers, as groups, are at increased risk for psychosocial adjustment problems compared to peers, but that there is considerable individual variation in outcome. Since the last review on this topic (Eiser, 1990a), many studies have been conducted to identify risk and resistance factors associated with differences in adjustment among these children and their mothers. Improvements are noted in the theoretical basis for this work, programmatic nature of some of the research, and efforts at producing clinically relevant information. Evaluations of interventions, however, are lagging. Critical issues and future directions regarding developmental approaches, theory, method, measurement, and intervention are discussed.     

Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin

This study examined the sleep and mood differences between premenopausal and perimenopausal women matched for age and sociodemographic variables. Wrist actigraphy, Profile of Mood State (POMS), State-Trait Anxiety Inventory (STAI), a sleep questionnaire, and responses to a sleep diary were recorded for a period of 1 week. It was found that the sleep disruption of perimenopausal subjects was significantly greater than that of the premenopausal group (p < 0.05). Overall, the perimenopausal group demonstrated a significant increase in sleep disruption and mood alterations when compared with the premenopausal group. Actigraphic data showed that perimenopausal subjects experienced longer and more numerous arousals resulting in significantly less sleep (p < 0.05). In addition, perimenopausal subjects scored significantly higher (p < 0.05) on the STAI and significantly lower on the Vigor subscale of the POMS (p < 0.01) than premenopausal subjects. Correlational analyses indicated that sleep and mood changes were significantly related in the perimenopausal group, but not in the premenopausal group. Taken together, these results suggest that the mood changes experienced by the perimenopausal group may be mediated by sleep disruption.     

Horseradish peroxidase and glycosylated BSA induce nitric oxide production in murine macrophages

The in vitro activation of murine macrophages by horseradish peroxidase (HRP) induced nitric oxide production in a dose-dependent manner, and increased the induction of NO-synthase by LPS. Nitrite production after HRP stimulation was inhibited by NG-monomethyl-L-arginine (NMMA), a specific inhibitor of NO-synthase. Equivalent amounts of nitrite were obtained with native and heat-inactivated HRP. High concentrations of mannose inhibited nitric oxide production, while the HRP inhibitor 3-aminotyrosine did not. Glycosylated serum albumin derivatives also induced murine macrophage NOS, probably by an interaction between carbohydrates and their specific cell membrane receptors. The inability of HRP apoprotein to stimulate NO production, and the specific inhibition of HRP-mediated activation of macrophages by hemin suggests that the heme moiety of this enzyme is involved in NO-synthase induction.     

Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages

Leishmania parasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand fly Phlebotomus papatasi is the vector of Leishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate that P. papatasi saliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that the P. papatasi salivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by which P. papatasi saliva exacerbates cutaneous leishmaniasis.     

Effect of prolactin on nitric oxide and interleukin-1 production of murine peritoneal macrophages: role of Ca2+ and protein kinase C

The Psychiatric Hospital at the Municipal (General) Hospital in G?rlitz, Germany, was the only Department of Psychiatry in a non-University (general) hospital in the newly integrated German provinces who were originally part of the so-called "German Democratic Republic" before the re-unification of Germany, to participate in the German collective study on "Psychiatric and Psychosomatic Consultation and Liaison Service in German General Hospitals-A Multicentre Empirical Study to Assess and Evaluate Existing Structures and Services". This study is an independent part project that includes specific questions forming part of the European collective study on "Effectiveness of Mental Health Consultation and Liaison Service Delivery in the General Hospital".     

The role of prostaglandin E2 and nitric oxide in cell death in J774 murine macrophages

We investigated the role of prostaglandin E2 (PGE2) and its interactions with nitric oxide (NO) on cell death and NO-mediated cytotoxicity in the murine macrophage cell line J774. Stimulation of the J774 cells with lipopolysaccharide together with interferon-gamma resulted in a dose-dependent cytotoxicity and production of PGE2 and NO, measured as nitrite. Our results showed a linear correlation between PGE2 release and cytotoxicity. The cyclooxygenase (COX) inhibitor indomethacin completely inhibited PGE2 biosynthesis, without affecting NO production or cell death. This supports previous reports suggesting that overproduction of endogenous PGE2 is mainly the consequence of cell death and does not cause it. In contrast, the NO synthase inhibitor N(omega)-monomethyl-L-arginine (L-NMMA) gave a significant, though incomplete suppression of NO release and cell death. This points to the presence of other cytotoxic factors besides NO. To evaluate the toxic effect solely due to NO, macrophages were exposed to the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Incubation with SNAP also resulted in a concentration-dependent cell injury and PGE2 production. When exogenously added, PGE2 protected against SNAP-mediated cytotoxicity and simultaneously increased PGE2 release into the medium, without inducing COX-2. The cytoprotection and the stimulation of PGE2 release were both reversed by indomethacin. In conclusion, PGE2 biosynthesis may represent a mechanism by which inflammatory macrophages protect themselves against the cytotoxic effects of NO.     

Aflatoxin B1-induced suppression of nitric oxide production in murine peritoneal macrophages

Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and non-specific immune responses. AFB1 mainly decreases lymphocyte functions and may also affect macrophages assisting lymphocyte functions. Macrophages play an important role in a host defense against tumors and bacteria. Furthermore, some macrophage products, including nitric oxide (NO), may be involved in cytotoxicity. The effect of aflatoxin B1 (AFB1) was investigated on NO production from murine peritoneal macrophages. Macrophages were pretreated with AFB1 for 24 h and then stimulated with lipopolysaccharide (LPS) for 24 h. AFB1 at 10 or 50 microM reduced the production of NO. Compared to vehicle control, there was a greater reduction of NO production with increased AFB1 pretreatment and LPS stimulation. AFB1 at 10 or 50 microM decreased inducible nitric oxide synthase (iNOS) activity about 24% and 28%, respectively, after stimulation with 1 microg/ml LPS and about 12% and 24%, respectively, after stimulation with 10 microg/ml LPS. AFB1 pretreatment also decreased the synthesis of iNOS protein and the mRNA of macrophages. Taken together, these results suggest that AFB1 pretreatment reduces NO production from murine peritoneal macrophages stimulated by LPS, which is mediated by the reduction of iNOS activity, mRNA, and protein.