Time-dependent distinct roles of Toll-like receptor 4 in a house dust mite-induced asthma mouse model.

House dust mites (HDMs) are a common source of allergens that trigger both allergen-specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll-like receptor 4 (TLR4) in the process of sensitization to house dust mite allergens in an HDM extract-induced asthma mouse model.　Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose-dependent manner from 2.5 to 30 µg/dose. In TLR4-knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild-type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR4-knockout mice than in wild-type mice. This suggests that the roles of TLR4 signaling are different between the early phase and the later phase of HDM allergen-induced inflammation. Thus, innate immune response through TLR4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.

Polo-like kinase 1 phosphorylates and regulates Bcl-x(L) during pironetin-induced apoptosis.

Bcl-x(L), an anti-apoptotic Bcl-2 family member protein, contributes to the resistance against chemotherapies such as tubulin-binder treatment in many human tumors. Although Bcl-x(L) is phosphorylated after tubulin-binder treatment, the role of the phosphorylation and its responsible kinase(s) are poorly understood. Here, we identified Plk1 (polo-like kinase 1) as a Bcl-x(L) kinase. Same location of Bcl-x(L) and Plk1 was revealed by immunocytochemical analyses at M-phase in situ. Plk1 phosphorylates Bcl-x(L) in vitro, and we identified Plk1 phosphorylation sites in Bcl-x(L). When all of these phosphorylation sites were substituted to alanines, the anti-apoptotic activity of the Bcl-x(L) mutant against the apoptosis induced by pironetin, but not against ultraviolet-induced apoptosis, was increased. These observations suggest that Plk1 is a regulator of Bcl-x(L) phosphorylation and controls the anti-apoptotic activity of Bcl-x(L) during pironetin-induced apoptosis.

N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.

MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.

F13459, a new derivative of mycophenolic acid. I. Taxonomy, isolation, and biological properties.

In the course of screening for inhibitors of intracellular trafficking of glycoprotein, a new inhibitor, F13459 was isolated from the culture broth of a Penicillium sp. It was purified using solvent extraction, silica gel, Sephadex LH-20 and ODS column chromatography. From structural analysis, F13459 was a derivative of mycophenolic acid, an inhibitor of inosine 5'-monophosphate dehydrogenase. F13459 inhibited hemagglutinin synthesis of NDV at concentrations more than 25 microg/ml. However, syncytium formation as a result of cell surface expression of F-glycoprotein of NDV was inhibited at concentrations of F13459 lower than those required for appreciable inhibition of glycoprotein synthesis.

F13459, a new derivative of mycophenolic acid. II. Physico-chemical properties and structural elucidation.

F13459 is a new inhibitor of synthesis and trafficking of virus glycoprotein isolated from the culture broth of a Penicillium sp. The molecular formula of F13459 was determined to be C27H28O11 by HRFAB-MS and NMR spectral analyses. The structure of F13459 was elucidated to be 3,4-dihydro-3,4,6,8-tetrahydroxy-3-methyl-1H-2-benzopyran-1-one 4-O-mycophenolate, an ester derivative of mycophenolic acid. F13459 was isolated as the optically inactive form. F13459 exists in epimeric mixtures at C-3' through relatively fast hemiacetal-ketone tautomerism and at C-4' through slow keto-enol tautomerism. Those epimerizations were confirmed by NOE differential experiments for fast chemical exchange and equilibrium and by deuteration experiments in NMR for slow chemical exchange.

Co-existing proteins interfere with the action of nordihydroguaiaretic acid on retrograde Golgi-to-ER protein trafficking in NRK cells and alpha-glucosidase reaction in vitro.

Induction of retrograde trafficking of mannosidase II and TGN38 in NRK cells and inhibition of alpha-glucosidase in vitro by nordihydroguaiaretic acid (NDGA) were strongly interfered with by serum, serum albumin, or other unrelated proteins added to the medium or incubation mixture. These observations indicate that NDGA interacts with diverse kinds of proteins, and therefore, pharmacological effects of NDGA at cellular levels should be carefully interpreted.

Pituitary adenylate cyclase-activating polypeptide may function as a neuromodulator in guinea-pig adrenal medulla.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in catecholamine secretion from dissociated adrenal chromaffin cells of the guinea-pig was investigated using amperometry, the patch clamp technique and immunochemistry. Pretreatment of adrenal chromaffin cells with 0.3-10 nM PACAP for 2 min resulted in enhancement of nicotine- and muscarine-induced secretions in either the presence of external Ca2+ ions or nominally Ca2+-free solution, with no change in basal secretion or the holding current at -60 mV in most of the cells tested. Pretreatment with PACAP augmented the muscarine-induced non-selective cation current, but did not affect the muscarine-induced outward current or nicotine-induced current. PACAP-induced enhancement of nicotine- and muscarine-induced secretions was suppressed by the simultaneous application of PACAP and the protein kinase inhibitors 100 microM HA1004 or 2 microM H89. Application of forskolin enhanced both muscarine- and nicotine-induced secretions, whereas application of a phorbol ester augmented the nicotine-induced secretion, but suppressed the muscarine-induced secretion in a reversible manner. Immunohistochemical analysis of adrenal medullae revealed that PACAP-like immunoreactivity was present in nerve fibres surrounding putative chromaffin cells. PAC1R-like immunoreactivity was distributed diffusely in the plasma membrane, whereas nicotinic ACh receptor-like immunoreactivity was concentrated at the plasma membrane near the nucleus, where the synapses were mainly localized. These observations suggest that PACAP in the guinea-pig adrenal medulla functions as a neuromodulator to facilitate ACh-induced secretion through a cAMP-protein kinase A-dependent pathway.

Lipopolysaccharide-triggered desensitization of TNF-alpha mRNA expression involves lack of phosphorylation of IkappaBalpha in a murine macrophage-like cell line, P388D1.

Activation of nuclear factor kappaB (NF-kappaB) is thought to be required for cytokine production by lipopolysaccharide (LPS)-responsive cells. Here, we investigated the contribution of NF-kappaB in preventing LPS-induced transcription of the tumor necrosis factor alpha (TNF-alpha) gene in a murine macrophage cell line, P388D1, when tolerance was induced in the cells with a short exposure to a higher dose of LPS. Electrophoretic mobility shift assays with the kappaB elements of the murine TNF-alpha promoter and enhancer revealed that nuclear mobilization of heterodimers of p65/p50, c-rel/p50 and p65/c-rel, and homodimers of p65 was markedly reduced in LPS-tolerant cells, whereas that of p50 homodimers was only slightly increased. Western blot analysis showed that the phosphorylation of Ser32 on IkappaBalpha and its transient degradation did not occur in LPS-tolerant cells. These results thus suggest that desensitization of TNF-alpha gene expression in this LPS-tolerant state is closely associated with down-regulation of transactivating NF-kappaB and may involve a defect in the LPS-induced IkappaBalpha kinase pathway.

Processing glucosidase inhibition by 1-azafagomine.

Natural azasugars have the ring oxygen substituted by nitrogen. They show potent inhibitory activity against glycosidases. The effect of substituting the ring carbon with nitrogen was examined with 1-azafagomine. 1-Azafagomine exhibited similar activity against processing glucosidase to that of fagomine.

Evidence for psychological refractory effect in motor inhibition for a dual-response Go/No-Go task.

Human subjects exhibit difficulty in initiating two independent, discrete responses in close succession, a difficulty known as the 'psychological refractory effect.' It is not yet known whether motor-inhibition processes are under the influence of this effect, as are motor-execution processes. This study examined the temporal changes of subjects' reaction times, interpreted in terms of motor programming for inhibition, in a dual-response Go/No-Go task that required two independent responses in close succession. Eight subjects performed the task with both a shorter (400 msec.) and a longer interstimulus interval (800 msec.). The mean reaction time for the second stimulus (RT2) in the Go response of the 400-msec. condition was significantly longer than that of the 800-msec. condition. For committed error responses during the No-Go trials, the mean RT2 in the 400-msec. condition was longer than that in the 800-msec. condition. The total number of these errors in the 400-msec. condition was significantly greater than that in the 800-msec. condition. These results suggested that both the motor-execution processes and motor-inhibition processes were influenced by the psychological refractory effect.

Novel blockade of cell surface expression of virus glycoproteins by leucinostatin A.

The nonapeptide leucinostatin A (LSA) inhibited syncytium formation without profoundly affecting HN glycoprotein synthesis in Newcastle disease virus (NDV)-infected BHK cells. At similar doses of LSA, cytopathic effect and infectious virus production were suppressed in vesicular stomatitis virus (VSV)-infected BHK cells. Blockade by LSA of cell surface expression of NDV-HN and VSV-G glycoproteins was demonstrated, accompanied by intracellular accumulation of these virus glycoproteins. LSA acts as an inhibitor of mitochondrial F-type H(+)-translocating ATPase, a key enzyme in the generation of ATP, but its action against cell surface expression of virus glycoproteins was independent of the depletion of intracellular ATP. LSA also acts as an ionophore, but its action on intoxication by ricin and diphtheria toxin was different from that of monensin. This novel action of LSA is expected to be useful in investigation of the mechanism of intracellular trafficking of proteins.

Efrapeptins block exocytic but not endocytic trafficking of proteins.

During the course of screening studies to identify inhibitors of intracellular protein trafficking, we isolated efrapeptins as active principles. These compounds arrested syncytium formation (SF) and cytopathic effect (CPE) in Newcastle disease virus (NDV)- and vesicular stomatitis virus (VSV)-infected BHK cells, respectively, without profoundly affecting glycoprotein synthesis. Efrapeptins blocked cell surface expression of NDV-HN and VSV-G glycoproteins, but did not suppress intoxication by ricin or diphtheria toxin even after prolonged pretreatment. Efrapeptins are inhibitors of F-ATPase, or ATP synthase, but their inhibitory effect on SF and CPE was independent of the amount of intracellular ATP.

Effects of mepanipyrim on intracellular trafficking: a comparative study on its effects on exocytic and endocytic trafficking of proteins, sphingolipids, and cholesterol.

Mepanipyrim, N-(4-methyl-6-prop-1-ynylpyrimidin-2-yl)aniline, diminished the cell surface expression of envelope glycoproteins of Newcastle disease and vesicular stomatitis viruses at concentrations where their synthesis was not profoundly affected. Intoxication by diphtheria toxin and ricin and recycling of transferrin were not affected even when cells were treated with mepanipyrim for 2 h before the addition of these probes, indicating that mepanipyrim does not act on the endocytic and recycling pathways of these proteins. Metabolic conversion of C6-NBD-ceramide to sphingomyelin and its back-exchange to the medium was also not affected, but synthesis and back-exchange of C6-NBD glucosylceramide were greatly influenced, and an accumulation of LDL-derived, unesterified cholesterol was induced by the drug. These results are discussed relating to the site(s) of action of mepanipyrim.

Nectrisine is a potent inhibitor of alpha-glucosidases, demonstrating activities similarly at enzyme and cellular levels.

Nectrisine, discovered as an immunomodulator, was found to inhibit alpha-glucosidase, alpha- and beta-mannosidases, beta-glucosidase and beta-N-acetylglucosaminidase, in that order of inhibition strength. Beta-Galactosidase, alpha-fucosidase, and neuraminidase were insensitive to this antibiotic. Also sensitive was the trimming glucosidase I which participates in the first step of modifying N-glycosidic oligosaccharide. Nectrisine demonstrated an inhibitory effect at the cellular level as strong as expected based on its action at enzyme levels; castanospermine and 1-deoxynojirimycin did not. Nectrisine and castanospermine suppressed syncytium formation and hemolytic activity in Newcastle disease virus (NDV)-infected BHK cells, without blocking the synthesis and cell-surface expression of HANA glycoprotein of NDV.

Two isoforms of acid phosphatase secreted by tobacco protoplasts: differential effect of brefeldin A on their secretion.

The effects of brefeldin A (BFA) on the secretion of acid phosphatase (APase) by tobacco protoplasts were investigated. Secretion of APase was inhibited by BFA in a dose-dependent manner, with a concomitant intracellular accumulation of the enzyme. The secreted APase was composed of two isoforms. BFA (10/ µg/ml) inhibited the secretion of one of the isoforms without inhibiting that of the other, and this phenomenon explains the partial inhibition of APase secretion as a whole. The inhibition of APase secretion was accompanied by changes in the morphology of the Golgi apparatus and also by an increment in massdensity of cells.

Depolarizing neuromuscular blocking action of coryneine derived from aconite root in isolated mouse phrenic nerve-diaphragm muscles.

The mode of the neuromuscular blocking action of coryneine (a quaternary ammonium derivative of dopamine) derived from aconite root was investigated in isolated phrenic nerve-diaphragm muscles and denervated diaphragm muscles of mice. Coryneine (20-150 microM) blocked the nerve-evoked twitch response without affecting the contraction evoked by electrical stimulation of the muscle. The blocking effect was reversed by neostigmine, a cholinesterase inhibitor. The electrical charge-response curve on depolarization produced by iontophoretically applied acetylcholine (ACh) at the endplate regions in normal muscles was shifted to the right on decreasing the maximal response by 40 microM coryneine. The double-reciprocal plot revealed that coryneine reduced the apparent affinity of ACh for its receptor on decreasing the maximal response. Coryneine (20 microM-2mM) itself depolarized the endplate membrane and this effect was reversibly suppressed by 1 and 5 microM pancuronium. Coryneine 30 microM-10mM) produced contractions of denervated muscles in a concentration-dependent manner and the effects were reduced by 70nM pancuronium. These results indicate that coryneine is a depolarizing agent and a mixed-type competitive and noncompetitive neuromuscular blocker.

[A clinicopathological study on extensive lymph node dissection for thoracic esophageal cancer].

In order to evaluate clinical effect of the extensive lymph node dissection for thoracic esophageal cancer, 78 cases with esophagectomy and extensive lymph node dissection were reviewed. Pathological depth of invasion was the submucosa (sm: 25 cases), the proper muscle (pm: 7 cases), al (8 cases) and a2 (38 cases). Incidence of operative death rate in 30 days was 2.8% of all cases and hospital death rate 5.1%. Lymph node metastasis was identified in 65% of all cases (cervical metastasis occupied 19.2% of all cases with metastasis, mediastinal metastasis 48.7% and abdominal metastasis 34.6%). Over all cumulative three year survival rate was 55.3% (sm 88.9%, pm & al 55.6% and a2 40%). Postoperative recurrence was analyzed on thirty eight cases with more than three years passed after esophagectomy with extensive lymph node dissection. Postoperative recurrence was detected in 50% of all cases (local recurrence occupied 16% of all cases with recurrence, distant organ metastasis 58%, lymph node metastasis 21% and dissemination 5%). Extensive lymph node dissection significantly elongated disease free interval and survival time of cases with recurrence and lymph node metastasis, but there was no significant improvement in cases with other mode of recurrence. Dissecting field was classified into four regions (cervical, paratracheal, periesophageal and abdominal regions). Number of lymph nodes with metastasis and number of regions with lymph node metastasis showed close relationship with incidence of post operative recurrence. In cases with number of lymph node metastasis more than six and in more than two regions, all cases resulted in recurrence. Extensive lymph node dissection have an effect improving survival rate on cases with number of lymph node metastasis less than five and in less than two regions.