Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Simultaneous Determination of Dermatan Sulfate and Oversulfated Dermatan Sulfate in Plasma by High-Performance Liquid Chromatography with Postcolumn Fluorescence Derivatization

A chemical method for the determination of dermatan sulfate (DS) and oversulfated dermatan sulfate has been developed and applied to the pharmacokinetic study of these polysaccharides in experimental animals. The analytical procedure includes a simple preparation step of administered DS and oversulfated DS from blood plasma, HPLC for the separation and detection of DS and oversulfated DS using an Asahipak NH₂P-50 column, fluorometric reaction of the polysaccharides with guanidine in a strong alkaline medium. DS and oversulfated DS were extracted from plasma by treating it with proteinase to remove plasma proteins and recovered with endogenous plasma glycosaminoglycans by ethanol precipitation. Finally, DS and oversulfated DS were analyzed by fluorometric HPLC. The detection limits of DS and oversulfated DS were 10 and 20 ng, respectively. Furthermore, we demonstrated that artificial oversulfation of DS increased its biological half-life after intravenous administration to rats.     

A morphological study and hybridization analysis of Caloglossa leprieurii (Ceramiales, Rhodophyta) from Japan, Singapore and Australia

Morphological and hybridization experiments were performed on Caloglossa leprieurii (Montagne) J. Agardh collected from Japan, Singapore and Australia in order to evaluate taxonomic characters of this species. Within C. leprieurii at least four mating groups were recognized from the Indo-Pacific region. These mating groups could be characterized by the blade width at the inter-node and the cell-row numbers on the opposite side derived from the first axial cell at the main axis, though these properties showed a certain variability even in the same plant under both field and culture conditions. The phylogenetic relationship and geographic distribution of the four mating groups are discussed.     

Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.     

Impairment of raw 264.7 macrophage function by antiarrhythmic drugs

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O ₂ ^– ) and H₂O₂. The production of O₂ was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H₂O₂ production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O₂ and H₂O₂ release in a dose dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O₂ production in zymosan and phorbol ester stimulated cells were found to be 250 M and 300 M, respectively and the amounts required to cause one-half optimum levels of H₂O₂ production in these cells were found to be 50 M and 100 M, respectively. The effect of these drugs on O₂ producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p<0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient differential phagocytosis assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O₂ and H₂O₂ by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.     

Evolution of restriction sites of ribosomal DNA in natural populations of the field mouse,Apodemus speciosus

An analysis by restriction endonuclease digestion of ribosomal DNA (rDNA) was carried out in natural populations of Apodemus speciosus, a field mouse that is endemic to Japan. Two restriction sites, the EcoRI (E3) and DraI (D4) sites, in the nontranscribed spacer region downstream from the gene for 28S RNA showed polymorphism within and between individuals in the populations from the Japanese main islands. By contrast, populations from the small adjoining islands which are thought to have separated from the main islands 1–2 × 10⁴ years ago showed relatively low levels of polymorphism within and between individuals, i.e., one of the polymorphic bands in the case of each enzyme was predominant in these populations, irrespective of the variants. These results indicate that the rate of fixation of site variations depends on population size and that the direction of fixation is random. Furthermore, each polymorphic restriction site seems to be fixed independently.Correspondence to: H. Suzuki     

Comparative study of inhibitory effects by murine interferon γ and a new bisphosphonate (alendronate) in hypercalcemic,nude mice bearing human tumor (LJC-1-JCK)

The inhibitory effect of murine interferon (muIFN) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), muIFN was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.     

Mutation analysis of a Sandhoff disease patient in the Maronite community in Cyprus

Sandhoff disease occurs in the Christian Maronite community in Cyprus, a community that established over a thousand years ago. Nowadays, this community comprises less than 1% of the whole population, and has been culturally and socially isolated. Cultured fibroblasts from a patient from this inbred group showed a -hexosaminidase subunit mRNA of apparently the normal size but of reduced quantity. A mutational analysis of cDNA obtained by polymerase chain reaction amplification of mRNA showed a deletion of A at nt 76 (counted from A of the initiation codon, ATG). The deletion results in a frame shift and a premature termination within 20 amino acids from the N-terminus of the normal mature enzyme protein. The patient was homozygous for the deletion. The 5-end of the gene showed many discrepancies from the previously published sequence. We consider that these differences are probably polymorphisms of little functional significance, because the patient's fibroblasts generate decreased but stable mRNA and because some of these base changes were also found in the genes from control fibroblasts. An extensive evaluation of the prevalence of this mutant allele in this community is being initiated.     

A Similar Expression Pattern of Adhesion Molecules between Intermediate TCR Cells in the Liver and Intraepithelial Lymphocytes in the Intestine

Two major populations of extrathymically differentiated T cells exist in the liver and intestine. Such T cells in the liver have TCR of intermediate intensity (i.e., intermediate TCR cells) and constitutively express IL-2 receptor β-chain (IL-2Rβ), whereas those in the intestine, especially intraepithelial lymphocytes, have TCR of bright intensity, consisting of a mixture of IL-2Rβ⁺ and IL-2Rβ^–. All mature thymocytes and thymus-derived T cells seen in the peripheral immune organs are TCR-bright⁺IL-2Rβ^– under resting conditions. When the expression pattern of adhesion molecules, including CD44, L-selectin, LFA-1 and ICAM-1, was compared among these T-cell populations, they displayed quite unique patterns of expression. All extrathymic T cells in the liver, intestine, and even other organs were CD44⁺L-selectin^– LFA-1⁺⁺ICAM-1⁺, whereas thymocytes and thymus-derived T cells were CD44^– L-selectin⁺LFA-1⁺ICAM-1^–. This inverted expression of adhesion molecules between extrathymic T cells and thymus-derived T cells might be associated with their unique tissue-localization.     

The Location of a Disease-Associated Polymorphism and Genomic Structure of the Human 52-kDa Ro/SSA Locus (SSA1)

Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exons were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race.