Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes

Stimulation of rat astrocytes in vitro by calcium ionophore A23187 and/or lipopolysaccharide results in the generation of a cytotoxic factor that is functionally similar to the previously described macrophage-derived cytotoxic factor, tumor necrosis factor. Like the macrophage product, the astrocyte cytotoxic factor kills murine L 929 cell targets. In addition, it kills rat oligodendrocytes, the myelin-producing cells of the central nervous system. Human recombinant tumor necrosis factor also has cytotoxic activity directed against rat oligodendrocytes.     

Tyrosine kinase activation in LPS stimulated rat kupffer cells

Kupffer cells, a majority of the body's fixed macrophages, are a major site of bacterial lipopolysaccharide (LPS) metabolism and are mediators in the body's response to sepsis. Uptake of LPS is different in Kupffer cells than other macrophages. Signal transduction in other macrophages in response to LPS involves phosphorylation of proteins in the 50–60 kDa range. We hypothesized that Kupffer cells may have unique signal transduction pathways in response to LPS. Rat Kupffer cells were exposed to LPS (1 μg/mL) for varying times ranging from 15 to 90 min. Cell lysates were Western blotted using an anti-phosphotyrosine antibody. The blots showed an increase in the amount of tyrosine phosphorylation on two proteins of 119 kDa and 83 kDa. The effects of varying LPS concentration (1 ng/mL-1 μg/mL) showed an increasing amount of phosphorylation with increasing LPS concentration. To associate the importance of tyrosine phosphorylation in the response of Kupffer cells to LPS, the tyrosine kinase inhibitors, tyrphostin, lavendustin, and genisten were used to study the effects of inhibiting phosphorylation on TNF-α production. Kupffer cells were preincubated in the presence of the inhibitor and exposed to LPS (1 μg/mL). TNF-α was measured in the conditioned media by ELISA. A 70% or greater decrease in TNF-α production was observed. When phagocytosis of latex beads by rat Kupffer cells was measured in vivo using intravital video microscopy, LPS treatment significantly increased uptake. This increase in phagocytosis was inhibited by tyrphostin. These results show what may be unique phosphorylation events in Kupffer cells that are related to LPS induced production of TNF-α. Presented in part at the American Association for the Study of Liver Diseases Annual Meeting, Chicago, IL (USA), November 3–7, 1995.     

Hyperforin promotes mitochondrial function and development of oligodendrocytes

St. John's wort has been found to be an effective and safe herbal treatment for depression in several clinical trials. However, the underlying mechanism of its therapeutic effects is unclear. Recent studies show that the loss and malfunction of oligodendrocytes are closely related to the neuropathological changes in depression, which can be reversed by antidepressant treatment. In this study, we evaluated the effects of hyperforin, a major active component of St. John's wort, on the proliferation, development and mitochondrial function of oligodendrocytes. The study results revealed that hyperforin promotes maturation of oligodendrocytes and increases mitochondrial function without affecting proliferation of an oligodendrocyte progenitor cell line and neural stem/progenitor cells. Hyperforin also prevented mitochondrial toxin-induced cytotoxicity in an oligodendrocyte progenitor cell line. These findings suggest that hyperforin may stimulate the development and function of oligodendrocytes, which could be a mechanism of its effect in depression. Future in vitro and in vivo studies are required to further characterize the mechanisms of hyperforin.     

Dual role of nNOS in ischemic injury and preconditioning

Background  

Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoK_ATP channels and p38MAPK, two key components of IP transduction pathway.     

腹腔注射LPS 对大鼠海马nNOS 和iNOS 表达的影响

目的：探讨外周免疫刺激对大鼠海马nNOS和iNOS表达的影响。方法：腹腔注射LPS600μg／kg建立免疫激发大鼠模型,对照组注射等量的生理盐水,2．5h后用RT—PCR技术,观察两组大鼠海马nNOS mRNA和iNOS mRNA的表达变化,检测OD值并进行统计学分析。结果：腹腔注射LPS组大鼠海马的nNOS和iNOS的mRNA水平较对照组高。结论：海马在大鼠机体的免疫应激反应中起重要调节作用,iNOS和nNOS来源的N0是该调节过程中的重要信使分子。     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.

     

Increased FITC fluorescence on LPS stimulated neutrophils cultured in whole blood

Recently, it has been observed that Annexin V labelling of phosphatidylserine (PS) on non-apoptotic cells can vary in different leukocyte populations and with the activation of cells, due to differences in the absolute level of exposed PS. We have also observed changes in the absolute level of Annexin V-FITC intensity, but under conditions where absolute PS expression did not change. In the present study, we have explored the effect of neutrophil cell activation on Annexin V-FITC fluorescence intensity by comparing alternatively labelled matched antibodies against Annexin V. Human venous whole blood was cultured with and without stimulation with lipopolysaccharide (LPS). Apoptosis in the neutrophil and lymphocyte populations was analyzed by flow cytometry and the intensity of FITC labelling was compared to matched fluorochromes conjugated to the same cell surface markers. There was an increase in the intensity of Annexin V-FITC in non-apoptotic neutrophils when stimulated with LPS, which did not correlate with increased apoptosis. Furthermore, CD65-FITC intensity also increased on activated neutrophils. Activated neutrophils exhibited higher amounts of FITC fluorescence that were not associated with changes in extracellular PS expression. This effect appears to be fluorochrome related, likely due to an increase in the pH surrounding activated neutrophils.     

LPS induces cardiomyocyte injury through calcium-sensing receptor

The development of atherosclerosis (AS) is a multifactorial process in which elevated plasma cholesterol levels play a central role. As a new class of players involved in AS, the regulation and function of microRNAs (miR) in response to AS remain poorly understood. This study analyzed the effects of miR-1 (antagomir and mimic) on endothelial permeability and myosin light chain kinase (MLCK) expression and activity in the artery wall of apoE knock-out mice after feeding them a high-cholesterol diet. Further, we tested to determine whether that effects are involved in ERK phosphorylation. Here, we show that a high-cholesterol diet induces a significant decrease of miR-1 expression. Histopathologic examination demonstrated that miR-1 antagomir enhances endothelial permeability induced by high cholesterol and miR-1 mimic attenuated endothelial barrier dysfunction. Consistent with endothelial permeability, Western blotting, qPCR, and γ-³²P-ATP phosphate incorporation showed that MLCK expression and activity were further increased in miR-1 antagomir-treated mice and decreased in miR-1 mimic-treated mice compared with those of mice receiving control miR. Further mechanistic studies showed that high-cholesterol-induced extracellular signal regulated kinase (ERK) activation was enhanced by miR-1 antagomir and attenuated by miR-1 mimic. Collectively, those results indicate that miR-1 contributes to endothelial barrier function via mechanisms involving not only MLCK expression and activity but also ERK phosphorylation.     

A yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide

Several DNase activities, with different substrate and pH requirements, have been identified in yeast mitochondria. One of them is active on double stranded DNA at neutral pH and stimulated by Ethidium Bromide and other DNA intercalating drugs. This activity could be responsible for the yeast mitochondrial DNA degradation induced during mutagenesis by Ethidium Bromide.     

Neutrophil mediated myocardial injury

• 1.1. Activated polymorphonuclear neutrophils (PMN) were shown to exacerbate ischemic myocardial injury and their activation is modulated by complement system, platelet activating factor, arachidonic acid metabolites, adenosine and nitric oxide.
• 2.2. Mechanisms of injurious PMN effect on ischemic myocardium are related to both mechanical and biochemical processes.
• 3.3. Activated PMN aggregate and adhere to endothelium that results in capillary plugging and subsequent impairment of coronary blood flow as well as participating in the development of endothelial cell edema.
• 4.4. PMN-related biochemical damage of ischemic myocardium is a result of the release of cytotoxic free oxygen radicals and proteolytic enzymes as well as vasoconstrictor leukotriene B4 and leukotoxin.

     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Silver nanoparticles augment releasing of pyrogenic factors by blood cells stimulated with LPS

Silver nanoparticles (AgNPs) have cytotoxic properties via generation of reactive oxygen species which are involved in the generalized sickness behavior of the host, including fever and lethargy among others. The aim of the present study was to investigate the impact of AgNPs on the ability of rat peripheral blood mononuclear cells (PBMCs) to release fever mediating factors after stimulation with lipopolysaccharide (LPS). Body temperature and motor activity of the Wistar rats were measured by biotelemetry system. Rat PBMCs were stimulated with LPS and after that the cells were washed and incubated alone or with AgNPs. The final supernatants were injected intraperitoneally. The levels of endogenous pyrogens such as interleukin-1β (IL^?1β), IL-6 and tumor necrosis factor-α (TNF-α) released from the PBMCs into the final supernatants were also estimated. The results indicated that injection of the supernatants from the cells stimulated with LPS induced fever and inhibited motor activity. These effects were potentiated by the presence of AgNPs during the final incubation. The presence of the AgNPs also resulted in significant increases in levels of endogenous pyrogens. The augmentation of fever in the rats by the AgNPs treatment of the cultures seemed to be primarily associated with the changes in interleukin-1β levels.     

Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein

Oligodendrocytes (OLs) extend arborized processes that are supported by microtubules (MTs) and microfilaments. Little is known about proteins that modulate and interact with the cytoskeleton during myelination. Several lines of evidence suggest a role for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in mediating process formation in OLs. In this study, we report that tubulin is a major CNP-interacting protein. In vitro, CNP binds preferentially to tubulin heterodimers compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces dramatic morphology changes in both glial and nonglial cells, resulting in MT and F-actin reorganization and formation of branched processes. These morphological effects are attributed to CNP MT assembly activity; branched process formation is either substantially reduced or abolished with the expression of loss-of-function mutants. Accordingly, cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. We propose that CNP is an important component of the cytoskeletal machinery that directs process outgrowth in OLs.     

Early functional muscle regeneration after myotoxic injury in mice is unaffected by nNOS absence

Nitric oxide (NO) is an important signaling molecule produced in skeletal muscle primarily via the neuronal subtype of NO synthase (NOS1, or nNOS). While many studies have reported NO production to be important in muscle regeneration, none have examined the contribution of nNOS-derived NO to functional muscle regeneration (i.e., restoration of the muscle's ability to produce force) after acute myotoxic injury. In the present study, we tested the hypothesis that genetic deletion of nNOS would impair functional muscle regeneration after myotoxic injury in nNOS(-/-) mice. We found that nNOS(-/-) mice had lower body mass, lower muscle mass, and smaller myofiber cross-sectional area and that their tibialis anterior (TA) muscles produced lower absolute tetanic forces than those of wild-type littermate controls but that normalized or specific force was identical between the strains. In addition, muscles from nNOS(-/-) mice were more resistant to fatigue than those of wild-type littermates (P < 0.05). To determine whether deletion of nNOS affected muscle regeneration, TA muscles from nNOS(-/-) mice and wild-type littermates were injected with the myotoxin notexin to cause complete fiber degeneration, and muscle structure and function were assessed at 7 and 10 days postinjury. Myofiber cross-sectional area was lower in regenerating nNOS(-/-) mice than wild-type controls at 7 and 10 days postinjury; however, contrary to our original hypothesis, no difference in force-producing capacity of the TA muscle was evident between the two groups at either time point. Our findings reveal that nNOS is not essential for functional muscle regeneration after acute myotoxic damage.     

Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes.

To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.     

Inhibition of microglial fatty acid amide hydrolase modulates LPS stimulated release of inflammatory mediators

Anandamide and other fatty acid amides are metabolised by the enzyme fatty acid amide hydrolase (FAAH), which thereby regulates their endogenous levels. Here we demonstrate that cultured rat cortical microglia express FAAH at low levels. The potent FAAH inhibitor URB597 reduced the LPS stimulated microglial expression of cyclo-oxygenase 2 and inducible nitric oxide, with concomitant attenuation of the release of PGE2 and NO. Additional of supplemental exogenous anandamide did not increase the magnitude of attenuation of mediator release. The effect of URB597 on LPS stimulated PGE2 release was not blocked by selective CB1 or CB2 receptor antagonists.     

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.