Molecular characterization and nuclear localization of rat timeless-like gene product.

Among three period genes (per1, per2, per3) in mammals, only per2 gene was shown to be involved in the core clock mechanism. To elucidate the molecular function of rat PERIOD2 (rPER2), we searched for binding proteins to the PAS domain of rPER2. We isolated a binding protein to this domain and identified it as a TIMELESS-like protein (TLP) on the basis of mass analyses. Then, we isolated a rat TLP cDNA from the rat hypothalamus library. RNA blot analysis and in situ hybridization indicates that rTLP mRNA was expressed in all rat tissues from whole brain, the suprachiasmatic nucleus, eye, lung, heart, liver, kidney, placenta, and testis. When rTLP gene product was expressed in COS-1 cells, nuclear localization of rTLP was detected in 99.6% of transfected cells. These results suggest that the interaction of rPER2 with rTLP may influence the regulation of circadian clock components in nucleus after rPER2 is translocated into the nucleus.

Suppression of tumor growth by the 3' untranslated region of mel-18 in 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18.

By introducing a cDNA library derived from rat embryonic fibroblast cells, we isolated several morphologically flat revertants of rat 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18 (HPV18). From one of the revertants, we recovered a 0.2-kb cDNA, N56, that suppresses the tumor growth of the transformed 3Y1 cells irrespective of the expression of the E6 and E7 genes. The nucleotide sequence of the cDNA was shown to be identical to that of the 3' untranslated region of a putative mammalian polycomb group gene, mel-18.

Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse.

Here we report the identification of a novel homeobox gene family Dbx in mouse, which consists of Dbx and Dbx2. The two genes share similar structural organization and are encoded by different chromosomes. The predicted Dbx and Dbx2 proteins share 85% identity in their homeodomain amino acid sequences, but otherwise showed no significant similarity. Characterization of the expression of these two genes in the embryos suggested their role in the development of the CNS. In the forebrain, Dbx is expressed in various regions, while Dbx2 showed a more restricted pattern of expression. In the midbrain, the expression domains of Dbx and Dbx2 overlap along the dorso-lateral wall of the ventricle. In the hindbrain and spinal cord, both genes are expressed in the boundary separating the basal and alar plates, which seems to correspond to the sulcus limitans. Expression of the Dbx/Dbx2 genes is restricted to the ventricular region of the embryonic CNS except for that of Dbx in the septum of the telencephalon. Together these observations indicate possible participation of the members of the Dbx family in regionalization of the CNS. While the expression of Dbx was restricted to the CNS, Dbx2 was also expressed in some of the mesenchymal cells, such as limb buds and tooth germs.

An in vivo model for receptor tyrosine kinase autocrine/paracrine activation: auto-stimulated KIT receptor acts as a tumor promoting factor in papillomavirus-induced tumorigenesis.

Constitutive overactivation of growth factor receptors through autocrine/paracrine mechanisms occurs frequently in cancer cells and are thought to play a critical role in carcinogenesis. In the present report, we propose a refined in vivo model which explains the significance of these mechanisms in tumour development. We have previously established transgenic mouse lines containing human papillomavirus type 16 (HPV16) E6E7 oncogenes, in male mice of which a Leydig cell tumor developes with a very high incidence. Not only HPV transgene but also the c-kit proto-oncogene receptor tyrosine kinase and its ligand Steel Factor (SLF) were coexpressed in all tumors analysed. This coexpression of c-kit/SLF was also found in two other Leydig cell tumor lines. Moreover, the proliferation of transgenic tumor cells was attenuated by treatment with a c-kit neutralizing antibody in vitro, strongly suggesting that tumorigenesis is closely related to stimulation of receptors through ligand induction. To confirm the significance of these findings, a defective mutation of the SLF gene in a laboratory mouse, the Steel-Dickey (Sld) mutation, was introduced into a line of transgenic mice showing 100% incidence of the tumor. In Sld-E6E7 transgenic mice, tumorigenesis was initiated but numbers of tumor cells were markedly reduced compared with transgenic mice carrying both wild type SLF allele, showing that c-kit activation through the induction of SLF is essential for testicular tumorigenesis, especially in tumour promotion. This transgenic mice system should be a useful in vivo model for clarifying the implication of growth factor autostimulation in carcinogenesis.

Establishment and further characterization of a line of transgenic mice showing testicular tumorigenesis at 100% incidence.

We have reported production of transgenic mice containing human papillomavirus type 16 (HPV16) E6 and E7 oncogenes in which a characteristic testicular tumor develops at a very high incidence. Three transgenic mice transmitted the transgene to their siblings, in which the same type of tumor developed. In one line, named line 181, this testicular tumor developed in all the 93 males obtained for 10 generations. In most cases, this tumor was detectable bilaterally in the testes 9 to 10 months postdelivery. On cross-matings with other inbred strains, the HPV transgene was dominant in all the genetic backgrounds examined. In the condition of experimental cryptorchidism, obvious delay of tumor formation was observed. In these testes, the tumor cells were seen to arise from the interstitium. Moreover, this tumor also manifested obvious expression of gonadal specific 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and other enzymes for androgen metabolism. These observations strongly suggest that this tumor has originated from Leydig cells. This transgenic mouse line, therefore, provides a novel system for investigating in vivo carcinogenesis and the mechanism of transformation of male gonadal cells.

Degradation of p53 only is not sufficient for the growth stimulatory effect of human papillomavirus 16 E6 oncoprotein in human embryonic fibroblasts.

Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein.

Regulation of early gene expression of human papillomavirus type 16 by inflammatory cytokines.

The host's immune reaction against human papillomavirus (HPV) infection remains poorly understood. Inflammatory cytokines undoubtedly play a key role through activating and coordinating the immune response. However, their direct interactions with the HPV genes remain unclear. In the present study, the effects of various inflammatory cytokines on HPV16 gene expression were investigated. In a CAT assay, tumor necrosis factor (TNF) alpha and interleukin-1 (IL-1) alpha were shown to repress HPV16 early gene expression at the transcriptional level through the noncoding region (NCR), whereas IL-6 and interferon-gamma did not. In Northern blot analysis, TNF and IL-1 were also shown to repress HPV16 E6/E7 mRNA expression in the HPV16-immortalized human keratinocyte cell line. The TNF- and IL-1-responsive elements in the HPV16 NCR were determined to lie within the cell-type-specific enhancer, where there are several binding sites for nuclear factors involved in HPV16 early gene regulation, suggesting the participation of these factors in TNF and IL-1 regulations. Thus, TNF and IL-1 were shown to have antiviral effects on HPV through down-regulation of its gene transcription. This is the first demonstration that TNF and IL-1 are involved in HPV gene regulation. These functions of inflammatory cytokines are presumed to contribute to the host's defense against HPV infection.

Isolation of flat revertants from human papillomavirus type 18 E6E7 transformed 3Y1 cells by transfection with a rat embryo fibroblast cDNA expression library.

A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes.

Regional mapping of the Rowett nude gene (RONU) to rat chromosome 10q24-->q32 by localizing linked SYB2 and GH loci.

The Rowett nude gene (RONU) has been mapped on rat chromosome (Chr) 10 by linkage analysis using (ACI x F344/N-RONU/RONU)F1 x F344/N-RONU/RONU backcross progeny. The gene order on the chromosome was RR92- (16.1 cM) - RR24 - (17.9 cM) - MYHSE (myosin heavy chain, embryonic) - (1.0 cM) - SYB2 (synaptobrevin 2) - (1.0 cM) - SHBG (sex hormone-binding globulin) - (4.0 cM) - RONU (Rowett nude) - (29.0 cM) - AEP (anion exchange protein), PPY (pancreatic polypeptide) - (3.0 cM) - ACE (angiotensin I converting enzyme), GH (growth hormone). The RONU locus was localized to 10q24-->q32 by fluorescence in situ hybridization of the closely linked SYB2 and loosely linked GH loci on the opposite side. Conserved linkage of homologous loci mapped to rat Chr 10 and mouse Chr 11 supports the hypothesis that the RONU locus is a rat homolog of the mouse nu locus.

[Hospital and district differences in susceptibilities to antibiotics of main clinical isolates].

A nationwide survey of susceptibilities of Staphylococcus aureus, Escherichia coli, Klebsiella sp. and Proteus mirabilis initiated in 1980 was continued in 1986. This report documents differences in susceptibilities of clinical isolates of the above microorganisms to ampicillin (ABPC), cefazolin (CEZ), cefmetazole (CMZ) and gentamicin (GM) among general hospitals throughout Japan. Clinical isolates of each species of microorganisms which were collected in 24 hospitals made up each study group and were collected at the Kosei General Hospital, Tokyo, from April 1980 to March 1986. We compared the variability in the resistant rates (MIC greater than or equal to 25 micrograms/ml) and MICs of each antibiotic for 50% and 80% of the isolates among the hospitals. MICs were determined by the serial 2 fold agar plate dilution method, standardized by the Japan Society of Chemotherapy, with an inoculum of approximately 10(6) CFU/ml or 10(8) CFU/ml. Susceptibility patterns of S. aureus to ABPC, CEZ and GM, of E. coli to ABPC, of Klebsiella sp. to CEZ and GM and of P. mirabilis to ABPC, CEZ and GM varied from hospital to hospital. On the other hand, the differences in the susceptibility patterns among the 6 districts of Japan were not obvious, because the differences were affected by different susceptibility patterns of each hospital located in each district.

Periapical cemental dysplasia with multiple lesions.

3 cases of periapical cemental dysplasia with multiple lesions in both maxilla and mandible are reported. All 3 patients are middle-aged females with an average age of 47 years; histological examination of excised tissue revealed that the lesions were composed of fibrous connective tissue and cementum-like hard tissue. The location of the teeth affected were mainly in the premolar-molar regions. A subsequent literature survey of previously described Japanese cases of periapical cemental dysplasia disclosed a similar distribution pattern for the location of PCD lesions. This evidence indicates that the occurrence sites of lesions of periapical cemental dysplasia is predominantly in premolar-molar regions in Japan, contrary to the location of PCD in other ethnic groups.

Derivation of a murine monoclonal antibody useful for immunohistochemical diagnosis of human adenoid cystic carcinoma and stomach cancer.

A monoclonal antibody, NCC-SG-007, was raised with a formalin-fixed adenoid cystic carcinoma of the salivary gland as the immunogen. The reactivity of the antibody was tested on paraffin sections using the avidin-biotin-peroxidase complex (ABC) method. NCC-SG-007 reacted with 36% of the adenoid cystic carcinomas, 42% of the pleomorphic adenomas and 14% of the mucoepidermoid tumors of the salivary gland which were tested. The antibody also reacted with some normal tissues and various other tumors. Surprisingly, NCC-SG-007 showed a reactivity as high as 94% with gastric carcinomas. The antigenic determinant was revealed to be a carbohydrate chain with no terminal sialic acid, the molecular weight of which was estimated to be 1.5 X 10(6) daltons. This antigen differed from other previously reported gastrointestinal cancer-associated antigens. The antibody should be useful for studying adenoid cystic carcinomas of the salivary gland and gastric cancers.