Biomedical subjects

C Kohchi

Publications and source records attributed to C Kohchi.

11 recordsLinked to original sources

PubMed1995

Molecular cloning of a cDNA that encodes the precursor to several exogastrula-inducing peptides, epidermal-growth-factor-related polypeptides of the sea urchin Anthocidaris crassispina.

Complementary DNA clones for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are related to epidermal growth factor (EGF), were obtained from a cDNA library of late gastrula embryos using, as probe, the partial cDNA for one of the EGIP (EGIP-D) obtained by the reverse-transcription PCR method. The longest cDNA was composed of 1662 bp, and encoded a protein of approximately 36 kDa with a region that resembled a signal sequence. The deduced protein contains the sequences of EGIP-C, EGIP-D, and EGIP-A in that order, followed by the sequence for an unidentified EGIP-like polypeptide. When expressed in Escherichia coli as a fusion protein with beta-galactosidase, the product for the cDNA was specifically recognized by a rabbit antibody raised against EGIP-D that had been purified from embryos. Characteristic amino acid residues were found around the N-terminus and the C-terminus of each EGIP sequence, suggesting a specific processing mechanism for the generation of the individual EGIPs from the precursor. RNA-blot analysis revealed the presence of EGIP mRNA in unfertilized eggs. The level of this mRNA decreased gradually after fertilization, began to increase dramatically after the onset of gastrulation, and continued to increase through the pluteus stage. Genomic Southern-blot analysis suggested that this gene is present as a single copy. A homology search showed that the EGIP cDNA has a similarity to the cDNA for SpEGF2 which was cloned as a gastrula-specific gene in another sea urchin, Strongylocentrotus purpuratus.

Amino Acid Sequence↗

PubMed1994

Constitutive expression of TNF-alpha and -beta genes in mouse embryo: roles of cytokines as regulator and effector on development.

1. Using the RT/PCR method, we examined mRNA expression of several inflammatory factors in mouse embryos during mid-late embryonal development. mRNAs of tumor necrosis factor (TNF)-alpha, TNF-beta, their receptors (TNF-RI, TNF-RII), transforming growth factor (TGF)-beta, were expressed constitutively in most of the embryonic tissues. 2. While mRNAs of other factors, interleukin (IL)-1 alpha, IL-1 beta, IL-3, IL-6, granurocyte-colony stimulating factor (G-CSF), leukaemia inhibitory factor (LIF), and interferon (IFN)-gamma were only limitedly expressed. 3. The mRNAs of several complement components (C2, C3, C4, C5) and receptors (CR1, CR2) were also detected. Among them, the expression of C3 and CR1 were prominent. These results strongly support our idea that inflammation-like system play an important role to regulate embryogenesis.

Animals↗

PubMed1992

Expression of transcripts of complement components and their receptors during differentiation of embryonal carcinoma cell lines.

Based on our previous finding that TNF-alpha and TNF-beta can be expressed constitutively during early embryonal development [1], we extended our work to identify factors which are generally known to take part in inducing inflammation in adults. They can be regarded as candidate molecules involved in ontogenic inflammation during embryonal development. In this study, we chose the factors which are constituents of either a classical or an alternative pathway of a complement system and found that mRNAs corresponding to those of C2, C3, C4, C5 and to those of receptors CR1 and CR2 were expressed. Among them, mRNA expression of C3, C4, and CR1 was especially constitutive. Contrary to these observations, expression of two kinds of scavenger receptors (SR-I, SR-II) proved to be negative. In this report, the framework of ontogenic inflammation as a regulatory mechanism in embryonal development at the molecular level is discussed.

Animals↗

PubMed1991

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.

Amino Acid Sequence↗

PubMed1991

Expression of TNF-alpha and TNF-beta transcripts in murine embryonal carcinoma cells and trophoblast cell.

Using a combination system of Transcription and PCR, we examined gene expression of TNF-alpha, TNF-beta and other cytokines (IL-1, G-CSF, IL-3 and IFN-gamma) in embryonal carcinoma cell lines (PCC3, PCC4, ECA2 and F9) and trophoblast cell line (PL/B6). We found that both TNF-alpha and TNF-beta transcripts were expressed in all of the embryonic cell lines. We also detected transcripts of two types of TNF receptors and transcription factor NF-kappa B in these embryonic cells.

Animals↗

PubMed1986

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae.

Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the beta-glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the beta-glucosidase gene originated from C. pelliculosa. beta-Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, beta-glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of beta-glucosidase synthesis in S. cerevisiae carrying the cloned beta-glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the beta-glucosidase gene negatively in S. cerevisiae.

Candida↗

PubMed1985

Nucleotide sequence of Candida pelliculosa beta-glucosidase gene.

The nucleotide sequence of the DNA fragment containing the beta-glucosidase gene of Candida pelliculosa was determined. Analysis of the sequence revealed three open reading frames which could encode 65,825, and 412 amino acid residues. The presence of the second frame was found to be sufficient for the expression of the beta-glucosidase gene in a heterologous host Saccharomyces cerevisiae. Putative protein encoded by this gene had hydrophobic amino acids, resembling a signal peptide, at its N-terminal region and 19 potential glycosylation sites. Codon usage of Candida genes had the similar pattern shown in S.cerevisiae. Codon bias of the beta-glucosidase gene of Candida was relatively low, compared with that of the highly expressed genes of S. cerevisiae.

Amino Acid Sequence↗

PubMedDate not supplied

Expression of tumor necrosis factor-alpha and -beta transcripts in embryonal carcinoma and trophoblast cell lines: inflammation-like state as possible regulatory mechanism for ontogenesis.

TNF-alpha and TNF-beta are both involved in inflammation which regulates homeostasis in adults. We examined the mRNA expression of TNF-alpha and TNF-beta in murine embryonal carcinoma (EC) cell lines (PCC3, PCC4, ECA2 and F9) and trophoblast cell line (PL/B6) using a combined system of reverse transcription and polymerase chain reaction. Four lines of EC cells and PL/B6 expressed mRNA of both TNFs. Moreover, mRNA expression of two types of TNF-receptor and transcription factor NF-kappa B, both of which mediate a part of the biological function of TNFs, were also detected in EC and trophoblast cells. In order to clarify whether other cytokines may form a network during embryonal development, we also examined the expression of transcripts of inflammatory cytokines produced by activated macrophages (IL-1 alpha, IL-1 beta and G-CSF) or by T lymphocytes (IL-3 and IFN-gamma) in adult. We found that no IL-1 alpha transcript was expressed in any of the cell lines examined whether differentiated or not. Contrary to this, the transcript of G-CSF was continuously expressed in all cell lines, and those of IL-1 beta, IL-3 and IFN-gamma were slightly expressed in some cell lines.

Animals↗

PubMedDate not supplied

Early embryonic cells activate the alternative complement system.

Murine embryonic stem cells, embryonic carcinoma cells and pre-implantation embryos were found to be extremely sensitive to cytolysis by normal human serum as compared to matured cells. The cytolytic activity to embryonic cells was not removed by pre-absorption of serum with spleen lymphocytes. Conditions which block both complement activation pathways or, selectively, the alternative pathway completely abrogated the activity of human serum against embryonic cells whereas the activity was retained under conditions which block the classical complement pathway, indicating that embryonic cells activate the alternative complement system (ACS). The cytotoxic effect to murine embryonic cells was reproduced using syngeneic murine serum. Concerning the mechanism of ACS-activation, the expression of regulators of complement activation and of membrane bound sialic acid was analysed. Embryonic cells express mRNA for Crry similarly to other cells but additionally express Cr2-transcripts not found in most adult cells. Embryonic cells have strikingly low levels of membrane-bound sialic acid compared to adult cells.

Animals↗

PubMedDate not supplied

Induction of differentiation in embryonic stem cells by 26-kD membrane-bound tumor necrosis factor (TNF) and 17-kD free TNF.

The biological activity of 26-kD membrane-bound tumor necrosis factor (TNF)-a in embryonal development was examined in an in vitro system using embryonic stem (ES) cells. ES cells were seeded on NIH3T3 feeder cells transformed with mouse precursor TNF-a gene to express membrane-bound TNF-a on their cell surface. The proliferation of the ES cells was reduced and differentiation was accelerated. The same effects were also observed when 17-kD free TNF-a was added to the culture medium of the ES cells. Since free TNF-a is not present during embryogenesis, these results suggest that membrane-bound TNF-a may play an important role in embryonal development through cell-cell contact.

3T3 Cells↗

PubMedDate not supplied

Selective lysis of early embryonic cells by the alternative pathway of complement--a possible mechanism for programmed cell death in embryogenesis.

Early embryonic cells and early mouse embryos were shown to activate the alternative pathway of complement, and to be highly sensitive to complement-mediated cytolysis (Kircheis et al, In Vivo 9: 85-98, 1995). Under further development embryonic cells become resistant. The induction of resistance to the alternative pathway of complement correlates with: a) altered splicing of Cr2-transcript and b) changes in the acidic glycolipids under differentiation. Early embryonic cells have low amounts of sialic acid-containing glycolipids or express mainly GM3. The induction of differentiation changes the glycolipid pattern leading to an increase in membrane-bound sialic acid. The importance of membrane-bound sialic acid in the restriction of complement activation is demonstrated by increased sensitivity to complement after pre-treatment of cells with neuraminidase. The results indicate that there is target-specific lysis of early embryonic cells by the alternative pathway of complement. Early embryonic cells activate the alternative pathway of complement by expressing activators and low levels of membrane-bound sialic acid. Induction of differentiation changes the glycolipid pattern, leading to an increase in membrane-bound sialic acid sufficient to restrict complement-activation on the cell surface.

Animals↗