Cholesterol-α-glucosyltransferase gene is present in most Helicobacter species including gastric non-Helicobacter pylori helicobacters obtained from Japanese patients.

BACKGROUND: Non-Helicobacter pylori helicobacters (NHPHs) besides H. pylori infect human stomachs and cause chronic gastritis and mucosa-associated lymphoid tissue lymphoma. Cholesteryl-α-glucosides have been identified as unique glycolipids present in H. pylori and some Helicobacter species. Cholesterol-α-glucosyltransferase (αCgT), a key enzyme for the biosynthesis of cholesteryl-α-glucosides, plays crucial roles in the pathogenicity of H. pylori. Therefore, it is important to examine αCgTs of NHPHs. MATERIALS AND METHODS: Six gastric NHPHs were isolated from Japanese patients and maintained in mouse stomachs. The αCgT genes were amplified by PCR and inverse PCR. We retrieved the αCgT genes of other Helicobacter species by BLAST searches in GenBank. RESULTS: αCgT genes were present in most Helicobacter species and in all Japanese isolates examined. However, we could find no candidate gene for αCgT in the whole genome of Helicobacter cinaedi and several enterohepatic species. Phylogenic analysis demonstrated that the αCgT genes of all Japanese isolates show high similarities to that of a zoonotic group of gastric NHPHs including Helicobacter suis, Helicobacter heilmannii, and Helicobacter ailurogastricus. Of 6 Japanese isolates, the αCgT genes of 4 isolates were identical to that of H. suis, and that of another 2 isolates were similar to that of H. heilmannii and H. ailurogastricus. CONCLUSIONS: All gastric NHPHs examined showed presence of αCgT genes, indicating that αCgT may be beneficial for these helicobacters to infect human and possibly animal stomachs. Our study indicated that NHPHs could be classified into 2 groups, NHPHs with αCgT genes and NHPHs without αCgT genes.

Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3.

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

Oligomerized CARD16 promotes caspase-1 assembly and IL-1ß processing.

Increasing evidence indicates that caspase recruitment domain (CARD)-mediated caspase-1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)-1ß release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo-oligomer. Oligomerized CARD16 formed a filament-like structure with CASP1CARD and a speck with apoptosis-associated speck-like protein containing a CARD. A filament-like structure formed by CARD16 promoted CASP1 filament assembly and IL-1ß release. In contrast, CARD17 did not form a homo-oligomer or filaments and inhibited CASP1-dependent IL-1ß release. Mutated CARD16D27G, mimicking the CARD17 amino acid sequence, formed a homo-oligomer but failed to form a filament-like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL-1ß release. These results suggest that oligomerized CARD16 promotes CARD-mediated molecular assembly and CASP1 activation.

Inflammasome activation by mitochondrial oxidative stress in macrophages leads to the development of angiotensin II-induced aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of AAA remains unclear. We previously showed that the inflammasome, which regulates the caspase-1-dependent interleukin-1ß production, mediates the sterile cardiovascular inflammatory responses. Therefore, we hypothesized that the inflammasome is a key mediator of initial inflammation in AAA formation. APPROACH AND RESULTS: Apoptosis-associated speck-like protein containing a caspase recruitment domain is highly expressed in adventitial macrophages in human and murine AAA tissues. Using an established mouse model of AAA induced by continuous infusion of angiotensin II in Apoe(-/-) mice, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice were shown to decrease the incidence, maximal diameter, and severity of AAA along with adventitial fibrosis and inflammatory responses significantly, such as inflammatory cell infiltration and cytokine expression in the vessel wall. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice also reduced elastic lamina degradation and metalloproteinase activation in the early phase of AAA formation. Furthermore, angiotensin II stimulated generation of mitochondria-derived reactive oxygen species in the adventitial macrophages, and this mitochondria-derived reactive oxygen species generation was inhibited by NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency. In vitro experiments revealed that angiotensin II stimulated the NLRP3 inflammasome activation and subsequent interleukin-1ß release in macrophages, and this activation was mediated through an angiotensin type I receptor/mitochondria-derived reactive oxygen species-dependent pathway. CONCLUSIONS: Our results demonstrate the importance of the NLRP3 inflammasome in the initial inflammatory responses in AAA formation, indicating its potential as a novel therapeutic target for preventing AAA progression.

ASC in renal collecting duct epithelial cells contributes to inflammation and injury after unilateral ureteral obstruction.

Inflammation plays a crucial role in the pathophysiological characteristics of chronic kidney disease; however, the inflammatory mechanisms underlying the chronic kidney disease process remain unclear. Recent evidence indicates that sterile inflammation triggered by tissue injury is mediated through a multiprotein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in the development of chronic kidney disease using a murine unilateral ureteral obstruction (UUO) model. Inflammasome-related molecules were up-regulated in the kidney after UUO. Apoptosis-associated speck-like protein containing a caspase recruitment domain deficiency significantly reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and improved subsequent renal injury and fibrosis. Furthermore, apoptosis-associated speck-like protein containing a caspase recruitment domain was specifically up-regulated in collecting duct (CD) epithelial cells of the UUO-treated kidney. In vitro experiments showed that extracellular adenosine triphosphate (ATP) induced inflammasome activation in CD epithelial cells through P2X7-potassium efflux and reactive oxygen species-dependent pathways. These results demonstrate the molecular basis for the inflammatory response in the process of chronic kidney disease and suggest the CD inflammasome as a potential therapeutic target for preventing chronic kidney disease progression.

Cutting edge: TLR signaling licenses IRAK1 for rapid activation of the NLRP3 inflammasome.

Activation of the NLRP3 inflammasome by diverse stimuli requires a priming signal from TLRs and an activation signal from purinergic receptors or pore-forming toxins. In this study, we demonstrate, through detailed analysis of NLRP3 activation in macrophages deficient in key downstream TLR signaling molecules, that MyD88 is required for an immediate early phase, whereas Toll/IL-1R domain-containing adapter inducing IFN-ß is required for a subsequent intermediate phase of posttranslational NLRP3 activation. Both IL-1R-associated kinase (IRAK) 1 and IRAK4 are critical for rapid activation of NLRP3 through the MyD88 pathway, but only IRAK1 is partially required in the Toll/IL-1R domain-containing adapter inducing IFN-ß pathway. IRAK1 and IRAK4 are also required for rapid activation of NLRP3 by Listeria monocytogenes, as deletion of IRAK1 or IRAK4 led to defective inflammasome activation. These findings define the pathways that lead to rapid NLRP3 activation and identify IRAK1 as a critical mediator of a transcription-independent,inflammasome-dependent early warning response to pathogenic infection.

Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western diet-fed apolipoprotein E-deficient mice.

OBJECTIVE: Recent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe(-/-)Casp1(-/-) mice were significantly reduced compared to those in Apoe(-/-) mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe(-/-)Casp1(-/-) mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1ß in the plaques as well as plasma levels of IL-1ß, IL-1α, IL-6, CCL2, and TNF-α, in Apoe(-/-)Casp1(-/-) mice were lower than those in Apoe(-/-) mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1ß and IL-1α in macrophages. CONCLUSION: Our findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis.

Anti-inflammatory activity of PYNOD and its mechanism in humans and mice.

Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat-containing protein (NLR) family play important roles in pathogen recognition and inflammation. However, we previously reported that human PYNOD/NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in reconstitution experiments using HEK293 cells. In this study, we investigated the molecular mechanism of PYNOD's anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1-mediated IL-1beta processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1-mediated IL-1beta secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF-kappaB activation and apoptosis and to reduce ASC's ability to promote caspase-1-mediated IL-1beta production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1beta processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1-mediated IL-1beta processing without inhibiting caspase-4 (mouse caspase-11)-mediated caspase-1 processing. Furthermore, PYNOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton.

Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

Critical role of bone marrow apoptosis-associated speck-like protein, an inflammasome adaptor molecule, in neointimal formation after vascular injury in mice.

BACKGROUND: Inflammatory cytokines such as interleukin (IL)-1 beta and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1-dependent IL-1 beta and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC(-/-)) mice. METHODS AND RESULTS: Wire-mediated vascular injury was produced in the femoral artery of ASC(-/-) and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC(-/-) mice after injury. IL-1 beta and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC(-/-) mice. To investigate the contribution of bone marrow-derived cells, we developed bone marrow-transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC(-/-) bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC(-/-) vascular smooth muscle cells was not impaired. CONCLUSIONS: These findings suggest that bone marrow-derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.

Regulatory molecules involved in inflammasome formation with special reference to a key mediator protein, ASC.

The recent identification of cytosolic pattern recognition receptors (PRRs) with leucine-rich repeats, which recognize pathogen-associated molecular patterns (PAMPs), has been garnering considerable attention. Activated PRRs form molecular complexes called inflammasomes, consisting of related proteins that include procaspase 1[interleukin (IL) 1beta converting enzyme (ICE)]. Inflammasomes have been shown to facilitate molecular proximity, stimulate activation of procaspase 1, which consequently produces inflammatory cytokines IL-1beta and IL-18 and ultimately lead to the initiation of innate immunity. An adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), which recruits PRRs carrying the pyrin homologous domain (PYD) and procaspase 1 through PYD and CARD, respectively, is responsible for the formation of some inflammasomes and also activation of procaspase 1. In this review, our main attention will be directed to PYD region analysis of ASC to understand the interaction between PYD-carrying PRRs and ASC. Taking into consideration the other aspects of the ASC gene in the proapoptotic ability and down-regulation by methylation, the biological function of ASC will be discussed in relation to the epigenetic aspects of infection, inflammation, and cancer.

Translocation of heparanase into nucleus results in cell differentiation.

We recently reported that heparanase, one of the extracellular matrix-degrading enzymes, which plays a critical role in cancer progression, is located not only in the cytoplasm but also in the nucleus. Here we identified nuclear translocation of heparanase as a key step in cell differentiation. We applied an in vitro differentiation model of HL-60 cells with 12-0-tetradecanoylphorbol-13-acetate (TPA), in which nuclear translocation of heparanase was observed using immunohistochemical analysis. In this system, nuclear translocation of heparanase was abolished by inhibitors of heat shock protein 90 (HSP90), suggesting the involvement of HSP90 in translocation of heparanase. We further confirmed that overexpression of active form of heparanase induced differentiation of HL-60 cells, although the catalytic negative form of heparanase did not. Therefore we speculate that nuclear translocation of enzymatically active heparanase may be involved in cellular differentiation. Our results suggest that a novel function of heparanase upon cell differentiation would raise a potential new strategy for cancer therapy of promyeloid leukemia and other types of cancer.

Dihydropyrimidine dehydrogenase expression predicts survival outcome and chemosensitivity to 5-fluorouracil in patients with oral squamous cell carcinoma.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme of 5-fluorouracil (5-Fu) catabolism. The aim of this study was to evaluate the prognostic value of DPD expression and the correlation between DPD expression and efficacy of 5-Fu. Retrospective analysis of DPD expression was performed immunohistochemically in 103 patients with oral squamous cell carcinoma (OSCC), in which staining intensity of DPD expression and degree of heterogeneity of DPD expression were categorized. Expression of DPD correlated with lymph node metastasis, mode of invasion and differentiation. Expression of DPD was an independent significant factor for survival outcome and was more predictive than conventional clinical factors. Furthermore, heterogeneous expression of DPD was more effective than homogeneous expression of DPD in neoplastic cells when evaluated in patients treated with chemotherapy including tegafur/uracil (UFT). Expression of DPD is an independent predictor for clinical outcome. Furthermore, heterogeneity of DPD expression may be a clue for predicting sensitivity to 5-Fu in patients with OSCC.

ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated signal adaptor molecules.

Toll-like receptors (TLRs) initiate a signalling cascade via association with an adaptor molecule, myeloid differentiation factor 88 (MyD88) and/or TIR domain-containing adaptor inducing-IFN-beta (Trif), to induce various pro-inflammatory cytokines for microbial eradication. After stimulation of TLR4 with lipopolysaccharide (LPS), both IL-1beta and IL-18 are processed, depending on the activation of caspase-1, although its mechanism remains unclear. ASC is an adapter protein possibly involved in the activation of procaspase-1. To unravel the requirement of ASC, we generated Asc(-/-) mice. Upon stimulation with LPS, Asc(-/-) macrophages failed in the processing of procaspase-1 and maturation of pro-IL-1beta and pro-IL-18, but normally produced other pro-inflammatory cytokines including TNF-alpha and IL-6. MyD88(-/-) and Trif(-/-) macrophages showed normal activation of caspase-1, demonstrating a dispensable role for MyD88 and Trif. After, LPS-challenged Asc(-/-) mice lacked serum elevation of IL-1beta and IL-18. Moreover, the Asc(-/-) mice exhibited neither acute liver injury nor lethal shock. These results demonstrate critical roles for ASC in the release of IL-1beta/IL-18 via activation of caspase-1 and provide new insights into the inflammatory responses for host defence and diseases.

Clinical significance of cellular distribution of moesin in patients with oral squamous cell carcinoma.

PURPOSE: Moesin is a linking protein of the submembraneous cytoskeleton and plays a key role in the control of cell morphology, adhesion, and motility. The aim of the present study was to elucidate the clinical significance of expression patterns of moesin in patients with oral squamous cell carcinoma (OSCC). EXPERIMENTAL DESIGN: Immunohistochemistry for moesin monoclonal antibody was performed on 103 paraffin-embedded specimens from patients with primary OSCC, including 30 patients with locoregional lymph node metastasis, and in the sections from nude mice transplanted with two cell lines derived from a single human tongue cancer (SQUU-A and SQUU-B). RESULTS: Expression patterns of moesin in OSCCs were divided into three groups: membranous pattern; mixed pattern; and cytoplasmic pattern. These expression patterns correlated with tumor size, lymph node metastasis, mode of invasion, differentiation, and lymphocytic infiltration. In about two-thirds of the patients with metastatic lymph node, homogeneous cytoplasmic expression was detected in the metastatic lymph nodes. In addition, SQUU-B with high metastatic potential showed more reduced levels of membrane-bound moesin than SQUU-A with low metastatic potential. A multivariate analysis demonstrated that expression patterns of moesin can be an independent prognostic factor. CONCLUSIONS: Our results suggest that moesin expression contributed to discriminating between patients with the potentiality for locoregional lymph node metastasis and those with a better prognosis and might improve the definition of suitable therapy for each.

ASC is a Bax adaptor and regulates the p53-Bax mitochondrial apoptosis pathway.

The apoptosis-associated speck-like protein (ASC) is an unusual adaptor protein that contains the Pyrin/PAAD death domain in addition to the CARD protein-protein interaction domain. Here, we present evidence that ASC can function as an adaptor molecule for Bax and regulate a p53-Bax mitochondrial pathway of apoptosis. When ectopically expressed, ASC interacted directly with Bax, colocalized with Bax to the mitochondria, induced cytochrome c release with a significant reduction of mitochondrial membrane potential and resulted in the activation of caspase-9, -2 and -3. The rapid induction of apoptosis by ASC was not observed in Bax-deficient cells. We also show that induction of ASC after exposure to genotoxic stress is dependent on p53. Blocking of endogenous ASC expression by small-interfering RNA (siRNA) reduced the apoptotic response and inhibited translocation of Bax to mitochondria in response to p53 or genotoxic insult, suggesting that ASC is required to translocate Bax to the mitochondria. Our findings demonstrate that ASC has an essential role in the intrinsic mitochondrial pathway of apoptosis through a p53-Bax network.

Methylation of ASC/TMS1, a proapoptotic gene responsible for activating procaspase-1, in human colorectal cancer.

ASC/TMS1, a proapoptotic activator of procaspase-1, was reported to be aberrantly methylated in human breast cancer. We found that ASC was methylated in three of five human colon cancer cell lines lacking ASC protein expression. Demethylation treatment of these cell lines lacking ASC with 5-aza-2'-deoxycytidine partially restored ASC expression. Methylated ASC was also detected in six of ten colorectal cancer tissues. Although clear down-regulation of ASC in the whole region of a tumor tissue was hardly observed by immunostaining with anti-ASC mAb, complete suppression of ASC was identified in a minor population of the colorectal tumor cells. The biological significance of ASC methylation inducible ASC suppression in colorectal cancer will be discussed.

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production.

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.