Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber

String-shaped reconstitutedsmooth muscle (SM) fibers were prepared in rectangular wells by thermalgelation of a mixed solution of collagen and cultured SM cells derivedfrom guinea pig stomach. The cells in the fiber exhibited an elongatedspindle shape and were aligned along the long axis. The fibercontracted in response to KCl (140 mM), norepinephrine (NE;10⁷ M), epinephrine (10⁷ M), phenylephrine(10⁶ M), serotonin (10⁶ M), and histamine(10⁵ M), but not acetylcholine (10⁵ M).Phentolamine (10⁷ M) produced a parallel rightward shiftof the NE dose-response curve. Moreover, NE-induced contractionwas partially inhibited by nifedipine and completely abolished by theintracellular Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, theRho kinase inhibitor Y-27632, and papaverine. A[³H]quinuclidinyl benzilate binding study revealed thatthe loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. Theseresults suggest that, although the fiber is not a model for fullydifferentiated SM, contractile mechanisms are maintained.

     

Reduced host resistance and Th1 response to Cryptococcus neoformans in interleukin-18 deficient mice

Using interleukin (IL)-18 deficient (IL-18(-/-)) mice, we examined the role of IL-18 in the host resistance and Th1 response against infection with Cryptococcus neoformans. Fungal clearance in the lung was reduced in IL-18(-/-) mice, although there was no significant change in the level of dissemination to the brain. The DTH response, as determined by footpad swelling, was also diminished in IL-18(-/-) mice compared to control wild-type (WT) mice. The levels of IL-12 and interferon (IFN)-gamma in the sera were significantly lower in IL-18(-/-) mice than in WT mice. Spleen cells from infected WT mice produced a high level of IFN-gamma upon stimulation with the microbe, while only a low level of IFN-gamma production was detected in spleen cells from infected IL-18(-/-) mice. Administration of IL-18 almost completely restored the reduced response in IL-18(-/-) mice, while IL-12 showed a marginal effect. These results demonstrated the important role of IL-18 in the resistance and Th1 response of mice to C. neoformans by potentiating the production of IFN-gamma.     

Similarities in function and gene structure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange proteins for ADP-ribosylation factors

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. A novel ARF GEP, designated cytohesin-4, was cloned from a human brain cDNA library. Deduced amino acid sequence of the 47-kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an approximately 200-amino acid Sec7 domain with an approximately 100-residue pleckstrin homology domain near the C terminus. The Sec7 domain sequence is 77% identical to those of other cytohesins. Structures of the cytohesin-4 and cytohesin-1 genes were remarkably similar, except for an extra 3-base pair (GAG) exon present in cytohesin-1. Two mRNAs with and without the 3-base pair sequence were found in brain in different ratios for cytohesin-1, -2, and -3 but not cytohesin-4. Recombinant cytohesin-4 stimulated guanosine 5'-3-O-(thio)triphosphate binding by human ARF1 and ARF5 but not ARF6. Like other cytohesins and unlike the approximately 200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of approximately 3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, approximately 90% of CD33(+) (monocytes), 80% of CD2(+) (NK/T), and 10-20% of CD19(+) (B) cells contained cytohesin-4 mRNA by in situ hybridization. Thus, in gene structure and brefeldin A-insensitive GEP activity, cytohesin-4 resembles other cytohesins, but its tissue distribution differs considerably, consistent with a different specific function.     

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.     

Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1DeltaN in PC12 cells. ASK1DeltaN-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1DeltaN induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1DeltaN-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.     

Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-alpha production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-alpha in response to LPS. LPS-induced activation of both NF-kappaB and c-Jun NH(2)-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.     

Synthesis of a model compound related to an anti-ulcer pectic polysaccharide

A stereocontrolled synthesis of the model compound for an anti-ulcer active polysaccharide (Bupleuran 2IIc) is described. Glycosidation of the disaccharide acceptor, 2-O-acetyl-3-O-benzyl-4-O-(p-methoxybenzyl)-alpha-L-rhamnopyranosyl-(1-- >4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranosyl trichloroacetimidate, with the disaccharide receptor, allyl 3,4-di-O-benzyl-alpha-L-rhamnopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta- D-galactopyranoside, using silver triflate (AgOTf) as a promoter gave the desired tetrasaccharide derivative, which was transformed into the acidic tetrasaccharide, corresponding to a segment of the rhamnogalacturonan (Bupleuran 2IIc) polysaccharide, propyl alpha-L-Rha-(1-->4)-alpha-D-GalA-(1-->2)-alpha-L-Rha-(1-->4)-beta-D-GalA , via removal of the corresponding ether and ester protecting groups, followed by oxidation.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

In Alzheimer's disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification

In this study, we compared the neuronal induction of the antioxidant heme oxygenase-1 (HO-1) in Alzheimer's disease with abnormalities in tau marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO-1-containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO-1. These findings suggest that tau phosphorylation precedes the HO-1 response and that HO-1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal tau and induces the Alz50 epitope in tau. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of tau but also requires tau phosphorylation because neither methylated, recombinant, nor dephosphorylated tau reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament-tau isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE-lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the tau conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of tau and neuronal oxidative stress-induced pathology is important in the formation of neurofibrillary tangles.     

Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5

The mature form of Alzheimer's beta-amyloid precursor protein (APP) is phosphorylated specifically at Thr(668) in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin-dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr(668). Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense-treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.