An 8-cM interstitial deletion on 4q21-q22 in DNA from an infant with hepatoblastoma overlaps with a commonly deleted region in adult liver cancers

We performed molecular analysis of a germline interstitial deletion of chromosome 4 [del(4)(q21.22q23)], which had been observed in a male infant manifesting early-onset hepatoblastoma (HBL). The chromosomal anomaly in this child was associated with a unique congenital syndrome including HBL, atrial septal defect, ventricular septal defect, patent ductus arteriosus, mental retardation, and seizures. However, the patient did not exhibit a megalencephaly typical of 4q21-22 deletions. His HBL was associated with an increasing serum alpha-fetoprotein level and rapid growth. To define the chromosomal deletion at the molecular level in this child, we analyzed his lymphoblasts with fluorescence in situ hybridization, using as probes a panel of BAC/PAC genomic clones containing STS markers covering the 4q12-27 region. The analysis revealed that the affected chromosome had an 8-cM deletion within 4q21-q22, flanked by markers D4S2964 and D4S2966. This microdeletion overlaps with the commonly deleted region at 4q21-q22 that was recently defined in adult hepatocellular carcinomas.     

Myelin Degeneration in Multiple System Atrophy Detected by Unique Antibodies

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.     

Vp130, a chloroviral surface protein that interacts with the host Chlorella cell wall

A protein, Vp130, that interacts with the host cell wall was isolated from Chlorovirus CVK2. From its peptide sequence, the gene for Vp130 was identified on the PBCV-1 genomic sequence as an ORF combining A140R and A145R. In Vp130, the N-terminus was somehow modified and the C-terminus was occupied by 23-26 tandem repeats of a PAPK motif. In the internal region, Vp130 contained seven repeats of 70-73 amino acids, each copy of which was separated by PAPK sequences. This protein was well conserved among NC64A viruses. A recombinant rVp130N protein formed in Escherichia coli was shown not only to bind directly to the host cell wall in vitro but also to specifically bind to the host cells, as demonstrated by fluorescence microscopy. Because externally added rVp130N competed with CVK2 to bind to host cells, Vp130 is most likely to be a host-recognizing protein on the virion.     

Loss of heterozygosity on chromosome 9q22.3 in microdissected basal cell carcinomas around the Semipalatinsk Nuclear Testing Site, Kazakhstan

A high incidence of skin cancers has been noted around the Semipalatinsk Nuclear Testing Site (SNTS) in Kazakhstan. Recently, basal cell carcinoma (BCC) susceptibility genes, human homolog of the Drosophila pathed gene (PTCH), and the xeroderma pigmentosa group A-complementing gene (XPA), have been cloned and localized on chromosome 9q22.3. To clarify the effect of low-dose irradiation on the occurrence of BCC, we used microdissection and polymerase chain reaction to identify loss of heterozygosity (LOH) at 9q22.3 using BCC samples obtained from this region. Ten Japanese samples were analyzed as controls. LOH with at least 1 marker was identified in 5 of 14 cases from around SNTS, whereas only 1 case with 1 marker was identified among the 10 Nagasaki cases. The total number of LOH alleles from SNTS (8 of 45) was significantly higher than the number from Nagasaki (1 of 26) (P = 0.03). The higher incidence of LOH on 9q22.3 in BCC from around SNTS suggests involvement of chronic low-dose irradiation by fallout from the test site as a factor in the cancers.     

Characterization of factor XII Tenri, a rare CRM-negative factor XII deficiency

Factor XII Tenri (Y34C), a rare cross-reacting material (CRM)-negative factor XII deficiency, was identified in a 71-yr-old Japanese woman with angina pectoris. In the patient's plasma, factor XII activity and antigen levels were only 1.6% and 5.0%, respectively, of those seen in a normal subject. Immunoblot analysis showed that the secreted factor XII Tenri existed not only as a monomer (76 kDa), but also in complexes with apparent molecular weights of approximately 115, 140, 190, 215, and 225 kDa. After reduction with 2-mercaptoethanol, the factor XII Tenri contained in the complexes was completely converted to monomeric form on immunoblot patterns. It appeared that some of the secreted factor XII Tenri formed several types of disulfide-linked complexes, including a factor XII-alpha1-microglobulin complex, through a newly generated Cys residue. The monomeric form of factor XII Tenri, like normal factor XII, was degraded into 2 major fragments with molecular weights of approximately 45 kDa and 30 kDa following mixing with activated partial-thromboplastin-time measuring reagent (cephalin and ellagic acid), whereas the factor XII Tenri that formed the complexes was not. This indicates that the factor XII Tenri present in disulfide-linked complexes with other proteins (and itself) is not converted to active forms, suggesting that attached proteins obstruct or delay the activation of factor XII via an inhibition of its binding to a negatively charged surface in vitro.     

<Emphasis Type="Italic">GPC5</Emphasis> is a possible target for the 13q31-q32 amplification detected in lymphoma cell lines

Comparative genomic hybridization (CGH) analyses have detected gains of copy number on 13q, especially at 13q31-q32, in cell lines and primary cases of various types of lymphoma. Since amplification of chromosomal DNA is one of the mechanisms that can activate tumor-associated genes, and because 13q amplification had been reported in various other types of tumors as well, we attempted to define by fluorescence in situ hybridization (FISH) a common region at 13q31-q32 in which to explore genes that might be targets for the amplification events. Although the commonly amplified region we defined was relatively large (approximately 4 Mb), only one true gene, GPC5, was found there. GPC5 was over-expressed in lymphoma cell lines that had shown amplification, in comparison with those that had not. Our findings suggest that GPC5 is a likely target for amplification, and that over-expression of this gene may contribute to development and/or progression of lymphomas and other tumors.     

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.     

Development of a particle agglutination assay system for detecting Japanese encephalitis virus-specific human IgM,using hydroxyapatite-coated nylon beads

Japanese encephalitis virus-specific IgM is a reliable indicator for serodiagnosis of Japanese encephalitis. A particle agglutination (PA) assay system was developed to detect anti-Japanese encephalitis virus IgM in human serum samples. The newly developed PA assay consisted of hydroxyapatite-coated nylon beads and V-bottom 96-well microplates. Hydroxyapatite-coated nylon beads were coated with Japanese encephalitis virus antigens. Japanese encephalitis virus antigen-coated, hydroxyapatite-coated nylon beads agglutinated in the IgM-captured wells when anti-Japanese encephalitis virus IgM-positive serum samples were used. A button pattern was formed at the bottom of the wells when anti-Japanese encephalitis virus IgM-negative serum samples were used. Thirty anti-Japanese encephalitis virus IgM-positive serum samples from Japanese encephalitis-confirmed cases were tested by the PA assay. All these serum samples were determined to be Japanese encephalitis virus IgM-positive. IgM titers determined by the PA assay corresponded to those determined by enzyme-linked immunosorbent assay. The titers were consistent in two independent PA assays. These results indicate that the newly developed PA assay is a reliable method for detecting anti-Japanese encephalitis virus IgM in human serum samples and that this assay will be a suitable diagnostic system especially in rural areas of Asia.     

Human arylhydrocarbon receptor repressor (AHRR) gene: genomic structure and analysis of polymorphism in endometriosis

The diversity of biological effects resulting from exposure to dioxin may reflect the ability of this environmental pollutant to alter gene expression by binding to the arylhydrocarbon receptor (AHR) gene and related genes. AHR function may be regulated by structural variations in AHR itself, in the AHR repressor (AHRR), in the AHR nuclear translocator (ARNT), or in AHR target molecules such as cytochrome P-4501A1 (CYP1A1) and glutathione S-transferase. Analysis of the genomic organization of AHRR revealed an open reading frame consisting of a 2094-bp mRNA encoded by ten exons. We found one novel polymorphism, a substitution of Ala by Pro at codon 185 (GCC to CCC), in exon 5 of the AHRR gene; among 108 healthy unrelated Japanese women, genotypes Ala/Ala, Ala/Pro, and Pro/Pro were represented, respectively, by 20 (18.5%), 49 (45.4%), and 39 (36.1%) individuals. We did not detect previously published polymorphisms of ARNT (D511N) or the CYP1A1 promoter (G-469A and C-459T) in our subjects, suggesting that these polymorphisms are rare in the Japanese population. No association was found between uterine endometriosis and any polymorphisms in the AHRR, AHR, ARNT, or CYP1A1 genes analyzed in the present study. Received: January 24, 2001 / Accepted: March 1, 2001     

Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung

A variety of pathological changes are seen in lymphoproliferative disorders of the lung but the histogenesis of these abnormalities is not yet fully understood. We previously showed that adenovirus vector-mediated transient expression of both the human interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes, but not the IL-6 gene alone, in the rat lung induced lymphocytic alveolitis. In the present study, we explored the lung pathology of human IL-6 and IL-6R double transgenic mice to elucidate the effects of prolonged IL-6 signalling on the lung. The transgenic animals developed mononuclear cell accumulation in peribronchovascular regions, but little infiltration into alveolar spaces. Immunohistochemical analysis revealed that the cellular accumulations contained not only mixtures of inflammatory cells but also lymphoid tissue-like structures. As the expression of CXCL13/BLC, the indispensable chemokine for lymphoid organogenesis, was recognized in the B cell follicles of the pulmonary lesions, we speculate that this chemokine plays an inductive role in the development of the lymphoid tissue-like structures. These structures were distinguished from bronchus-associated lymphoid tissues (BALTs) by their location and by the lack of lymphoepithelium, which is a characteristic of BALT. These findings imply that IL-6 signalling may play a role in the pathogenesis of lymphoproliferative disorders of the lung.