Induction of inflammatory arthropathy resembling rheumatoid arthritis in mice transgenic for HTLV-I.

Human T cell leukemia virus type-I (HTLV-I) is the etiologic agent of adult T cell leukemia and has also been suggested to be involved in other diseases such as chronic arthritis or myelopathy. To elucidate pathological roles of the virus in disease, transgenic mice were produced that carry the HTLV-I genome. At 2 to 3 months of age, many of the mice developed chronic arthritis resembling rheumatoid arthritis. Synovial and periarticular inflammation with articular erosion caused by invasion of granulation tissues were marked. These observations suggest a possibility that HTLV-I is one of the etiologic agents of chronic arthritis in humans.

Transformation of mouse BALB 3T3 cells by enterobacterial plasmid misrepair gene mucAB.

The enterobacterial plasmid misrepair gene mucAB, ligated to the metal-inducible mammalian MT-1 promoter, was introduced into the genome of mouse BALB 3T3 cells. In the presence of zinc ions, MucA but not MucB protein was produced, and the whole-cell population of each mucAB+ clone started to show the transformation phenotype in a few days. Foci appeared in the transformed cell population after 4 weeks, and cells from the foci produced tumors in nude mice, indicating malignant transformation by the mucA product. Growth of mucAB+ cells was stimulated by zinc-induced expression of mucA. The transformation phenotype was reversed by removing zinc ions from the culture, indicating that the transformation was due not to MucA-mediated mutation in the mouse genome but to the direct transforming activity of MucA protein.

The induction of cataracts by HIV-1 in transgenic mice.

OBJECTIVE: To elucidate the tissue specificity of the expression of HIV-1 genes in an animal and its pathological effects on these tissues. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were bred in order to overcome the host-range barrier of this virus. RESULTS: mRNA specific to the transgene was detected in the eyes and the spleen, and, in smaller quantities, in the thymus and the brain. Interestingly, many of the transgenic mice developed cataracts at 3-6 months of age. Swelling and vacuolation of the lens fiber cells were marked, but the epithelial cells of the lens were less affected. HIV antigens were detected in the lens fiber cells and the retina by immunological staining. Accumulation of large amounts of p24 Gag antigen was demonstrated in the affected lens by immunoblot analysis, while negligible Env or other viral proteins was detected. Although accumulation of the Gag protein was also detected in the skin and the brain, no apparent abnormality was observed in these tissues. CONCLUSIONS: Preferential expression of the HIV genes in the eyes, skin, brain and lymphoid tissues was demonstrated. The accumulation of the Gag protein is suggested to have detrimental effects on lens fiber cells, causing cataracts.

A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor.

We have demonstrated a simple yet direct method for determining the kinetic parameters in DNA-DNA interactions using biosensor technology based on the surface plasmon resonance phenomenon; a technique that does not require complex DNA labeling. To determine the effect of mismatches on the kinetics involved in DNA-DNA interactions, DNA hybridization kinetics were monitored in real time using synthetic oligonucleotides less than 20 bases in length which contained either a complementary sequence or mismatched bases. Upon analysis of the kinetic parameters obtained in oligonucleotide hybridization, we found that they were significantly affected by the presence of mismatches as well as by their number and location in a DNA duplex. In addition, the presented biosensor method is sensitive enough to detect kinetic effects caused by the presence of a single-mismatched base pair. Our findings strongly suggest that analysis of kinetic parameters involved in DNA-DNA interactions is advantageous for detecting the presence of mismatch base pairs in a DNA duplex.

Use of a biosensor based on surface plasmon resonance and biotinyl glycans for analysis of sugar binding specificities of lectins.

We have developed a new method for analysis of the interaction between lectins and biotin-derivatized oligosaccharides involving a biosensor based on surface plasmon resonance. Complex type asialo-bi-, tri-, and tetra-antennary oligosaccharides were quantitatively converted into their biotin derivatives by incubating them with 6-(D-biotinyl)-aminohexanoyl hydrazide. This method was also applicable to sialyl sugar chains without any removal of sialic acid residues. The reaction mixture could be directly injected onto the streptavidin pre-immobilized surface of a sensor chip without any purification because of its fairly low reagent/carbohydrate molar ratio. The amounts of sugar chains required for interaction analysis by this method were as low as 1 pmol. The binding specificities of Sambucus sieboldiana lectin, Maackia amurensis lectin, Ricinus communis agglutinin-120 (RCA120), and concanavalin A could be rapidly determined qualitatively by this method. Furthermore, kinetic analysis of the interaction between RCA120, and complex type asialo-bi-, tri-, and tetra-antennary oligosaccharides revealed that both the association rate constant and the dissociation rate constant (kdiss) decreased with increasing numbers of terminal galactosyl residues. Because the tendency observed for kdiss paralleled the elution order of these oligosaccharides on RCA120 immobilized affinity chromatography, kiss might hold the key to determination of the elution order on affinity chromatography.

[Gene diagnosis with an affinity sensor, BIACORE--principle and applications].

We are developing new techniques for detecting point mutations by DNA-DNA hybridization and DNA-protein interaction analysis with an affinity sensor, BIACORE. To detect point mutations by the hybridization method using synthetic oligonucleotides, we already found that the length of the probe and the location of mismatches were important. The PCR products of the N-ras gene derived from Hep G2 cells, which have heterozygous point mutations at codon 61 in the gene, were analyzed and the point mutations were detected with 13-mer probes. We suggest that the detection method using DNA-DNA hybridization is useful for detecting known point mutations. However, detect unknown mutations, E. coli mismatch recognition protein, MutS, was employed. All mismatches in immobilized 20 base pairs of double-strand DNA could be detected by MutS binding. We have started to apply of the MutS to the detection of point mutations in PCR products.

Extrachromosomal circular DNAs in interspecific mouse-rat reconstituted cells.

Several clones of reconstituted cells were isolated by fusion of karyoplasts of mouse melanoma B16 cells with cytoplasts of rat myoblastic L6 cells and processed by the mica-press-adsorption method for electron microscopy. Extrachromosomal small circular DNAs of less than 1 micron in contour length (smaller circular DNA) were present in both parental cells, and circles larger than 1 micron (larger circular DNA) were found more frequently in reconstituted cells at an early stage after the clonal isolation. Mitochondrial DNA was not released from mitochondria by this method. The new phenotypes of reconstituted cells were stable, but larger circles were lost after prolonged cultivation of the cells. The possibility that the larger circular DNAs result from intrachromosomal DNA rearrangement induced by rat cytoplasts is discussed.

Histone expressions in mouse-rat somatic reconstituted cells.

A critical analysis of histone expression was performed on the four interspecific and the two intraspecific reconstituted cells formed between karyoplast from mouse B16 cells and the cytoplast from rat cells (L6TG.CAPr) or mouse cells (B82.CAPr). All the reconstituted cells had the same pattern of mouse histones and the same amount of mouse-specific H2B. 2 histone as that of mouse nuclear donor cells. A hybrid between B16 and L6TG.CAPr contained both mouse and rat-specific H1b subtypes, whereas no rat-specific H1b was detected in the interspecific reconstituted cells. In both intra- and interspecific reconstituted cells, the proportion of H1b content was lower than that of B16 cells but that of H1 degree was higher, indicating that the mouse H1 patterns from these cells slightly resembled the pattern of slower growing and differentiated cytoplast donor cells. As an effect of the tumor promoter, the H1 pattern tended to revert to that of the nuclear donor cells in agreement with the phenotypic reversion, without any significant change in cell growth.

Clonal isolation and characterization of myoblast-like reconstituted cells formed by fusion of karyoplasts from mouse teratocarcinoma cells with rat myoblast cytoplasts.

To examine the roles of the cytoplasms of differentiated somatic cells on nuclear gene expression, reconstituted cells (RC-cells) were isolated clonally by fusing karyoplasts (isolated nuclei) from neomycin-resistant mouse teratocarcinoma PCC4-neor cells with cytoplasts (isolated cytoplasms) of chloramphenicol (CAP)-resistant rat myoblasts L6TG.CAPr cells, and after double selection in the medium containing 400 micrograms/ml of neomycin and 100 micrograms/ml of CAP (G418 plus CAP medium). The RC-cells were characterized by the presence of two genetic markers, neomycin- and CAP-resistance, by the absence of latex beads which had incorporated into karyoplast donor PCC4-neor cells as a cytoplasmic physical marker, and by the similar karyotypes as that of parental PCC4-neor cells. In contrast to the teratocarcinoma cybrids previously isolated, all the isolated RC-clones expressed myoblast-like morphologies of three types. The phenotypic expression of these RC-cells was compared with that of PCD-1 cells, a teratocarcinoma-derived myoblast line. RC-cells and PCD-1 cells did not express alkaline phosphatase (ALPase) activity while parental PCC4-neor expressed it strongly. After induction of myogenic differentiation by treatments with excess thymidine and conditioned medium, two clones were capable of forming short multinucleated cells. The protein synthetic patterns of RC-cells analysed by two-dimensional polyacrylamide gel were different from PCC4-neor cells, and quite resembled those of PCD-1 cells. Particularly, multinucleated RC-clones expressed alpha-tropomyosin, and contained 10 nm filaments, characteristic markers of early myogenic cells. These results suggest that the RC-cells are myoblast-like cells, that a few of them maturate to partially differentiated myogenic cells, that the rat myoblast cytoplasm contains regulatory factor(s) able to determine the myogenic cell lineage of the undifferentiated stem cells, and that this factor is continuously expressed in these myoblasts.

Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor based on surface plasmon resonance.

Kinetic measurements of the interaction between an oligosaccharide and various lectins were performed using a biosensor based on surface plasmon resonance (SPR). A glycopeptide, prepared from asialofetuin and having a nearly homogeneous N-linked sugar chain, was immobilized on the surface of a sensor chip via the amino groups of its peptide moiety. The interactions of this bound glycopeptide with six lectins [Sambucus sieboldiana lectin, Maackia amurensis lectin, Aleuria aurantia lectin, Ricinus communis agglutinin-120 (RCA120), Datura stramonium lectin (DSA) and Phaseolus vulgaris leukoagglutinating lectin] were monitored in real-time with the change in the SPR response. Of these lectins, only RCA120 and DSA showed an increase in the SPR response, indicating that these two lectins bound specifically to the immobilized glycopeptide. The other lectins did not show any significant changes in the SPR response. These results are in good agreement with the binding specificity previously demonstrated with affinity chromatography. The association-rate constant (kass) and the dissociation-rate constant (kdiss) for the glycopeptide-RCA120 interaction were 3.4 x 10(5) M-1 s-1 and 2.1 x 10(-3) s-1, respectively. The kass and kdiss determined for DSA were 5.7 x 10(5) M-1 s-1 and 1.3 x 10(-3) s-1, respectively. Furthermore, the relative binding molar ratio to the glycopeptide was three times higher for RCA120 than for DSA, suggesting that this sugar chain possesses three binding sites for RCA120 and one for DSA. These parameters are expected to provide useful information for defining the interaction between oligosaccharides and lectins.

Suppression of tumorigenicity in interspecific reconstituted cells and cybrids.

The effect of cytoplasms on the expression of tumorigenicity was examined in interspecific reconstituted cells and cytoplasmic hybrids (cybrids) formed by fusions between karyoplasts (or intact cells) from highly tumorigenic mouse melanoma B16 cells and cytoplasts from non-tumorigenic, hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-deficient and chloramphenicol (CAP)-resistant rat myoblastic cells, L6TG X CAPr. The reconstituted clones and cybrid clones isolated in the double selection medium, HAT and CAP, acquired CAP-resistance, and each showed unique morphology and cellular arrangement. Tumorigenicity was suppressed in all the reconstituted clones and cybrid clones examined at an early stage after their isolation, but re-appeared in some clones after prolonged cultivation of the cells.

Mitochondrial DNA analysis of mouse-rat hybrid cells: effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs.

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAPr) and CAP-sensitive (CAPs) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAPr mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAPr parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAPr parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1-4 times that of rat mtDNAs inthe mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.

Structure of HTLV and its biological function in leukemogenesis of adult T-cell leukemia.

There is a high homology of nucleotide sequence between 3' two-thirds of the X (or pX) regions of human T-cell leukemia virus (HTLV)-I, and of HTLV-II. Monoclonal antibody against p41 coded from X-IV, an open reading frame of X region of HTLV-I, was established. Two proteins coded by Xb, one of the open reading frames in X region of HTLV-II, were newly identified as p24 and p26. The expression of X protein of HTLV-II in the reconstituted mouse embryonal carcinoma cell line, which shows myoblastic morphology, reverted the morphology to that of the original embryonal carcinoma cells. This suggests that the function of X protein is to disturb the regulation of cell lineage determination. Leukemogenesis of adult T-cell leukemia (ATL) is also considered to consist of multisteps, in which HTLV-I constitutes one step, other factors also being involved. Even the role of HTLV-I factor could be similarly played by other factor(s). In agreement with this hypothesis, there are patients with ATL without associated HTLV-I.

Rapid method for detection of point mutations using mismatch binding protein (MutS) and an optical biosensor.

This paper describes a new method for detecting DNA point mutations using a mismatch binding protein. The interactions of mismatches and mismatch binding proteins are detected by the optical biosensor technology based on surface plasmon resonance (SPR).

Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter.

Chloramphenicol-resistant (CAPr) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B16 cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) -deficient, CAPr rat myoblastic cells, L6TG.CAPr, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B16 cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.

Bifunctional labeling reagent for oligosaccharides to incorporate both chromophore and biotin groups.

We have developed a convenient and effective method for biotinylation of oligosaccharides at their reducing ends. A novel biotin hydrazide having a phenyl group produced the biotin adduct of N-acetyllactosamine (LacNAc) by simple incubation at 90 degrees C for 1 h. Although the biotin adduct was obtained as a mixture of several stereoisomers, one of the isomers, cyclic beta-glycoside, became predominant upon letting the reaction mixture stand in a weakly acidic state (pH 3.5). This conversion may be very advantageous for functional analysis of oligosaccharides because natural N-linked oligosaccharides exist in the cyclic beta form. The limit of detection of labeled LacNAc in reversed-phase chromatography was 330 fmol and showed good linearity in the range from 330 fmol to 261 pmol. When this procedure was applied to complex type and high mannose type N-linked oligosaccharides, the labeled oligosaccharides were easily detected and separated by reversed-phase, gel filtration, and anion exchange chromatographies. Furthermore, these labeled oligosaccharides were able to be immobilized onto the solid phase using avidin-biotin technology and were stable enough to allow the binding assay to be performed repeatedly and under the conditions for in situ exoglycosidase digestion. These results suggest that this derivatization technique might be useful for both separation and functional analysis of oligosaccharides.

Augmentation of c-fos and c-jun expression in transgenic mice carrying the human T-cell leukemia virus type-I tax gene.

To analyze the effect of human T-cell leukemia virus type I (HTLV-I) on cellular gene expression and its relation to tumorigenesis, two lines of transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR regions of the HTLV-I genome were produced. The transgene was expressed in many organs, including the brain, salivary gland, spleen, thymus, skin, muscle, and mammary gland. We found that the expression of the c-fos and c-jun genes, but not of the lyn and c-myc genes, was augmented 2- to 20-fold in histologically normal skin and muscle of these mice. The augmentation was tissue specific, suggesting the involvement of a cellular factor in the transgene action. In these mice, a three to seven times higher incidence of tumors was seen as compared with the control mice. These tumors included mesenchymal tumors, such as fibrosarcoma, neurofibroma, and lipoma, and adenocarcinomas of the mammary gland, salivary gland, and lung. The c-fos and c-jun genes were also activated in these tumors. The possible roles of elevated c-fos and c-jun gene expression in tumorigensis are discussed.

Autoimmunity induction by human T cell leukemia virus type 1 in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans.

We recently reported on an inflammatory arthropathy resembling rheumatoid arthritis that develops in high incidence among transgenic mice that carry the env-pX region of the human T cell leukemia virus type 1 genome. In an effort to elucidate the pathogenesis of this disease, we found that genes for inflammatory cytokines, including IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, transforming growth factor-beta 1, IFN-gamma, and IL-2, as well as MHC genes were activated in transgenic joints. Serum levels of IL-1 beta and IL-6 were also elevated. Interestingly, these mice produced Ab against IgG, type II collagen (IIC), and heat shock proteins accompanied by IgG hypergammaglobulinemia. The cellular immune response to IIC as well as that to heat shock proteins were activated. Moreover, these mice became immunologically responsive to exogenously administered IIC and developed arthritis, in contrast to their nontransgenic littermates, which showed little response to IIC. Taken together, the results suggest that human T cell leukemia virus type 1 can cause immune system hyperreactivity and induce autoimmunity. The possibility that elevated cytokine and/or MHC gene expression are involved in the development of autoimmunity and arthropathy are discussed.