Membrane fusion between liposomes containing SNARE proteins involved in mast cell exocytosis.

OBJECTIVE AND DESIGN: We monitored the membrane fusion of liposomes to determine if the minimal components of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE), which is involved in mast cell exocytosis, have fusogenic activity. METHODS: Three core components of SNARE were reconstituted into liposomes. Membrane fusion between liposomes containing vesicle associated membrane protein (VAMP) -7 or -8 and liposomes containing synaptosomal-associated protein 23 kDa (SNAP23) and syntaxin-3 or -4 was monitored by fluorescence resonance energy transfer. RESULTS: The combination of SNAP23/syntaxin-3/VAMP-8 showed the most efficient liposome-liposome fusogenic activity. Liposomes with VAMP-7 exhibited poor fusogenic activity regardless of the syntaxin isoform. CONCLUSION: The core components of SNAP23, syntaxin-3, and VAMP-8 appear to be minimal machinery to induce membrane fusion, while VAMP-7 appears to be unessential for membrane fusion.

Hexacyanocobaltate(III) anions as precursors of Co(II)-Ni(II) cyano-bridged multidimensional assemblies: hydrothermal syntheses, crystal and powder X-ray structures, and magnetic properties.

Three novel cyanide-bridged heterobimetallic coordination polymers have been synthesized by hydrothermal routes, in superheated water solutions, by using K3[Co(CN)6], NiCl2.6H2O, and alpha-diimine ligands: [Ni(CN)4Co(phen)] (1; phen = 1,10-phenanthroline), [Ni(CN)4Co(2,2'-bipy)] (2; 2,2'-bipy = 2,2'-bipyiridine), and [Ni(CN)4Co(2,2'-bipy)2] (3). The isostructural compounds 1 and 2 contain a two-dimensional network with Co(II) centers octahedrally coordinated by one chelating 2,2'-bipy ligand and four cyanide groups of four distinct [Ni(CN)4]2-, through crystallographically equivalent, bridging units. Compound 3 contains one-dimensional zigzag chains in which the Co(II) ion is coordinated by two chelating 2,2'-bipy ligands and two cyanides from two different [Ni(CN)4]2- units cis to each other. These compounds have been fully characterized by single-crystal or unconventional powder X-ray diffraction analyses and variable-temperature magnetic measurements.

Establishment and characterization of macrophage-like cell lines expressing osteoclast-specific markers.

Osteoclasts differentiate from hematopoietic precursors of the monocyte/macrophage lineage to mononuclear preosteoclasts and multinuclear mature osteoclasts. In the present study, we report on the establishment of macrophage like cell lines from mouse bone marrow by coculturing bone marrow cells with mouse chondrocytes. Isolated clones (MLC-6 and MLC-7 cells) expressed fully differentiated osteoclast markers, such as calcitonin receptors, vitronectin receptors, tartrate-resistant acid phosphatase and vacuolar H+ -ATPase, in the absence of osteoclast differentiation factor/osteoprotegerin ligand/RANKL/TRANCE, which was essential for osteoclast differentiation. Most clones also maintained expression of a macrophage-associated protein, namely non-specific esterase. Both MLC-6 and MLC-7 cells released cathepsin K into the culture medium. Both clones resolved dentine slices when cocultured with the osteoblast cell line ST2. The cloned cell lines are considered to be useful tools in the study of osteoclast differentiation.

Establishment and characterization of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cell lines.

Morphologically macrophage-like cells were cloned from hamster bone marrow cells by coculturing bone marrow cells with hamster chondrocytes. One of the clones (CCP-2) was characterized in the present study. CCP-2 cells were positive in an osteoclast marker enzyme, tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and non-specific esterase (NSE). We showed CCP-2 cells degraded cartilage matrix and hydroxyapatite coated on Osteologic disks. A gelatinase secreted from CCP-2 cells was observed and purified from serum-free conditioned medium of the cells. N-terminal amino acid sequencing of the purified enzyme revealed it was matrix metalloproteinase-9. However, CCP-2 cells failed to express calcitonin receptors, a mature osteoclast marker, even after coculture with osteoblast ST2 cells in the presence of 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3]. The cells showed high affinity to types X and I but not to type II collagen. In addition, histochemical studies have shown the presence of tartrate-resistant acid phosphatase and alkaline phosphatase double positive cells at the secondary ossification site of the hamster humerus. From these observations, we concluded that CCP-2 cells are similar to osteoclast but not the same. CCP-2 cells are therefore important tools for investigating chondroclastogenesis/osteoclastogenesis and endochondral ossification.

Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene.

A novel dehydratase that catalyzes the stoichiometric dehydration of Z-phenylacetaldoxime to phenylacetonitrile has been purified 483-fold to homogeneity from a cell-free extract of Bacillus sp. strain OxB-1 isolated from soil. It has a M(r) of about 40 000 and is composed of a single polypeptide chain with a loosely bound protoheme IX. The enzyme is inactive unless FMN is added to the assay, but low activity is also observed when sulfite replaces FMN. The activity in the presence of FMN is enhanced 5-fold under anaerobic conditions compared to the activity measured in air. The enzyme has maximum activity at pH 7.0 and 30 degrees C, and it is stable at up to 45 degrees C at around neutral pH. The aerobically measured activity in the presence of FMN is also enhanced by Fe(2+), Sn(2+), SO(3)(2)(-), and NaN(3). Metal-chelating reagents, carbonyl reagents, electron donors, and ferri- and ferrocyanides strongly inhibit the enzyme with K(i) values in the micromolar range. The enzyme is active with arylalkylaldoximes and to a lesser extent with alkylaldoximes. The enzyme prefers the Z-form of phenylacetaldoxime over its E-isomer. On the basis of its substrate specificity, the enzyme has been tentatively named phenylacetaldoxime dehydratase. The gene coding for the enzyme was cloned into plasmid pUC18, and a 1053 base-pair open reading frame that codes for 351 amino acid residues was identified as the oxd gene. A nitrilase, which participates in aldoxime metabolism in the organism, was found to be coded by the region just upstream from the oxd gene. In addition an open reading frame (orf2), whose gene product is similar to bacterial regulatory (DNA-binding) proteins, was found just upstream from the coding region of the nitrilase. These findings provide genetic evidence for a novel gene cluster that is responsible for aldoxime metabolism in this microorganism.

Synthesis and pharmacological activity of 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2-hydroxymethyl-4- pyrrolidinyl]benzamide (TKS159) and its optical isomers.

Of 4-amino-5-chloro-2-methoxy-N-(1-ethyl-2-hydroxymethyl-4- pyrrolidinyl)benzamide, four optical isomers, (2S,4S)-1 (TKS159), (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27, were prepared from optically active 4-amino-1-ethyl-2-hydroxymethylpyrrolidine di-p-toluenesulfonate [(2S,4S)-14, (2S,4R)-17, (2R,4S)-20 and (2R,4R)-23, respectively]. The requisites, (2S,4S)-14, (2S,4R)-17, (2R,4S)-20 and (2R,4R)-23, were prepared from a commercially available trans-4-hydroxy-L-proline. The absolute configurations of (2S,4S)-1 (TKS159), (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27 were spectroscopically determined. Of the benzamide derivatives, four optical isomers, (2S,4S)-1, (2S,4R)-25, (2R,4S)-26 and (2R,4R)-27, showed a relatively potent affinity for 5-hydroxytryptamine 4 (5-HT4) receptors in a radioligand binding assay ([3H]GR113808). The activities of 25-27 were less effective than that of 1 for the gastric emptying of a phenol red semisolid meal in rats. All this suggests that the most potent of the isomers was 4-amino-5-chloro-2-methoxy-N-[(2S,4S)-1-ethyl-2- hydroxymethyl-4-pyrrolidinyl]benzamide (1).

Metabolic activities of partially degenerated hypertrophic chondrocytes: gene expression of hyaluronan synthases.

Ultrastructural aspects of hypertrophic chondrocytes in hamster and mouse epiphysial cartilage were examined in relation to their metabolic activities. With the hypertrophic change, cytoplasmic vacuolization proceeded leaving the partially intact endoplasmic reticulum (ER). In the hypertrophic cells, cytoplasmic hyaluronan was stained with the biotinylated hyaluronan-binding region (b-HABR) of aggrecan, and mRNAs of hyaluronan synthase (Has 1, Has 2 and Has 3) were detected by in situ hybridization. When the epiphysial cartilage was cultured in the presence of 35S, 3H-GlcNAc, 3H-proline or 14C-palmitic acid, vacuolated late hypertrophic chondrocytes were labeled with these radioactive precursors. The evidence indicates that late hypertrophic chondrocytes are metabolically active, which appears to be essential for the enlargement of chondrocytes.

Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody.

OBJECTIVE: The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA). METHODS: Normal articular cartilage (n = 6) and cartilage obtained from joints with RA (n = 15) and osteoarthritis (OA, n = 10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA. RESULTS: In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor alpha. CONCLUSION: These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.

Pertussis toxin-sensitive signal controls the trafficking of thymocytes across the corticomedullary junction in the thymus.

We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4-CD8- cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8- or CD4-CD8+ single-positive (SP) cells. Positively selected CD69+CD3intermediate cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69-CD3high cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.

Selective complement C1s deficiency caused by homozygous four-base deletion in the C1s gene.

The complement system plays an important role in defense mechanisms by promoting the adherence of microorganisms to phagocytic cells and lysis of foreign organisms. Deficiencies of the first complement components, C1r/C1s, often cause systemic lupus erythematosus-like syndromes and severe pyogenic infections. Up to now no genetic analysis of the C1r/C1s deficiencies has been carried out. In the present work, we report the first genetic analysis of selective C1s deficiency, the patient having a normal amount of C1r. C1s RNA with a normal size was detected in patient's subcutaneous fibroblasts (YKF) by RNA blot analysis and RT-PCR. The amount of C1s RNA was approximately one-tenth of the RNA from the human chondrosarcoma cell line, HCS2/8. In contrast, the levels of C1r and beta-actin RNA of YKF were similar to that of HCS2/8. Sequence analysis of C1s cDNA revealed a deletion at nucleotides 1087-1090 (TTTG), creating a stop codon (TGA) at position 94 downstream of the mutation site. Direct sequencing of the gene between the primers designed on intron 9 and exon 10 indicated the presence of the deletion on exon 10 of the gene. Quantitative Southern blot hybridization suggested the mutation was homozygous. The 4-bp deletion on exon 10 was also found in the patient's heterozygous mother who had normal hemolytic activity.

Inhibition of bFGF activity by complement C1s: covalent binding of C1s with bFGF.

The first complement component C1s formed large aggregates with bFGF when bFGF and C1s were incubated at 37 degrees C overnight. Under non-reducing conditions, a part of the aggregates did not penetrate into 5% polyacrylamide gel in the presence of SDS, and the rest penetrated into 5% gel but not into 12% gel. The aggregates were dissociated into monomers by reducing with 2-mercaptoethanol. Both active and inactive C1s formed aggregates with bFGF. In addition, a portion of bFGF was degraded by active C1s but not by inactive C1s. Aggregates were not formed when 2-mercaptoethanol (2 mM) was added to the incubation mixture. After the incubation with C1s the growth-stimulating activity of bFGF was measured by using human umbilical vein endothelial cells (HUVEC) as indicator cells. The aggregate formation between C1s and bFGF significantly reduced the activity of bFGF.

Coordinated change between complement C1s production and chondrocyte differentiation in vitro.

In vitro synthesis of the first component of complement C1s was examined by using hamster epiphyseal chondrocytes (HAC) and human chondrosarcoma cell line HCS-2/8. Hamster and human C1s produced by the cells were quantified by immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA), respectively. It was possible to measure active and inactive C1s by sandwich ELISA, when we used anti-human C1s monoclonal antibodies, M241 recognizing only active C1s, and M365 and M81 recognizing both active and inactive C1s. Approximately 40% of C1s secreted from HCS-2/8 was found to be activated in the culture medium, whereas C1s from HAC was not. C1s production increased in accordance with chondrocyte differentiation induced by ascorbic acid. In contrast, transforming growth factor-beta1 and basic fibroblast growth factor, which inhibited differentiation, suppressed C1s production. These results confirmed our previous observation showing that C1s synthesis increased with differentiation into hypertrophic chondrocytes in vivo.

Complement Cls, a classical enzyme with novel functions at the endochondral ossification center: immunohistochemical staining of activated Cls with a neoantigen-specific antibody.

The secondary ossification center of 14- to 16-day-old hamster tibiae was examined immunohistochemically with active and inactive Cls-specific antibodies, RK5 and RK4, respectively. At the ossification center, chondrocytes differentiate from proliferating and hypertrophic to degenerating stages, and their site is occupied by the bone marrow. Cls was strongly immunostained in hypertrophic chondrocytes. In order to discover whether Cls is activated at a particular site, the cartilage was immunostained with RK5 and RK4. RK5 mainly reacted with degrading matrix around invading vessels. In contrast, RK4 strongly stained hypertrophic chondrocytes. Immunoelectron microscopy revealed Cls on degrading fragments of chondrocytes and fibers of cartilage matrix. Decorin, one of the major matrix proteoglycans, was dose and time dependently degraded by Cls. Type II collagen and type I gelatin were also degraded. Articular cartilage from patients with rheumatoid arthritis was positively immunostained (11/12 cases) with an anti-Cls monoclonal antibody (mAb) PG11, whereas normal articular cartilage (5/5 cases) was negative, suggesting Cls participation in the etiology of rheumatoid arthritis.

Density dependent change of myristoylated proteins in C3H10T1/2 fibroblasts and their transformants.

We have examined the pattern of protein myristoylation in C3H10T1/2 fibroblasts during cell growth. During the growing phase of 10T1/2 cells, several proteins were radiolabelled with [3H]myristate, and among them proteins with molecular masses of 22, 35, a doublet of 42-45 and 67 kDa were labelled predominantly. The extent of myristoylation in each of these proteins changed with cell density. The amount of radioactivity incorporated into the 22 kDa protein in 10T1/2 cells decreased with increasing cell density and remained at a low level during the stationary phase. In contrast, the incorporation into the 67 kDa protein increased parallel to cell density. The density-dependent change of myristoylation was not observed in any of the transformants of 10T1/2 cells thus far examined. The 67 kDa protein was identified as MARCKS (myristoylated alanine-rich C kinase substrate) by immunoprecipitation with an anti-MARCKS antibody. By Western blot analysis, we found that the amount of MARCKS in 10T1/2 cells increased significantly analogous with cell density. Therefore, it is possible that MARCKS and the 22 kDa protein play a role in contact-mediated cell signalling in 10T1/2 cells, but the mechanism is lost in transformed cells.

Change of complement C1s synthesis during development of hamster cartilage.

Expression of the first complement component (C1s) has been examined in chondrocytes of hamster epiphyseal cartilage during development and fracture healing. C1s is immunostained with anti-hamster C1s monoclonal antibody, PG11. The C1s staining increases in accordance with chondrocyte differentiation and reaches a maximal level in hypertrophic chondrocytes. This change is observed at both the tibia ossification center and at the callus in which the replacement of cartilage by bone marrow takes place. The concomitant increase of C1s and chondrocyte hypertrophy has been confirmed by RNA blot and by in situ hybridization. These results, in addition to previous findings on C1s collagenolytic and gelatinolytic activities, suggest C1s participation in cartilage remodeling.

Complete removal of sphingolipids from the plasma membrane disrupts cell to substratum adhesion of mouse melanoma cells.

GM-95, a mutant cell line derived from mouse melanoma MEB-4 cells, is deficient in glycosphingolipids (GSLs) due to the lack of ceramide glucosyltransferase-1 activity (Ichikawa, S., Nakajo, N., Sakiyama, H., and Hirabayashi, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2703-2707). In this study, we examined the involvement of the complex sphingolipids in cell to substratum adhesion. Immunofluorescent and chemical analyses revealed that the complex sphingolipids were significantly concentrated in the detergent-insoluble substrate attachment matrix of both GM-95 and MEB-4 cells. In spite of the absence of GSLs, GM-95 cells retained the ability to adhere to extracellular matrix (ECM) proteins such as fibronectin, collagen, and laminin. When both GM-95 and MEB-4 cells were treated with neutral sphingomyelinase, GM-95 cells were rounded up and detached from all ECM proteins examined. In contrast, neither the morphology nor the adherence of MEB-4 cells was altered. Under this treatment, sphingomyelin (SM) became undetectable in both cells. A similar inhibition was observed upon pretreatment of cells with fumonisin B1 or ISP-1, both of which block the synthesis of ceramide, a common precursor of both GSLs and SM. Stable transfectants expressing GSLs, which were established by transfection of glucosyltransferase-1 cDNA into GM-95 cells, became resistant to neutral sphingomyelinase-mediated rounding up and detachment from ECM proteins. In conclusion, the complex sphingolipids play critical roles in cell to substratum adhesion, and the presence of either GSLs or SM is sufficient for the adhesion.

Site-directed mutagenesis of hamster complement C1S: characterization with an active form-specific antibody and possible involvement of C1S in tumorigenicity.

We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster C1s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. C1s produced from these transfectants lost their enzyme activity. Transfectants of these mutated C1s cDNA did not form tumors in nude mice, To distinguish between active and inactive C1s in situ, we have developed novel antibodies, one directed to the NH2-terminal neoepitope of the L chain and the other specific for uncleaved inactive C1s. These antibodies were used to characterize C1s produced by transfectants, so as to determine whether or not it was cleaved at the right position.

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

Identification of putative 5-hydroxytryptamine(4) (5-HT4) receptors in guinea pig stomach: The effect of TKS159, a novel agonist, on gastric motility and acetylcholine release.

We investigated the existence and the function of 5-hydroxytryptamine(4) (5-HT4) receptors and the effect of TKS159, a novel agonist of 5-HT4 receptor, on guinea pig stomach. The mechanical activity and the release of [3H]ACh were measured using preparations of muscle layers attached to intramural plexus from guinea pig stomach. 5-HT in the presence of 1 microM methysergide, 1 microM ketanserin and 1 microM granisetron, 5-methoxytryptamine or TKS159 enhanced the electrical transmural stimulation-evoked contraction and [3H]ACh release in strips of the stomach in a concentration-dependent manner. This enhancement by 5-HT, 5-methoxytryptamine or TKS159 was antagonized by SDZ 205-557 or atropine. Cisapride, metoclopramide and TKS159 enhanced the electrical transmural stimulation-evoked contraction and release of [3H]ACh in a concentration-dependent manner. We conclude that the pharmacological characteristics of the receptor, which mediates contraction of the guinea pig stomach by the activation of cholinergic nerves, are consistent with its being of the putative 5-HT4 receptor type and that TKS159 is an agonist at 5-HT4 receptors.