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Abstract : The protein product of the retinoblastoma susceptibility gene (Rb) is a tumor suppressor implicated in controlling tumorigenesis in a subset of human cancers. Rb also functions to regulate cell differentiation, apoptosis, and senescence and is a target of important cell cycle regulatory proteins. Although the mechanism through which Rb mediates these diverse biological effects on the cell is unclear, the ability of Rb to regulate transcription is most likely important for its biological effects. Rb regulates transcription by interacting directly with specific upstream and basal transcription factors, resulting in either positive or negative regulation of expression of a subset of genes encoding important cell cycle regulatory proteins. A description of the biological activites of Rb and the mechanisms through which Rb modulates transcription is presented.

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Abstract : A wound-inducible cDNA clone (AWI 34) was obtained from the cDNA library of Arabidopsis thaliana by differential screening. The corresponding gene expression was induced rapidly reaching a maximum level in approximately 1.5 h and then progressively reducing after wounding. The predicted amino acid sequence of AWI 34 has 57% homology with the DC 1.2 gene product. The DC 1.2 product is known to be temporarily induced during the initiation of somatic embryogenesis in carrot cells cultured in an auxin(2,4-D)-free medium. The AWI 34 gene was induced by an exogenous 2,4-D treatment on normal plant tissue. The AWI 34 gene was also induced by other environmental stresses such as drought, high-salt, and lowtemperature conditions.

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Abstract : A Drosophila melanogaster gene (dvha) encoding a vacuolar ATPase subunit A has been isolated and characterized. The gene encodes a protein which is 607 amino acids long. The deduced amino acid sequence of the cloned gene is 86.5 and 82.1% identical to those of Manduca sexta and bovine vacuolar ATPase subunit A, respectively. Analysis of a genomic clone revealed that there are three coding exons. RNA blot analysis detected two transcripts; a 2.9 kb transcript present in both adult heads and bodies; a 0.5 kb transcript present predominantly in adult heads.

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Abstract : Primary human oral keratinocytes were previously transformed by transfection with cloned human papillomavirus type 16 (HPV-16) DNA, and a transformed cell line named human oral keratinocyte-16B (HOK-16B) was established. This line contained intact HPV-16 DNA in an integrated form, expressed viral genes, and demonstrated immortality. However, the cells proliferated only in keratinocyte growth medium (KGM) containing a low level of calcium and were not tumorigenic in nude mice. To determine the effect of glucocorticoid on cell proliferation in HPV-immortalized oral keratinocytes and to investigate the mode of cell proliferation, the HOK-I6B cells were exposed to 10-7 M of dexamethasone for 3 or 180 days. We determined the degree of cell proliferation and expression of c-myc, c-fos, transforming growth factor-α (TGF-α), epidermal growth factor receptor (EGFR), and HPV-16 E6/E7 genes from these cells. Dexamethasone increased cell proliferation in a time-dependent manner, and enhanced the 'transcription of c-myc and EGFR. Furthermore, the level of HPV-16 E6/E7 transcripts in the cells treated with dexamethasone for 180 days was notably higher than that of the parental counterparts. These data indicate that overexpression of EGFR, c-myc, and HPV-16 E6/E7 genes may be, in part, responsible for dexamethasone-induced cell proliferation in HPV- 16-immortalized human oral keratinocytes.

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Abstract : IL-6 is known to inhibit growth and induce differentiation of several myeloid leukemia cell lines. With the recombinant human interleukin-6 (IL-6) as a target protein, DNA aptamers specifically binding to IL-6 were isolated from a random oligodeoxyribonucleotide pool. A large pool of 96-mer oligodeoxyribonucIeotides, made up of 60 bases of random sequences flanked by the defined regions of primer binding sites at the . 5' and 3' ends, was synthesized. This randomly generated oligodeoxyribonucIeotide pool was denatured to generate single-stranded DNA and subjected to in vitro selection with affinity chromatography and in vitro amplification with polymerase chain reaction (PCR) for enrichment in single-stranded DNA aptamers specifically binding to IL-6. Multiple rounds of successive affinity chromatography and PCR resulted in the continued purification of binding species. The gel retardation experiment showed that aptamers had affinity to IL-6 with dissociation constants of the order of magnitude of 10^-6 M. The sequences of the aptamers were identified. A homology search revealed that they were categorized into at least four groups according to the consensus sequences among them.

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Abstract : We established a Chinese hamster ovary (CHO) cell line that stably expressed E2 envelope glycoprotein of hepatitis C virus. E2 glycoprotein was stably expressed in the cell, but not secreted into culture medium. To examine the extent of cellular processing, biochemical properties of the E2 glycoprotein were examined by deglycosylation analysis, lectin affinity chromatography, and sedimentation analysis. Our results showed that (i) the E2 glycoprotein was heavily glycosylated, (ii) it was endo-H sensitive, (iii) it bound to a Galanthus nivalus agarose lectin column, and (iv) it existed as a monomer in the cell as evidenced by sedimentation analysis. These biochemical properties indicated that the processing of E2 glycoprotein was blocked at the endoplasmic reticulum. It will be of interest to determine whether E2 glycoprotein derived from the CHO cell line could elicit neutralizing antibody in an animal model.

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Abstract : We report here two peripheral blood dendritic cell (PBDC)-specific class II-related antigen genes identified by differential screening of the PBDC-cDNA library. Dendritic cells (DC) were isolated and purified from the peripheral blood leukocytes (PBL) by employing negative selection procedure and monoclonal antibody sorting with magnetic beads. The DC-cDNA library was constructed using a λgt11 system. The cDNA library was screened by two steps of differential hybridization with [³²P]-labeled DC-cDNA probe, and T-and monocyte-cDNA probes. Isolated clones which were potentially specific to DC were sequenced and then searched for their sequence homology in Genbank database. Novel or interesting genes were .confinned for their specificity to DC by checking the relative amounts of the mRNA expressed in Tcell, B-cells, monocytes and DC by polyperase chain reaction (PCR) and Nothern blot hybridization. Through these experiments, two MHC classs II-related genes, DMα and DMβ, were found to be expressed only in DC among the PBL. Biological functions of these genes remain to be solved by producing monoclonal antibodies against these gene products.

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Abstract : Accumulating evidance indicates that the products of the FGF gene family function as signaling molecules in diverse processes during vertebrate embryogenesis. In tring to further understand the function of the FGFs during early vertebrate development, we attempted to isolate and clone chicken cognate of the mouse and human FGF1 gene using a polymerase chain reaction (PCR) technique. A single pair of degenerate oligonucleotide primer set was used for amplification of chicken FGF1 gene. About 340 base pairs of expected FGF1 sequences between the primers were amplified. Southern blot hybridization using probes for a mouse FGF1 was performed to confirm that the chicken FGF1 cDNA of interest had been amplified. Sequences upstream and downstream of the amplified products were obtained by means of the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that chicken FGF1 is highly homologous to that of its mammalian counterparts. mRNA in situ hybridization analysis studies demonstrate that FGF1 transcripts are highly expressed in developing eyes, suggesting that FGF1 may be an important signaling molecule for vertebrate eye development.

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Abstract : In order to secrete the human pancreatic secretory trypsin inhibitor (hPSTI) from Bacillus subtilis, three different plasmids for the secretion of hPSTI were constructed using the B. subtilis α-amylase promoter and signal sequence. When B. subtilis LKS87 was transformed with each hPSTI secreting plasmid, biologically active hPSTI was secreted into the culture broth. The average amount of the secreted hPSTI was about 1,215 mg/L and the specific inhibitory activities of the secreted hPSTIs were very similar in all three cases. We wondered whether the signal peptide of hPSTI can function in B. subtilis. Therefore, plasmid pUA₂ TID was constructed containing the amyR₂ promoter followed by hPSTI signal peptide and structural gene. However, no hPSTI activity was detected in the culture broth of the transformant harboring pUA₂TID.

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Abstract : We evaluated the in vivo antitumor activity of interleukin 12 (IL-12) in a murine sarcoma model using gene transfer techniques. M-MSV-BALB/3T3 clone engineered to secrete IL-12 was rejected when transplanted into syngeneic BALB/c mice. The tumors rejected in vivo not only induced protective immunity against subcutaneous growth or pulmonary metastases of the parental, nontransfected tumor cells, but also induced curative immunity against established subcutaneous tumors. A generation of CTL activity was significantly induced by injection of IL-12-secreting tumor cells. The wild-type and IL-12-secreting tumor cells grew equally well in nude mice, implying that T-lymphocytes could playa major role in mediating the antitumor effects of IL-12. Adoptive transfer experiments subsequently identified both CD4⁺ and CD8⁺ T-cells as the major mediators. It was also shown that tumor cells transiently expressing IL-12 were as potent as stable transformants in causing the antitumor activity of IL-12. The results described in the present studies indicate that IL-12 secreted from tumor cells is a potent mediator in induction of protective as-well-as curative immune responses against the parental cells in vivo.