Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs.

In eukaryotes, specific DNA-protein structures called telomeres exist at linear chromosome ends. Telomere stability is maintained by a specific capping protein complex. This capping complex is essential for the inhibition of the DNA damage response (DDR) at telomeres and contributes to genome integrity. In Drosophila, the central factors of telomere capping complex are HOAP and HipHop. Furthermore, a DDR protein complex Mre11-Rad50-Nbs (MRN) is known to be important for the telomere association of HOAP and HipHop. However, whether MRN interacts with HOAP and HipHop, and the telomere recognition mechanisms of HOAP and HipHop are poorly understood. Here, we show that Nbs interacts with Mre11 and transports the Mre11-Rad50 complex from the cytoplasm to the nucleus. In addition, we report that HOAP interacts with both Mre11 and Nbs. The N-terminal region of HOAP is essential for its co-localization with HipHop. Finally, we reveal that Nbs interacts with the N-terminal region of HOAP.

CHEK1 coordinates DNA damage signaling and meiotic progression in the male germline of mice.

The continuity of life depends on mechanisms in the germline that ensure the integrity of the genome. The DNA damage response/checkpoint kinases ATM and ATR are essential signaling factors in the germline. However, it remains unknown how a downstream transducer, Checkpoint Kinase 1 (CHEK1 or CHK1), mediates signaling in the male germline. Here, we show that CHEK1 has distinct functions in both the mitotic and meiotic phases of the male germline in mice. In the mitotic phase, CHEK1 is required for the resumption of prospermatogonia proliferation after birth and the maintenance of spermatogonia. In the meiotic phase, we uncovered two functions for CHEK1: one is the stage-specific attenuation of DNA damage signaling on autosomes, and the other is coordination of meiotic stage progression. On autosomes, the loss of CHEK1 delays the removal of DNA damage signaling that manifests as phosphorylation of histone variant H2AX at serine 139 (γH2AX). Importantly, CHEK1 does not have a direct function in meiotic sex chromosome inactivation (MSCI), an essential event in male meiosis, in which ATR is a key regulator. Thus, the functions of ATR and CHEK1 are uncoupled in MSCI, in contrast to their roles in DNA damage signaling in somatic cells. Our study reveals stage-specific functions for CHEK1 that ensure the integrity of the male germline.

FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis.

Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline.

Genomewide identification of target genes of histone methyltransferase dG9a during Drosophila embryogenesis.

Post-translational modification of the histone plays important roles in epigenetic regulation of various biological processes. Among the identified histone methyltransferases (HMTases), G9a is a histone H3 Lys 9 (H3K9)-specific example active in euchromatic regions. Drosophila G9a (dG9a) has been reported to feature H3K9 dimethylation activity in vivo. Here, we show that the time required for hatching of a homozygous dG9a null mutant and heteroallelic combination of dG9a null mutants is delayed, suggesting that dG9a is at least partially responsible for progression of embryogenesis. Immunocytochemical analyses of the wild-type and the dG9a null mutant flies indicated that dG9a localizes in cytoplasm up to nuclear division cycle 7 where it is likely responsible for di-methylation of nucleosome-free H3K9. From cycles 8-11, dG9a moves into the nucleus and is responsible for di-methylating H3K9 in nucleosomes. RNA-sequence analysis utilizing early wild-type and dG9a mutant embryos showed that dG9a down-regulates expression of genes responsible for embryogenesis. RNA fluorescent in situ hybridization analysis further showed temporal and spatial expression patterns of these mRNAs did not significantly change in the dG9a mutant. These results indicate that dG9a controls transcription levels of some zygotic genes without changing temporal and spatial expression patterns of the transcripts of these genes.

The conserved P body component HPat/Pat1 negatively regulates synaptic terminal growth at the larval Drosophila neuromuscular junction.

The temporal and spatial regulation of protein synthesis plays an important role in the control of neural physiology. In axons and dendrites, translationally repressed mRNAs are actively transported to their destinations in a variety of ribonucleoprotein particles (RNPs). A subset of these neuronal RNPs has been shown to contain proteins associated with mRNA processing bodies (P bodies). P bodies are a class of highly conserved cytoplasmic granules that have been linked to both mRNA decay and translational repression via general and miRNA-mediated pathways. Here, we characterize functions for HPat/Pat1 (also known as Patr-1), a core component of P bodies, at the glutamatergic larval Drosophila neuromuscular junction (NMJ). We show that hpat mutants exhibit a strong synaptic hyperplasia at the NMJ. The synaptic defects observed in hpat mutants are associated with rearrangement of the axonal microtubule cytoskeleton suggesting that HPat negatively regulates presynaptic microtubule-based growth during NMJ development. Consistent with this, overexpression of HPat also blocks the rapid growth of presynaptic boutons induced by spaced depolarization. Finally, we demonstrate that HPat interacts genetically with the catalytic subunit of the deadenylase complex (twin/CCR4) and the miRNA pathway (Argonaute 1) to control bouton formation. We propose that HPat is required to target mRNAs involved in the control of microtubule architecture and synaptic terminal growth for repression, presumably in P bodies, via both general and miRNA-mediated mechanisms.

Drosophila Mon2 couples Oskar-induced endocytosis with actin remodeling for cortical anchorage of the germ plasm.

Drosophila pole (germ) plasm contains germline and abdominal determinants. Its assembly begins with the localization and translation of oskar (osk) RNA at the oocyte posterior, to which the pole plasm must be restricted for proper embryonic development. Osk stimulates endocytosis, which in turn promotes actin remodeling to form long F-actin projections at the oocyte posterior pole. Although the endocytosis-coupled actin remodeling appears to be crucial for the pole plasm anchoring, the mechanism linking Osk-induced endocytic activity and actin remodeling is unknown. Here, we report that a Golgi-endosomal protein, Mon2, acts downstream of Osk to remodel cortical actin and to anchor the pole plasm. Mon2 interacts with two actin nucleators known to be involved in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. We also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. We propose that, in response to the Osk-mediated endocytic activation, vesicle-localized Mon2 acts as a scaffold that instructs the actin-remodeling complex to form long F-actin projections. This Mon2-mediated coupling event is crucial to restrict the pole plasm to the oocyte posterior cortex.

Effects of aliskiren on the fibrinolytic system in patients with coronary artery disease receiving angiotensin-converting enzyme inhibitor or angiotensin II type 1 receptor blocker.

Aliskiren is a novel blood pressure-lowering agent acting as an oral direct renin inhibitor. We evaluated the effects of aliskiren on the fibrinolytic system in patients with coronary artery disease who were receiving angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II type 1 receptor blockers (ARBs). We studied 17 patients with coronary artery disease whose systolic blood pressure was more than 130 mmHg despite treatment with ACEIs or ARBs. Aliskiren (150 mg) was added to ACEIs or ARBs, and was continued for 6 weeks. Aliskiren significantly decreased systolic blood pressure (140 ± 6-128 ± 8 mmHg, P < 0.001) and plasma renin activity (1.8 ± 2.3-0.6 ± 0.9 ng/ml/h, P < 0.01) after 6 weeks. However, it did not affect plasminogen activator inhibitor-1 (28.8 ± 14.5-30.6 ± 13.6 ng/ml, P = 0.84), fibrinogen (305 ± 72 vs 301 ± 71 mg/dl, P = 0.33), or D-dimer (0.49 ± 0.24-0.51 ± 0.28 µg/ml, P = 0.70) levels. Our data suggested that patients receiving ACEIs or ARBs would not be expected to have any changes in biomarkers of the fibrinolytic system with additional pharmacologic inhibition of the renin-angiotensin-aldosterone system.

Effects of lipid-lowering therapy with strong statin on serum polyunsaturated fatty acid levels in patients with coronary artery disease.

Residual risk of cardiovascular events after treatment with stain might be explained in part because patients have low levels of n-3 polyunsaturated fatty acids (PUFA). We examined how lipid-lowering therapy with strong statin affected serum PUFA levels in patients with coronary artery disease. The study population consisted of 46 patients with coronary artery disease whose low-density lipoprotein (LDL) cholesterol was more than 100 mg/dl. Lipid-lowering therapy was performed with a strong statin including atorvastatin (n = 22), rosuvastatin (n = 9) or pitavastatin (n = 15). Serum PUFA levels were determined by gas chromatography. The treatment with strong statin decreased the sum of dihomo-γ-linolenic acid (DGLA) and arachidonic acid (AA) levels (195 ± 41 to 184 ± 44 µg/ml, P < 0.05) as well as the sum of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels (233 ± 71 to 200 ± 72 µg/ml, P < 0.001). These effects of strong statin resulted in a significant decrease in ratio of the sum of EPA and DHA levels to the sum of DGLA and AA levels (1.20 ± 0.27 to 1.10 ± 0.35, P < 0.05). The percent decrease in the LDL cholesterol level correlated significantly with that in the sum of EPA and DHA levels (r = 0.38, P < 0.01). In conclusion, our results showed that lipid-lowering therapy with strong statin mainly reduced n-3 PUFAs in proportion to the decrease in the LDL cholesterol level in patients with coronary artery disease.

Sensory input regulates spatial and subtype-specific patterns of neuronal turnover in the adult olfactory bulb.

Throughout life, new neurons are added and old ones eliminated in the adult mouse olfactory bulb. Previous studies suggested that olfactory experience controls the process by which new neurons are integrated into mature circuits. Here we report novel olfactory-experience-dependent mechanisms of neuronal turnover. Using two-photon laser-scanning microscopy and sensory manipulations in adult live mice, we found that the neuronal turnover was dynamically controlled by olfactory input in a neuronal subtype-specific manner. Olfactory input enhanced this turnover, which was characterized by the reiterated use of the same positions in the glomeruli by new neurons. Our results suggest that olfactory-experience-dependent modification of neuronal turnover confers structural plasticity and stability on the olfactory bulb.

Roles of cytoplasmic RNP granules in intracellular RNA localization and translational control in the Drosophila oocyte.

Intracellular mRNA localization and translation are ways to achieve asymmetric protein sorting in polarized cells, and they play fundamental roles in cell-fate decisions and body patterning during animal development. These processes are regulated by the interplay between cis-acting elements and trans-acting RNA-binding proteins that form and occur within a ribonucleoprotein (RNP) complex. Recent studies in the Drosophila oocyte have revealed that RNP complex assembly in the nucleus is critical for the regulation of cytoplasmic mRNA localization and translation. Furthermore, several trans-acting factors promote the reorganization of target mRNAs in the cytoplasm into higher-order RNP granules, which are often visible by light microscopy. Therefore, RNA localization and translation are likely to be coupled within these RNP granules. Notably, diverse cytoplasmic RNP granules observed in different cell types share conserved sets of proteins, suggesting they have fundamental and common cellular functions.

Orally available pyridinylpyrimidine derivatives as novel RANKL-induced osteoclastogenesis inhibitors.

An HTS campaign led to the identification of 4-pyrroldino-2-(pyridin-2-yl)pyrimidine compound 1 as an RANKL-induced osteoclastogenesis inhibitor. The compound 1 showed high clearance values in microsomes, however. Modification of the pyrrolidino group to a benzylamino group improved human microsomal stability with a slight loss of in vitro activity. Substitution at the ortho position of the benzyl group ameliorated in vitro activity, and further fluorination of the benzyl group improved microsomal stability in rodents. Representative members of this series, compounds 20 and 23, exhibited efficacy in RANKL-induced osteopenic mice when administered orally at 0.3 mg/kg.

Pharmacological characterization of AS2690168, a novel small molecule RANKL signal transduction inhibitor.

Pathological osteolysis is associated with excessive bone resorption by activated osteoclasts. Given that receptor activator of NF-kB and its ligand (RANKL) are key players in the differentiation and activation of osteoclasts, the RANKL/RANK signaling pathway is considered a promising target for the development of effective osteoclastogenesis inhibitors. We previously found that the orally available compound, AS2690168, suppresses RANKL-induced osteoclastogenesis of RAW264 cells. In this report, we further characterized the pharmacological profiles of AS2690168 in vitro and in vivo. AS2690168 suppressed soluble RANKL (sRANKL)-induced NFATc1 mRNA expression in RAW264 cells at 0.3 and 3.0 µM. It also suppressed calcium release from parathyroid hormone-stimulated mouse calvaria with an IC50 value of 0.46 µM. Oral administration of AS2690168 completely suppressed the decrease in femoral bone mineral content in an sRANKL-induced osteopenic mice model at 3.0 mg/kg. It also significantly suppressed the decrease in femoral bone mineral density and increase in serum tartrate-resistant acid phosphatase-5b levels in ovariectomized rats at doses of 0.3, 1 and 3 mg/kg. Finally, AS260168 suppressed the increase in urine deoxypyridinoline in a rat prednisolone-induced osteoporosis model at 10 mg/kg. These results suggest that AS2690168 is a promising treatment for bone disorders with excessive bone resorption.

A new allele of engrailed, enNK14, causes supernumerary spermathecae in Drosophila melanogaster.

A spontaneous mutation, enNK14, was a new allele of engrailed (en) in Drosophila melanogaster. Females of enNK14 have three spermathecae, instead of two in wild type, under a wide range of developmental temperatures, while the males show no abnormal phenotype. Spermathecae of the mutant female can accept inseminated sperms, albeit with a delay of at least an hour until full acceptance compared with wild type. The time course of decrease in the number of stored sperms was thoroughly similar between the mutant and wild type. enNK14 females produced fewer progeny than wild type females despite storing a larger number of sperms. The delay of sperm entry and lower fecundity suggested some functional defects in secretory products of the spermathecae. In addition, some spermathecae in the mutant were accompanied by a mass of brown pigments in the adipose tissue surrounding the capsule. Six contiguous amino acids, Ser340-Ala345, were replaced by one Thr in enNK14. In another mutant, enspt, Ser325 was also shown to be substituted by a Cys. These amino acid changes were located within a serine-rich region, in which Ser325, Ser340 and Thr341 were suggested as targets of Protein Kinase C by an in silico analysis. The splicing pattern of en mRNA did not differ between enNK14 and wild type in embryo, larva, pupa or adult. Our results suggest that en plays an important role in determining the number of spermathecae as well as in sperm storage function in the Drosophila female.

Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity.

The role of processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. Here, we report that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4, and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly. Loss of Patr-1-dependent P-bodies leads to a loss of stem cells that is associated with inappropriate expression of EC-fate gene nubbin. Transcriptomic analysis of progenitor cells identifies a cadre of such weakly transcribed pro-differentiation transcripts that are elevated after P-body loss. Altogether, this study identifies a P-body-dependent repression activity that coordinates with known transcriptional repression programs to maintain a population of in vivo stem cells in a state primed for differentiation.

Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase.

G9a is one of the well-characterized histone methyltransferases. G9a regulates H3K9 mono- and dimethylation at euchromatic region and consequently plays important roles in euchromatic gene regulation. Mammalian G9a contains several distinct domains, such as GHD (G9a homology domain), ANK, preSET, SET and PostSET. These domains are highly conserved between mammals and Drosophila. Although mammalian G9a has nuclear localization signal (NLS) in its N-terminal region, the amino acid sequences of this region are not conserved in Drosophila. Here we have examined the subcellular localization of a series of truncated forms of Drosophila G9a (dG9a). The identified region (aa337-aa470) responsible for nuclear localization of dG9a contains four short stretches of positively charged basic amino acids (NLS1, aa334-aa345; NLS2, aa366-aa378; NLS3, aa407-aa419; NLS4, aa461-aa472). Each of NLS1, NLS3 and NLS4 is sufficient for the nuclear localization when they are fused with the enhanced green fluorescent protein. These NLSs of dG9a are distinct from those of mammalian G9a in their positions and amino acid sequences.

Exercise-independent wheat-induced anaphylaxis caused by ω-5 gliadin in mice.

BACKGROUND: ω-5 Gliadin is known as a major allergen in wheat-dependent exercise-induced anaphylaxis. The characteristics that make ω-5 gliadin an allergen remain unclear. METHODS: Mice were sensitized by means of intraperitoneal injection of gliadin fractions together with the adjuvant alum. Anaphylactic responses were assessed by measuring body temperature and voluntary physical activity. Specific IgE levels in mouse serum were evaluated by ELISA. After oral administration of proteins to mice, concentrations of administered proteins in their portal blood were also analyzed by competitive inhibition ELISA. RESULTS: Oral administration of ω-5 gliadin evoked significant decreases in body temperature and physical activity of sensitized mice, whereas the gliadin fraction did not induce these effects at the same dose. These responses were exercise independent. ELISA analysis revealed that IgE antibodies from sensitized mice react to ω-5 gliadin with great efficacy. After oral administration of either the gliadin fraction or ω-5 gliadin, blood levels of ω-5 gliadin were much higher than those of the gliadin fraction. CONCLUSIONS: ω-5 Gliadin caused anaphylaxis in sensitized mice, whereas the gliadin fraction did not at the same dose. The anaphylactic response was exercise independent. It is likely that IgE of sensitized mice reacts strongly to ω-5 gliadin and that ω-5 gliadin is better absorbed from the gastrointestinal tract than other components of the gliadin fraction. These results indicated that ω-5 gliadin has prominent characteristics as an allergen and that exercise might be an indirect factor in anaphylaxis induction.

External side-compression of radial artery: a simple technique for successful advancement of guidewires through the radial approach.

BACKGROUND: The transradial approach has several pitfalls that include problems regarding the radial puncture and difficulties with the catheter technique. We evaluated whether external side-compression of radial artery was helpful to yield the success rate for advancement of guidewires under the presence of side branches or arterial tortuosity. METHODS AND RESULTS: The study population consisted of 11 patients with unsuccessful advancement of guidewires into the brachial artery. In 7 patients, the J-tip hydrophilic guidewire was not advanced into the brachial artery because it always directed into the side branch. During external side-compression of radial artery at the culprit site with a finger of the second operator, the guidewire was successfully advanced into the brachial artery in all patients. In 4 patients, the guidewire was not advanced into the brachial artery because the radial artery was tortuous. During external side-compression of radial artery at the culprit site, the guidewire was successfully advanced into the brachial artery in 2 patients. In the remaining 2 patients in whom this attempt was unsuccessful, coronary angiography was performed through the right brachial artery. Overall success rate of this technique was 82%. CONCLUSION: External side-compression of radial artery is an easy and feasible technique for difficulties in the advancement of guidewires due to the presence of side branches or arterial tortuosity.

Impact of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis in mice.

The effects of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis (WDEIA) were investigated by using a mouse model. The gliadin fraction was prepared as a 70% ethanol-soluble solution, and ω-5 gliadin was purified by chromatography. Purified ω-5 gliadin was run on SDS-PAGE gel to reveal three bands with a molecular mass of 53-60 kDa and had the characteristic N-terminal sequence of ω-5 gliadin. The mice were sensitized to the gliadin fraction, and the anaphylactic response was assessed by measuring the body temperature and voluntary physical activity. An oral administration of ω-5 gliadin evoked a significant drop in both the body temperature and voluntary physical activity, similar to the effects of the whole gliadin fraction. ELISA and immunoblotting analyses revealed that the IgE expression from sensitized mice reacted most strongly to ω-5 gliadin. Taken together, these results indicate ω-5 gliadin to be a major allergen responsible for stimulating WDEIA in mice, with the characteristic potential for stimulating IgE production.

L-carnitine is essential to beta-oxidation of quarried fatty acid from mitochondrial membrane by PLA(2).

Mitochondrial beta-oxidation is an important system involved in the energy production of various cells. In this system, the function of L-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O(2) consumption without substrates, is caused by L-carnitine treatment. In this study, we investigated whether L-carnitine is essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A(2) (PLA(2)) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and L-carnitine. The effect of L-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with L-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of L-carnitine. Moreover, the L-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA(2) inhibitors were treated before ADP treatment. The L-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that L-carnitine might be essential to the beta-oxidation of quarried fatty acids from the mitochondrial membrane by PLA(2).

Exhaustive exercise reduces tumor necrosis factor-alpha production in response to lipopolysaccharide in mice.

OBJECTIVE: Stressful exercise reduces the plasma pro-inflammatory cytokine concentration in response to lipopolysaccharide (LPS). The aim of this study was to clarify the mechanism of exhaustive exercise-induced suppression of the plasma tumor necrosis factor (TNF)-alpha concentration in response to LPS. METHODS: Male C3H/HeN mice (n = 66) were randomized to treadmill running to exhaustion (Ex) or a sedentary (Non-Ex) condition. Monocytes and splenic macrophages were collected from some animals, and other animals were injected with LPS (1 mg/kg) immediately after the exercise. The liver, lung and spleen tissues in the mice were removed 30 min after the LPS injection for determination of TNF-alpha mRNA expression. Blood and tissue samples were collected for determination of TNF-alpha and TNF receptors (TNFR) 1 h after the LPS injection. RESULTS: Although there was a significant suppression in LPS-induced plasma TNF-alpha in the Ex mice when compared to the Non-Ex mice (p < 0.01), soluble TNFR in plasma was not affected by the exercise. There was no change in cell-surface expression of Toll-like receptor 4 (TLR4) and in LPS-induced TNF-alpha mRNA expression and TNFR content in tissues between the Ex and Non-Ex groups. Interestingly, TNF-alpha contents in the liver, lung and spleen of the Ex mice were significantly lower than those of the Non-Ex group (p < 0.01, p < 0.01 and p < 0.05, respectively). CONCLUSION: These data suggest that exhaustive exercise-induced suppression of the plasma TNF-alpha concentration despite LPS stimulation might depend on translation of TNF-alpha in tissues.