LncRNA HOTTIP promotes proliferation and inhibits apoptosis of gastric carcinoma cells via adsorbing miR-615-3p.

OBJECTIVE: To explore the role of long-noncoding ribonucleic acid HOXA transcript at the distal tip (lncRNA HOTTIP) in the proliferation and apoptosis of gastric carcinoma cells. MATERIALS AND METHODS: The expressions of lncRNA HOTTIP in gastric carcinoma cell lines MGC-803, HGC-27, SNU-1, and SGC-7901 and normal gastric mucosa cell line RGM-1 were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and compared. The effects of lncRNA HOTTIP on proliferation and apoptosis of gastric carcinoma cells were detected by cell counting kit-8 (CCK-8), colony formation assay, and flow cytometry, respectively. StarBase v2.0 website was adopted to predict the relationship between lncRNA HOTTIP and target miRNAs. Dual-Luciferase reporter assay was performed to verify the sponge effect of lncRNA HOTTIP on miR-615-3p. CCK-8 experiment was conducted to detect its effect on proliferation of gastric carcinoma cells after co-silencing lncRNA HOTTIP and miR-615-3p. RESULTS: LncRNA HOTTIP was highly expressed in gastric carcinoma cell lines MGC-803, HGC-27, SNU-1, and SGC-7901 than in normal gastric mucosa cell line RGM-1. After knockdown of lncRNA HOTTIP, the proliferation function of gastric carcinoma cells was markedly weakened, and the proportion of apoptotic cells increased. LncRNA HOTTIP was able to adsorb miR-615-3p via a sponge effect. Notably, knockdown of miR-615-3p restored the effect of silenced lncRNA HOTTIP on the proliferation function of gastric carcinoma cells. CONCLUSIONS: LncRNA HOTTIP is highly expressed in gastric carcinoma cells. It affects cell proliferation and apoptosis in gastric carcinoma by adsorbing miR-615-3p via a sponge effect.

Role of the polycytin-primary cilia complex in bone development and mechanosensing.

Pkd1 encodes PC1, a transmembrane receptor-like protein, and Pkd2 encodes PC2, a calcium channel, which interact to form functional polycystin complexes that are widely expressed in many tissues and cell types. The study of autosomal dominant polycystic kidney disease (ADPKD), caused by inactivating mutations of PKD1 or PKD2 genes, has elucidated the functions of polycystins and their interdependence on primary cilia in renal epithelial cells. We have found that Pkd1 and Pkd2, as well as primary cilia, are present in osteoblasts and osteocytes. In addition, we have found that loss of polycystin-1 (Pkd1) function in mice results in abnormal bone development and osteopenia due to the impaired differentiation of osteoblasts. It is likely that the polycytin/primary cilia complex responds to a multitude of environmental clues affecting skeletal development and bone formation postnatally. Overall, polycystins in bone may define a new target for developing anabolic agents to treat osteoporotic disorders.

Effect of asymmetric dimethylarginine on osteoblastic differentiation.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) that accumulates in renal insufficiency and may be a uremic toxin. To determine whether ADMA inhibits bone metabolism, we investigated the in vitro effect of ADMA on osteoblastic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). METHODS: The effect of ADMA on nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit. The temporal sequence of osteoblastic differentiation in BMSCs was assessed in the presence and absence of ADMA by measuring alkaline phosphatase (ALP) activity, mineralization, and osteoblast gene expression at 0, 4, 8, 12 days of culture. RESULTS: ADMA (5, 50, 500 micromol. L-1) resulted in a dose-dependent decrease in nitrite formation in conditioned media of BMCS cultures, consistent with inhibition of NOS. ADMA treatment was associated with reduced ALP activity, calcium deposition and osteoblast-related gene expression in BMSCs cultures. Concurrent treatment with l-arginine (3600 micromol. L-1) reversed the ADMA (500 micromol. L-1)-mediated decrease in NO production, restored the differentiation potential of BMSCs, and significantly attenuated the down-regulation of Cbfa1 and osteocalcin gene expression by ADMA. CONCLUSIONS: ADMA inhibition of the NO-NOS pathway in BMSCs impairs osteoblastic differentiation of mouse BMSC cultures. These studies further support a role of NO in the local regulation of bone metabolism and the possibility that ADMA may act as uremic toxin on bone through its effect to inhibit NO actions in osteoblasts.

Characterization of the upstream mouse Cbfa1/Runx2 promoter.

Cbfa1 (or Runx2/AML-3/PEPB2alpha) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 5'-flanking region of the Cbfa1 gene containing its "bone-related" or P1 promoter and exon 1. We identified additional coding sequence in exon 1 and splice donor sites that potentially give rise to a novel Cbfa1 isoform containing an 18 amino acid insert. In addition, primer extension mapping identified in the Cbfa1 promoter a minor mRNA start site located approximately 0.8 kb 5' upstream of the ATG encoding the MASN/p57 isoform and approximately 0.4 kb upstream of the previously reported start site. A luciferase reporter construct containing 1.4 kb of the mouse Cbfa1 promoter was analyzed in Ros 17/2.8 and MC3T3-E1 osteoblast cell lines that express high levels of Cbfa1 transcripts. The activity of this construct was also examined in non-osteoblastic Cos-7 and NIH3T3 cells that do not express Cbfa1 and mesenchymal-derived cell lines, including CH3T101/2, C2C12, and L929 cells, that express low levels of mature Cbfa1 transcripts. The 1.4 kb 5' flanking sequence of the Cbfa1 gene directed high levels of transcriptional activity in Ros 17/2.8 and MC3T3-E1 osteoblasts compared to non-osteoblasts Cos-7 cells, but this construct also exhibited high levels of expression in C310T1/2, L929, and C2C12 cells as well as NIH3T3 cells. In addition, Cbfa1 mRNA expression, but not the activity of the Cbfa1 promoter, was upregulated in a dose-dependent manner in pluripotent mesenchymal C2C12 by bone morphogenetic protein-2 (BMP-2). These data indicate that Cbfa1 is expressed in osteogenic as well as non-osteogenic cells and that the regulation of Cbfa1 expression is complex, possibly involving both transcriptional and post-transcriptional mechanisms. Additional studies are needed to further characterize important regulatory elements and to identify additional regions of the promoter and/or post-transcriptional events responsible for the cell-type restricted regulation of Cbfa1 expression.

[Microstructure of erbium-doped Si-rich thermal oxidation SiO2/Si luminescent thin films].

Er ions with 108 keV to a dose of 1 x 10(17) cm-2 were implanted into Si-rich thermal oxidation SiO2/Si thin film using metal vapor vacuum arc (MEVVA) ion source implanter. Er concentration in as-implanted sample, which was attained to -10 at % correspond to the level of -10(21) atoms.cm-3, was analyzed by Rutherford back-scattering (RBS) and X-ray photoelectron spectroscope (XPS). Contents of Er, Si and O in annealed samples were investigated using XPS at room temperature. It showed that Si content was increased and SiO2 content was decreased with increasing the fluence of Si ions. It was found by use dof reflective high energy electron diffraction (RHEED) and atomic force microscope (AFM) that needle micro-crystalline Si in the surface of annealed samples had been formed and Er segregation and precipitation were formed little. Photoluminescence around 1.54 microns from Er-doped Si-rich thermal oxidized SiO2/Si thin film were studied with a He-Cd laser pumping at 414.6 nm at the low temperature of 77 K and room-temperature (RT).

Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells.

The mouse Cbfa1 gene potentially encodes several proteins that differ in their N-terminal sequences, including an osteoblast-specific transcription factor, Cbfa1/Osf2, a Cbfa1 isoform (Cbfa1/iso), and the originally described Cbfa1 gene product (Cbfa1/org). Uncertainty exists about the function of these potential isoforms of the Cbfa1 gene. To examine the transactivation potential of different Cbfa1 gene products, we compared the ability of Cbfa1/Osf2, Cbfa1/iso, and Cbfa1/org overexpression to activate an osteocalcin promoter/reporter construct in NIH3T3 fibroblasts, C3H10T1/2 pluripotent cells and MC3T3-E1 pre-osteoblasts. These three cell lines were transiently cotransfected with a 1.3-kb mouse osteocalcin promoter luciferase-fusion construct (p1.3OC-luc) and different amounts of expression vectors containing the respective full-length Cbfa1 isoform cDNAs. Using transfection protocols with lower amounts of expression plasmid DNAs, we found that all three Cbfa1 isoforms stimulated osteocalcin promoter activity in each of the cell types, consistent with the their ability to induce expression of an osteoblast-specific gene both in non-osteoblast cells and in osteoblast cell lines. However, using transfection protocols with higher amounts of expression plasmids containing Cbfa1 cDNAs, we found that the Cbfa1/Osf2 and Cbfa1/org had less transactivating potential compared with Cbfa1/iso. Our studies suggest that the 87-amino acid N-terminus of Cbfa1/Osf2 is not crucial for optimal transactivation, whereas the 19-amino acid N-terminal sequence of Cbfa1/iso augments transcriptional activation only at high doses of the expression plasmid. The physiological significance of these in vitro findings remain to be determined.

Coordinated maturational regulation of PHEX and renal phosphate transport inhibitory activity: evidence for the pathophysiological role of PHEX in X-linked hypophosphatemia.

The mechanism by which inactivating mutations of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) cause X-linked hypophosphatemia remains unknown. However, recent reports suggest errant PHEX activity in osteoblasts may fail to inactivate a phosphaturic factor produced by these cells. To test this possibility, we examined coordinated maturational expression of PHEX and production of phosphate transport inhibitory activity in osteoblasts from normal and hyp-mice. We assessed the inhibitory activity in conditioned medium by examining the effects on opossum kidney cell phosphate transport and osteoblast PHEX expression by reverse transcriptase-polymerase chain reaction during a 17-day maturational period. Inhibitory activity increased as a function of osteoblast maturational stage, with no activity after 3 days and persistent activity by 6 days of culture. More significantly, equal phosphate transport inhibitory activity in conditioned medium from normal and hyp-mouse osteoblasts (control 1.90 +/- 0.12, normal 1.48 +/- 0.10, hyp 1.45 +/- 0.04 nmol/mg of protein/minute) was observed at 6 days. However, by 10 days hyp-mouse osteoblasts exhibited greater inhibitory activity than controls, and by 17 days the difference in phosphate transport inhibition maximized (control 2.08 +/- 0.09, normal 1.88 +/- 0.06, hyp 1.58 +/- 0.06 nmol/mg of protein/minute). Concurrently, we observed absent PHEX expression in normal osteoblasts after 3 days, limited production at 6 days, and significant production by day 10 of culture, while hyp-mouse osteoblasts exhibited limited PHEX activity secondary to an inactivating mutation. The data suggest that the presence of inactivating PHEX mutations results in the enhanced renal phosphate transport inhibitory activity exhibited by hyp-mouse osteoblasts.

Frequency of functionally important beta-2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals.

There are ethnic differences in the prevalence and severity of hypertension and asthma and in beta-2 adrenergic receptor (BAR2)-mediated vascular responses. Two common polymorphisms of the human BAR2, Arg16 to Gly and Gln27 to Glu, are associated with alterations in BAR2 response, both in vitro and in vivo. Ethnic differences in disease manifestations and responses to treatment may be explained by the altered frequency of BAR2 polymorphisms. To determine the relative frequencies of the Arg16 to Gly and Gln27 to Glu BAR2 polymorphisms in different ethnic groups we studied 415 (123 African-American, 188 Caucasian-American and 104 Chinese) healthy individuals. There was a marked interethnic difference in the frequency of the BAR2 polymorphisms among the ethnic groups. The Glu27 allele was more frequent in Caucasian-American (34.8%) than in African-American individuals (20.7%) (P = 0.0001) and much less frequent in Chinese individuals (7.2%) (P = 0.0001 versus African-American or Caucasian-American). The homozygous Glu27 genotype was more frequent in Caucasian-American (15.4%) than African-American individuals (4.9%) (P = 0.003) and was not observed in Chinese. The Gly16 allele (54.3% versus 41.3%) and homozygous genotype (35.1% versus 18.3%) were more common in Caucasian-American than Chinese individuals (P = 0.003 for both). There is a marked ethnic difference in the frequency of these two common BAR2 polymorphisms among African-American, Caucasian-American and Chinese individuals, with a markedly reduced frequency of the Glu27 polymorphism, the polymorphism associated with resistance to desensitization and increased BAR2 responses, in African-American and Chinese individuals. Such ethnic genotypic differences may explain previously observed alterations in the response to the BAR agonists in different ethnic groups.

Intrinsic mineralization defect in Hyp mouse osteoblasts.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of PEX, an endopeptidase of uncertain function. This defect is shared by Hyp mice, the murine homologue of the human disease, in which a 3' Pex deletion has been documented. In the present study, we report that immortalized osteoblasts derived from the simian virus 40 (SV40) transgenic Hyp mouse (TMOb-Hyp) have an impaired capacity to mineralize extracellular matrix in vitro. Compared with immortalized osteoblasts from the SV40 transgenic normal mouse (TMOb-Nl), osteoblast cultures from the SV40 Hyp mouse exhibit diminished 45Ca accumulation into extracellular matrix (37 +/- 6 vs. 1,484 +/- 68 counts . min-1 . microgram protein-1) and reduced formation of mineralization nodules. Moreover, in coculture experiments, we found evidence that osteoblasts from the SV40 Hyp mouse produce a diffusible factor that blocks mineralization of extracellular matrix in normal osteoblasts. Our findings indicate that abnormal PEX in osteoblasts is associated with the accumulation of a factor(s) that inhibits mineralization of extracellular matrix in vitro.

Genomic structure and isoform expression of the mouse, rat and human Cbfa1/Osf2 transcription factor.

Although the CBFA1 gene encodes an osteoblast-specific transcription factor that regulates osteoblast differentiation, uncertainty exists about the organization of its 5' end and the relevance of a novel N-terminal sequence identified in the mouse Cbfa1/Osf2 isoform. We found the novel 5' Cbfa1/Osf2 sequence is encoded by a previously unrecognized upstream exon, designated exon -1, which is highly conserved in mouse, rat and human. In addition, two splice donor sites may be utilized to generate Cbfa1/Osf2 cDNAs containing different N-terminal sequences. The first ATG and splice donor site in exon -1 is predicted to transcribe a cDNA containing the unique Osf2 5' sequence, whereas a second donor splice site gives rise to cDNAs that contain sequences encoding an 11 amino acid insert. In the human CBFA1 gene, an additional 2-bp nucleotide insert shifts the reading frame and results in stop codons in the cDNA sequence derived from exon -1. The 5'-most exon of the human CBFA1 gene, therefore, contains the 5' non-coding region rather than a human OSF2 homolog. The absence of a homologous OSF2 coding sequence in the human CBFA1 cDNA suggests that the novel mouse N-terminal Osf2 sequence is not essential for functioning of the CBFA1 gene product. In addition, multiple transcripts derived from a single CBFA1/Cbfa1 gene were detected in osteoblasts by Northern analysis and RT-PCR, including additional Cbfa1/Osf2 isoforms containing deletions of exons 1 and 4. Thus, the alternative use of transcription start sites and splicing leads to the genesis of CBFA1/Cbfa1 isoforms with possible differences in transactivation potentials.

Evidence for the effect of gender on activity of (S)-mephenytoin 4'-hydroxylase (CYP2C19) in a Chinese population.

There is evidence that the sex-dependent expression of individual forms of the human cytochrome P450s (CYPs) results in gender-related differences in the hepatic metabolism of certain drugs. Previous work has shown that conflicting evidence exists relating to the sex differences in the activity of (S)-mephenytoin 4'-hydroxylase (CYP2C19). Accordingly, we assessed the effect of gender on CYP2C19 activity in a phenotyped and genotyped healthy unrelated Chinese population for further evidence of such a gender-based differentiation. One hundred and sixteen females and 129 males took one tablet of 100 mg racemic mephenytoin (Mesantoin, Sandoz) after emptying their urinary bladders. Amounts of (S)- and (R)-mephenytoin and its metabolite 4'-hydroxymephenytoin (4'-OH-M) excreted in the postdose 0-8 h urine collection were determined by GC and HPLC methods, respectively. The CYP2C19 activity was expressed as the ratio of S/R-mephenytoin (S/R-ratio), the percentage of the dose excreted as 4'-OH-M (D%), and the log10 of the hydroxylation index which was defined as the ratio of micromoles of (S)-mephenytoin dose to micromoles of 4'-OH-M excreted in urine (1g HI). From all the subjects studied, 53 extensive metabolizers (EMs) and 19 poor metabolizers (PMs) phenotyped were randomly selected and the DNA extracted from their blood samples was utilized for genotyping analysis according to the previously developed standard procedures. In this population, the phenotype PMs were identified in 10.9% (14/128) of the males, as compared with 11.2% (13/116) of the females (chi 2 = 0.0045, df = 1; p > 0.05). In all phenotyped subjects, the S/R-ratio of EM males was significantly higher than that of EM females (mean +/- SD; 0.28 +/- 0.17 vs. 0.24 +/- 0.15; p = 0.030), but no sexual differentiation was observed (p > 0.05) in 4'-OH-M excreted among all EMs and PMs, or the S/R-ratio among all PMs. In all genotyped EMs, the frequency of homozygous EMs was 18.4% higher in females (51.7%, 15/29) than in males (33.3%, 8/24) although there was no significant difference (chi 2 = 1.1370, df = 1, p > 0.05), but the S/R-ratio was lower in homozygous females than in homozygous males (0.22 +/- 0.14 vs. 0.33 +/- 0.09; p = 0.046). Thus, we conclude that the higher CYP2C19 activity in females exists among both the phenotyped EMs and the genotyped homozygous EMs compared with that in males, and that the defect frequency of the enzyme activity is equal between the genders. We also conclude that the S/R-ratio is more a sensitive metabolic marker of CYP2C19 enzyme activity than the D% and 1g HI.

Differences in the incidence of the CYP2C19 polymorphism affecting the S-mephenytoin phenotype in Chinese Han and Bai populations and identification of a new rare CYP2C19 mutant allele.

The incidence of the S-mephenytoin polymorphism was compared in two Chinese ethnic groups, Han (n = 101) and Bai (n = 202) by phenotype and genotype analysis. The frequency of poor metabolizers (PMs) in Han vs. Bai subjects was 19.8% vs. 13.4%. Han subjects had a higher frequency of the mutant CYP2C19m1 allele (0.366 vs. 0.257, P < .01) and a lower frequency of the wild-type allele (0.559 vs. 0.688, P < .01) than Bai subjects, which is consistent with the difference in the frequencies of PMs between the two ethnic groups. This results in a lower percentage of homozygous wild-type extensive metabolizers of mephenytoin (EMs) in Han subjects than in Bai subjects (40% vs. 59%, P = .005). Therefore, Han subjects may be more susceptible than Bai subjects to the drugs metabolized by the CYP2C19 enzyme. Ratios of urinary S/R-mephenytoin in homozygous EMs were lower than those of heterozygous EMs for both Han and Bai subjects, which shows a gene-dosage effect. Genotype analysis identified all but one PM as homozygous or heterozygous for the two known mutant CYP2C19m1 and/or CYP2C19m2 alleles. A single Bai PM outlier was shown to be heterozygous for CYP2C19m1 and a new mutant CYP2C19 allele containing a single amino acid change of Arg433 --> Trp433. A genotyping test demonstrated that only this one individual carried this rare allele (frequency of 0.0025 in Bai subjects).

Calcitonin gene-related peptide-induced preconditioning protects against ischemia-reperfusion injury in isolated rat hearts.

Our previous work has suggested that the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) can abolish the protective effect of ischemic preconditioning in the isolated rat heart. Therefore we tested the hypothesis that CGRP- or capsaicin-induced preconditioning protects against ischemia-reperfusion injury in the isolated perfused rat heart. Thirty minutes of global ischemia and 30 min of reperfusion caused a significant cardiac contractile dysfunction, ventricular arrhythmia, and an increased release of creatine phosphate kinase. Pretreatment with CGRP or capsaicin, which evokes release of CGRP from cardiac sensory nerves, for 5 min produced a significant improvement of cardiac function, a reduction in the incidence of ventricular arrhythmia, and a decrease in the release of creatine phosphate kinase. However, the cardioprotection provided by CGRP- or capsaicin-induced preconditioning was abolished by CGRP-(8-37) and ruthenium red, respectively. These findings suggest that CGRP- or capsaicin-induced preconditioning protects against ischemic myocardial injury. The present results also suggest that CGRP may be an endogenous myocardial protective substance in the rat.

Ischemic preconditioning mediated by calcitonin gene-related peptide in isolated rat hearts.

AIM: To study the mediating effect of calcitonin gene-related peptide (CGRP) in ischemic preconditioning in the isolated perfused rat heart. METHODS: Isolated rat hearts were subjected to 3 cycles of a 5-min ischemia and a 5-min reperfusion before a 30-min global ischemia followed by a 30-min reperfusion. RESULTS: Ischemic preconditioning caused an improvement of heart functions, reduced the incidence of ventricular arrhythmias and decreased the release of creatine kinase (CK) during reperfusion (CK activities = 0.30 +/- 0.07, 2.03 +/- 0.49, and 0.92 +/- 0.40 U.min-1/g wet wt for control, ischemia-reperfusion, and preconditioning, respectively). However, pretreatment with CGRP8-37 (0.1 mumol.L-1) abolished the improvement of cardiac contractivity, the reduction of the incidence of arrhythmias, and the inhibition of CK release by preconditioning (CK activities = 0.9 +/- 0.4 vs 2.55 +/- 0.32 U.min-1/g wet wt, P < 0.01). CONCLUSION: CGRP is an endogenous myocardial protective substance that played an important role in mediation of ischemic preconditioning.

Genetic analysis of the S-mephenytoin polymorphism in a Chinese population.

The 4'-hydroxylation of S-mephenytoin exhibits a polymorphism in humans, with the poor metabolizer phenotype exhibiting a lower frequency in white (3% to 5%) than in Oriental populations (13% to 23%). Two mutations in CYP2C19 (CYP2C19m1 and CYP2C19m2) have recently been described that account for approximately 85% of white and 100% of Japanese poor metabolizers. This study examines whether these mutations account for the poor metabolizer phenotype in the Chinese population. The metabolism of S-mephenytoin exhibited a bimodal distribution in 244 unrelated Chinese subjects, although the distribution of the two phenotypes overlapped. In 75 selected Chinese subjects, CYP2C19 genotype analysis predicted the phenotype with 100% accuracy. The frequency of the poor metabolizer phenotype was approximately 11% (95% confidence interval 7% to 15%). The frequency of the CYP2C19m1 allele was 0.289, whereas that of CYP2C19m2 was 0.044. Homozygous extensive metabolizers had slightly lower ratios of S/R-mephenytoin compared with heterozygous extensive metabolizers, showing a gene-dosage effect. These data show the advantages of genotype analysis in investigations of the mephenytoin phenotype in Oriental subjects.

Application of enzyme immunoassay on infected cells (EIA-IC) for arboviruses.

Comparative titrations of alpha-, flavi- and Bunyamwera viruses were made by EIA-IC and according to cytopathic effect (CPE). Specific enzymatic reactions appeared earlier and in higher titres than CPE. The titres of dengue type 1, Mayaro, Powassan and Langat viruses measured by EIA-IC were comparable to those measured by intracerebral inoculation of mice. The cross-reactivity testing of EIA-IC among alphaviruses (Chikungunya, Sindbis and Mayaro), flaviviruses (Japanese encephalitis, Murray valley encephalitis, Kunjin, West Nile, yellow fever and louping ill, Powassan, Langat) and Bunyamwera arboviruses using polyclonal immune ascitic fluids confirmed the high specificity of EIA-IC. Homologous reactions mostly showed higher titres than heterologous ones. No cross-reactivity was seen between alpha-, flavi- and bunyaviruses, among the three alphaviruses, between mosquito-borne and tick-borne flaviviruses, or between JE complex and YF viruses. However, a cross-reactivity to different extent was observed among the four JE complex viruses and among louping ill, Powassan and Langat viruses. The results of EIA-IC cross tests showed that this method can distinguish togavirus group- or species-specific antigens, more precisely than conventional ELISA.