Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF- secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF- detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF- is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

The sensitivity of head and neck squamous cell carcinomas to tumor necrosis factor-α

Sensitivities to recombinant human tumor necrosis factor- (TNF-) and chemotherapeutic agents (cisplatin, peplomycin, methotrexate) were evaluated in 20 tumor cells of head and neck squamous cell carcinomas, using a dye uptake method. Also, numbers of TNF receptors of these tumor cells were measured by Scatchard plot analysis. There was no relationship between the number of TNF- receptors and the sensitivity to TNF-. Furthermore, there was no correlation between the sensitivity to TNF- and that to chemotherapeutic drugs, nor between the sensitivity to TNF- and the clinical response to chemotherapy including of cisplatin and peplomycin. The sensitivity to TNF- was higher in poorly differentiated carcinomas than in well differentiated ones.Abbreviations BSA Bovine serum albumin - CDDP Cisplatin - 5-Fu 5-fluorouracil - IC50 Inhibition concentration 50 - MTX Methotrexate - PLM Peplomycin - TNF- Tumor necrosis factor-     

Interleukin-6 Induced Suppression of Bovine Parathyroid Hormone Secretion

Calcium disturbances in the critically ill coincide with elevations of proinflammatory cytokines. The effects of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) on parathyroid hormone (PTH) secretion were investigated. IL-6 and TNF- had no acute effect on PTH secretion in extracellular Ca²⁺ concentrations of 0.5, 1.25 and 3.0 mM. In contrast to TNF-, cultures for 24 h in the presence of l0 ng/mL of IL-6 showed decreased PTH secretion by 51% and 29% in 0.5 mM and 1.25 mMCa²⁺ respectively. Neither IL-6 nor TNF-, affected cytoplasmic Ca²⁺ of the cells. We conclude that PTH secretion in vitro can be suppressed by IL-6 at clinically relevant concentrations. This suppression may aggravate hypocalcemia of the critically ill and attenuate the conventionally strong stimulation of the PTH release by reduction in serum calcium.     

Cytoprotective mechanisms in cultured cardiomyocytes

Tumor necrosis factor- (TNF-), a potent cytokine mainly secreted by macrophages exerts pleiotropic effects on different cell types. However, the intracellular mediators of its action are not yet well characterized. To get an insight into endogenous cytoprotective mechanisms, we developed an in vitro model based on cultured cardiomyocytes treated with TNF- at which we examined gene expression of heat shock proteins (HSP-27, HSP-70 and ubiquitin). Cardiomyocytes were isolated from the hearts of 18 day old fetal mice by enzymatic dissociation and grown in minimum essential medium containing 10% fetal calf serum. Spontaneously contractile cells were serum deprived for 24 h and treated with TNF- (25 ng/ml) for 1, 2, 4, 6, 8, 12, and 24 h After each incubation, cells were processed to extract total proteins for Western and total RNA for Northern blot analyses. TNF- induced arrhythmias and cessation of spontaneous contractions in a concentration and time dependent manner. Steady state (ubiquitin) or undetectable mRNA levels (HSP-27, HSP-70) were drastically induced (> 4 fold for all three genes vs untreated control cells) by TNF-, reaching maximal values between 6–8 h of stimulation. Thereafter, the expression of these stress genes declined but remained elevated as compared to control. By Western blot analysis, we found increased multiple bands of ubiquitin protein conjugates in TNF-a treated cells whereas no significant change in HSP-27 protein accumulation until 12 h was observed as compared to control. 24 h of TNF- incubation resulted in partial cellular necrosis. Our results indicate that TNF- induces in cardiomyocytes transiently gene expression for cytoprotective molecules like HSP-27, HSP-70 and ubiquitin, suggesting these stress proteins to participate in subsequent defense mechanisms, for example in postischemic myocardial recovery. (Mol Cell Biochem 160/161: 217–224, 1996)     

Contribution of cytokines on the suppression of lung metastasis

Weekly injection of a protein-bound polysaccharide PSK in mice with Lewis Lung Cancer (LLC) significantly decreased the number of lung metastatic foci concomitant with enhancement of cytostatic activity in the bronchoalveolar lavage (BAL) cells. These effects were more marked when the agent was given intratracheally, inducing a larger number of pulmonary macrophages, lymphocytes and neutrophils concomitant with increases in BAL tumor necrosis factor- (TNF-), mouse inflammatory protein- (MIP-1), mouse inflammatory protein- (MIP-1), interleukin-1 (IL-1) and interleukin-6 (IL-6), but not interleukin-2(IL-2) and interleukin-4 (IL-4). Pre-treatment with anti TNF- antibody reduced these effects. The time course and production of PSK-induced cytokines were similar between the tumor-bearing mice and control mice. BAL neutrophils in mice with LLC showed a tendency toward acceleration of O2- production compared with circulating neutrophils. Pulmonary macrophage phagocytosis was also significantly higher in the LLC mice.These results suggest that enhancement of cytostasis appears to be induced by activation and/or improvement of function in inflammatory and immune cells through cytokines under immunomodulator treatment in lung metastasis, possibly via a TNF--dependent mechanism.     

Ischemic preconditioning ameliorates microcirculatory disturbance through downregulation of TNF-alpha production in a rat cremaster muscle model

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IB--NF-B-TNF- (tumor necrosis factor-) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n=8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF- expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF- protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IB- phosphorylation and NF-B (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-B activation and subsequently reduced TNF- expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.     

FR167653 suppresses the progression of experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) induced in rats by injection of cardiac myosin is an animal model of human myocarditis and post-myocarditis dilated cardiomyopathy. It has been reported that proinflammatory cytokines play crucial roles in the induction of EAM and in the progression of myocardial injury in this disease. FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl) pyrazolo [5,1-c] [1,2,4] triazin-2-yl]-2-phenylethanedione sulfate monohydrate) as been reported to suppress tumor necrosis factor-alpha (TNF-). We hypothesized that FR167653 would suppress the progression of EAM if TNF- and/or interleukin-1 beta (IL-1) were the culprit cytokines in EAM. To investigate the effects of FR167653 in EAM, FR167653 was given to rats for 4 weeks, immediately after they had been immunized with cardiac myosin. The ratio of heart weight to body weight and the area of inflammatory lesions were less in the FR167653 groups than in the control rats. FR167653 reduced serum sialic acid levels significantly. The control group showed a deterioration in cardiac function. The FR167653 groups had significantly better hemodynamic parameters, including improved left ventricular end-diastolic pressure, central venous pressure, aortic pressure, and positive and negative left ventricular pressure derivatives. mRNA expression of IL-1 in the heart was significantly lower in rats given FR167653. However, mRNA of TNF- was not detected in any groups. Our results suggest that FR167653 suppresses the development of myocarditis by suppression of IL-1.     

Metabolisme du γ-aminobutyrate chez Agaricus bisporus Lge.

Summary Transamination between -aminobutyrate and -ketoglutarate provides a pathway for the utilization of -aminobutyrate in fruit-bodies of Agaricus bisporus Lge. This reaction leads to the formation of succinic semialdehyde, a metabolic intermediate in the metabolism of -aminobutyrate to succinate in the cell. -aminobutyrate: -ketoglutarate aminotransferase (E.C. 2.6.1.19) was sonically extracted from the mitochondrial fraction and partially purified by DEAE-cellulose column chromatography. Aminotransferase had a pH optimum between 8.1 and 8.5 and did not require pyridoxal-phosphate in vitro; however, the enzyme was inhibited by carbonyl-trapping reagents such as pyridoxal-phosphate activated enzymes. The Km values for -aminobutyrate and -ketoglutarate calculated from Lineweaver-Burk plots were 2.2×10^-4 M and 2.5×10^-3 M, respectively. The transaminase was specific for -ketoglutarate but not for -aminobutyrate; aspartate, -alanine and -aminovalerianate also functioned as amino-group donors. Activity of the enzyme was not influenced by the addition of carboxylic acids of the Krebs cycle. The reversal of the transamination reaction showed optimal rates at pH 9.0–9.3. Some considerations on the physiological significance of these results are given.

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Abréviations DEAE diéthylaminoéthyl - EDTA éthylène diamine tétraacétate - DCIP 2,6-dichlorophénol-indophénol - GABA acide -aminobutyrique - GABA-T -aminobutyrate: -cétoglutarate aminotransférase - GAD L-glutamate décarboxylase - Glu acide glutamique - -KG -cétoglutarate - MBTH 3-méthyl-2-benzothiazolinone hydrazone - PLP pyridoxal-5-phosphate - PMS phénazine méthosulfate - SSA acide semialdéhyde succinique - TCA acide trichloracétique - Tris 2-amino-2-(hydroxyméthyl)-1,3-propanediol     

Interaction of Phomopsin A with Normal and Subtilisin-Treated Bovine Brain Tubulin

Tubulin, the major component of microtubules, has a tendency to lose its ability to assemble or to bind to ligands in a time-dependent process known as decay. The decay process also causes tubulin to expose sulfhydryl groups and hydrophobic areas. The antimitotic drug phomopsin A strongly protects the tubulin molecule from decay. Here we have studied the interaction of phomopsin A with tubulin and tubulin which has been treated with subtilisin to remove selectively the C-termini of the and chains (_ss). The binding of phomopsin A to tubulin decreases the sulfhydryl titer by approximately 1.0 mol/mol. Selective removal of the peptides from the C-terminal ends does not affect phomopsin A's interaction with tubulin. Moreover, the _ss tubulin–phomopsin A complex appears to be more stable than the tubulin–phomopsin A complex as determined by the time-dependent increase in exposure of sulfhydryl groups and hydrophobic areas on tubulin. In fact, phomopsin A inhibits the decay process of _ss tubulin completely. This observation raises the possibility of determining the conformtion of this configuration of tubulin.     

Cytidine 5′-Diphosphocholine (CDP-Choline) in Stroke and Other CNS Disorders

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor- (TNF-) and interleukin-1 (IL-1/) activate phospholipase A₂ (PLA₂) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA₂ releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA₂ stimulation and loss of CCT activity. TNF- also stimulates proteolysis of CCT. TNF- and IL-1 are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA₂ and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF- and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.     

TNF-α induced altered signaling mechanism in human neutrophil

In the present study we investigated the TNF- induced signal transduction mechanism in human neutrophil. Exogenously added TNF- affects both PKC activity and its translocation from cytosol to the membrane. Endogenous protein phosphorylation pattern is inhibited in TNF- induced neutrophil in Ca-dependent and Ca-independent manner, including a major 47 and 66 kDa cytosolic proteins, which may be implicated in superoxide anion generation. However TNF- dose dependently enhances the expression of -PKC isotype but not the -PKC. Morphology and cell cytotoxicity are studied in TNF- treated neutrophil to understand the TNF- induced cell death or apoptosis and these experiment is further confirmed by DNA fragmentation analysis. These results clearly demonstrate that TNF- induces cellular death of human neutrophil at least in part by enhanced expression of Ca-independent -PKC. These observations provide an insight towards understanding the function of -PKC in apoptotic pathway.     

Human blood monocyte activation byNocardia rubra cell wall skeleton for productions of interleukin 1 and tumor necrosis factor-α

Human blood monocytes were obtained from peripheral blood of healthy donors by counter-flow centrifugal elutriation. Functional integrity of monocytes for production of interleukin 1 (IL-1) and tumor necrosis factor (TNF-) in response toNocardia rubra cell wall skeleton (N-CWS) was examined by bioassay and enzyme immunoassay. Monocytes treated with N-CWS at more than 0.5 g/ml produced IL-1 and TNF- extracellularly. Extracellular TNF activity appeared within 4 h, and maximally, 16 h after N-CWS stimulation, whereas longer time was needed for IL-1 activity to appear, the peak production being at 24 h. The neutralizing experiment also showed that anti TNF- antibody did not affect IL-1 production by the monocytes treated with N-CWS, suggesting independen cy of IL-1 production of TNF-.These results suggest that the therapeutic antitumor effect of N-CWS is due, in part at least, to the augmented production of these monokines.     

Palmitates of Isomeric 15-Oxygenated Δ8(14)-Sterols

3-Hexadecanoyloxy-5-cholest-8(14)-en-15-one, 3-hexadecanoyloxy-5-cholest-8(14)-en-15-one, 15-hexadecanoyloxy-5-cholest-8(14)-en-3-ol, 15-hexadecanoyloxy-5-cholest-8(14)-en-3-ol, 15-hexadecanoyloxy-5-cholest-8(14)-en-3-one, and 15-hexadecanoyloxy-5-cholest-8(14)-en-3-one were synthesized and their chromatographic and ¹H NMR characteristics were determined.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

The molecular weight of the basic polypetide chain αB2 of α-crystallin

The molecular weights calculated from the amino acid sequences of the A and B chains of the lens protein -crystallin differ only slightly (19830 and 20070, respectively). SDS gel electrophoresis of these chains and comparison with marker proteins yield apparent molecular weights of 19500 for A and 22500 for B. The discrepancy between the value of 22500 and the real molecular weight of 20070 for B vanishes by the combined use of SDS and 6 M urea in the polyacrylamide gels.     

Continuous culture ofEscherichia coli for extracellular production of recombinant amylase

Summary A recombinantEscherichia coli, grown in continuous culture, expressed aBacillus stearothermophilus -amylase at 100-fold higher activities than theB. stearothermophilus itself. Excretion of the -amylase to the supernatant was shown and found to be independent of the growth rate of the organism. Eleven to eighteen percent of the -amylase was found in the supernatant. Dilution rates, or cell growth rates, ranging from 0.1 to 1.0 hours^–1 were shown not to affect the compartmentation of the amylase and -galactosidase.     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

N-Acetylcysteine ameliorates lipopolysaccharide-induced organ damage in conscious rats

Lipopolysaccharide is strongly associated with septic shock, leading to multiple organ failure. It can activate monocytes and macrophages to release proinflammatory mediators such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and nitric oxide (NO). The present experiments were designed to induce endotoxin shock by an intravenous injection ofKlebsiella pneumoniae lipopolysaccharide (LPS, 10 mg/kg) in conscious rats. Arterial pressure and heart rate (HR) were continuously monitored for 48 h after LPS administration. N-Acetyl-cysteine was used to study its effects on organ damage. Biochemical substances were measured to reflect organ functions. Biochemical factors included blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate transferase (GOT), alanine transferase (GPT), TNF-, IL-1, methyl guanidine (MG), and nitrites/nitrates. LPS caused significant increases in blood BUN, Cre, LDH, CPK, GOT, GPT, TNF-, IL-1, MG levels, and HR, as well as a decrease in mean arterial pressure and an elevation of nitrites/nitrates. N-Acetylcysteine suppressed the release of TNF-, IL-1, and MG, but enhanced NO production. These actions ameliorate LPS-induced organ damage in conscious rats. The beneficial effects may suggest a potential chemopreventive effect of this compound in sepsis prevention and treatment.