Specific messenger RNA expression for signal transduction molecules by lipopolysaccharide in intestinal macrophages.

Intestinal macrophages are known to display profound inflammatory anergy in response to lipopolysacchraide (LPS). To study the mechanisms of unresponsiveness of intestinal macrophages to LPS, we compared the mRNA expression of molecules associated with signal transduction of intestinal macrophages with those of other tissue macrophages. Also cellular localization of CD14 protein was examined. Intestinal, alveolar and peritoneal macrophages were isolated from rats or mice. The expression of mRNA was assessed by real-time PCR, and cellular localization of CD14 protein was examined by flow cytometry. Cellular responses to LPS were examined by production of TNF and NO. The expression of CD14 mRNA in intestinal macrophages was lower than for peritoneal macrophages but higher than for alveolar macrophages. The mRNA expression of other molecules corresponding to intracellular signal transduction in intestinal macrophages was similar with alveolar and peritoneal macrophages. Despite the presence of CD14 mRNA, proteins of CD14 were not detected on cell surfaces of intestinal macrophages, and induction of TNF or NO responding to LPS were not detected. Flow cytometric analysis demonstrated that CD14 protein was not expressed on the cell surface but was expressed inside intestinal macrophages. The unresponsiveness of intestinal macrophages after LPS exposure is considered to be largely attributed to the lack of CD14 protein on their cell surfaces. However, CD14 protein was expressed inside of the cells, suggesting that post-transcriptional regulation rather than transcriptional suppression may play a dominant role in determining the phenotype of the intestinal macrophages.

Characterization of 5-HT2A receptor desensitization and the effect of cycloheximide on it in C6 cells.

Effect of prolonged pretreatment with serotonin (5-HT) on 5-HT2A receptor desensitization was examined by the measurement of intracellular calcium ([Ca2+]i) mobilization in C6 cells. 5-HT-induced desensitization of [Ca2+]i mobilization was in a time and dose dependent manner and reached a plateau after 3 hr. After 1 and 3 hr 5-HT pretreatment, 5-HT concentration in the medium little changed. 5-HT pretreatment with cycloheximide, a protein synthesis inhibitor, produced an enhancement of the desensitization for 3 and 6 hr pretreatment. However, 5-HT pretreatment for 3 and 6 hr caused no marked change in the 5-HT2A receptor mRNA level or Galphaq/11 protein in this study, suggesting that 5-HT may decrease 5-HT-induced [Ca2+]i mobilization independent of 5-HT2A receptor mRNA or G-proteins. Endothelin-1-induced [Ca2+]i mobilization did not alter after 5-HT and/or cycloheximide pretreatment. These results showed that activation of the 5-HT2A receptor induced homologous desensitization and pretreatment with 5-HT and/or cycloheximide did not change the efficacy of the second messenger pathway from Gq to a [Ca2+]i rise.

Existence of galanin in lumbosacral sympathetic ganglionic neurons that project to the quail uterine oviduct.

Oviposition in birds is conducted by vigorous contractions of the uterine oviduct. We recently isolated an oviposition-inducing peptide that was identified as avian galanin from mature quail oviducts. This peptide was localized in neuronal fibers terminating in muscle layers in the uterine oviduct and evoked vigorous uterine contractions through binding to receptors located in the uterus. However, no cell bodies that express avian galanin were detected in the uterus or other oviduct regions. To understand the control mechanism of avian oviposition by galanin, we identified the neurons that synthesize galanin and project to the uterus with the combination of retrograde labeling with neurobiotin and immunocytochemistry for galanin in mature Japanese quails. Retrograde labeling with neurobiotin from the uterus revealed that lumbosacral sympathetic ganglionic neurons located in the uterine side projected their axons to the uterine muscle layer. Abundant elementary granules were observed in somata of the retrogradely labeled sympathetic ganglionic neurons, suggesting that labeled neurons may function as a neurosecretory cell. Immunocytochemical analysis with the antiserum against avian galanin showed an intense immunoreaction restricted to somata of the retrograde-labeled ganglionic neurons. Preabsorbing the antiserum with avian galanin resulted in a complete absence of the immunoreaction. Competitive enzyme-linked immunosorbent assay using antigalanin serum confirmed that avian galanin existed in the sympathetic ganglionic neurons. Expression of the avian galanin messenger RNA in the neurons was further verified by Northern blot analysis. In addition, both avian galanin and its messenger RNA in the neurons were highly expressed in mature birds, unlike in immature birds. These results suggest that lumbosacral sympathetic ganglionic neurons innervating the uterine muscle produce avian galanin in mature birds. Because this peptide acts directly on the uterus to evoke oviposition through a mechanism of the induction of vigorous uterine contraction, galaninergic innervation of the uterine oviduct may be essential for avian oviposition.

Cloning of cDNA encoding ethylene-responsive element binding protein-5 in the cultured cells of Nicotiana tabacum.

We have isolated a full-length cDNA clone from the cultured cells of Nicotiana tabacum, of which the deduced 225 amino acid sequence showed significant homology to the known ethylene-responsive element binding proteins (EREBPs) from tobacco and Arabidopsis. Although the clone shared substantial homology with an Arabidopsis ethylene-responsive element binding factor-3 (AtERF-3) throughout the full length, such a clone had not been found in N. tabacum cells yet; the clone was found to be 5th homologue of EREBPs on N. tabacum.

Avian galanin: cloning of complementary DNAs and characterization of transcripts in different tissues.

Avian galanin was first isolated from the chicken intestine, and subsequently from the quail oviduct. Avian galanin is a 29-residue peptide including an amidated threonine at the C-terminus and differs at several positions from the C-terminal part of mammalian galanins. In contrast to extensive mammalian studies, no information is available on the structure of precursor molecule of avian galanin. In this study, therefore, we conducted complementary DNA (cDNA) cloning of the avian galanin gene from quail brain RNA using degenerated 3'RACE and 5'RACE techniques. A cloned cDNA for avian galanin contained an open reading frame consisting of 117 amino acids which had overall amino acid homology of 63%, 60%, 54%, 58%, and 62% with bovine, human, mouse, porcine, and rat galanins, respectively. Another cDNA containing a 69-nucleotide insertion, which gave an additional 23 amino acids to mature avian galanin, was also cloned, suggesting the presence of two transcripts by alternative splicing. Northern blot analysis revealed that avian galanin mRNA was expressed, as a shorter transcript, in the quail brain, ovary, and intestine, unlike the liver and oviduct. A larger mRNA of avian galanin may be further expressed only in the ovary.

Expression and activity of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase in different regions of the avian brain.

Recently, we have demonstrated, using biochemical and immunochemical methods, that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and produces pregnenolone and its sulfate ester. To clarify progesterone biosynthesis in the avian brain, therefore, we examined the expression of messenger RNA (mRNA) encoding for the enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) and its enzymatic activity using the quail. RT-PCR analysis together with Southern hybridization indicated the expression of 3beta-HSD mRNA in the brain of sexually mature birds but with no clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of pregnenolone to progesterone was found in brain slices of mature males. Progesterone biosynthesis was increased in a time dependent manner and completely abolished by trilostane, a specific inhibitor of 3beta-HSD. The enzymatic activity of 3beta-HSD was greatest in the cerebrum and lowest in the mesencephalon. A specific RIA indicated that progesterone concentrations in the different brain regions closely followed the level of 3beta-HSD activity. High levels of progesterone concentration were observed in the diencephalon and cerebrum with lowest values in the mesencephalon. Progesterone levels in the brain regions were significantly higher than those in the plasma. These results suggest that the avian brain possesses not only cytochrome P450scc but also 3beta-HSD and produces progesterone. It is also indicated that progesterone biosynthesis in the avian brain may be region-dependent.

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life.

Recently, we demonstrated that cytochrome P450 side-chain cleavage enzyme (P450scc) occurs in the rat cerebellar Purkinje cell after differentiation and remains during neonatal development and into adulthood. 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3betaHSD) is also an essential enzyme for progesterone biosynthesis not only in peripheral steroidogenic glands but also in the nervous system. In the present study, therefore, the expression of 3betaHSD in the rat cerebellum was investigated during neonatal development and in the adult. RT-PCR analysis showed that the expression of 3betaHSD messenger RNA (mRNA) in the cerebellum was higher at 7-14 days of age than at other times. Biochemical studies together with HPLC analysis revealed that cerebellar slices at 10 days of age converted pregnenolone to progesterone, suggesting enzymatic activity of 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. A specific RIA indicated that progesterone concentrations in the cerebellum were higher at 3 and 10 days of age than at 60 days of age. The progesterone level in the cerebellum was significantly higher than that in plasma at 10 days of age. In contrast, the concentrations in both cerebellum and plasma at 3 and 60 days of age were similar. In the present study, the site of 3betaHSD mRNA expression in the cerebellum was further examined in neonatal and adult rats using in situ hybridization. The cerebellar expression of 3betaHSD mRNA was obscure at 3 days of age, whereas intense expression occurred in Purkinje cells and external granule cells throughout the cerebellum at 10 days of age. 3BetaHSD mRNA was also expressed in Purkinje cells and granule cells at 60 days of age, but a restricted expression was observed along the cerebellar meninges. These results suggest that the steroidogenic enzyme 3betaHSD as well as P450scc are expressed at least in the cerebellar Purkinje cell. The expression of 3betaHSD, however, may increase for a limited period around 10 days of age, unlike P450scc.

Neurosteroid biosynthesis in vertebrate brains.

In mammals, neurosteroids are now known to be synthesized de novo in the brain as well as other areas of the nervous system through mechanisms at least partly independent of the peripheral steroidogenic glands. However, limited information is available on neurosteroids in non-mammalian vertebrates. We therefore have attempted to demonstrate neurosteroid biosynthesis in the brain of birds and amphibians. These vertebrate brains possessed the steroidogenic enzymes, cytochrome P450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD), and produced pregnenolone, pregnenolone sulfate ester and progesterone from cholesterol. Significant seasonal changes in neurosteroids in the brain were observed in seasonally breeding vertebrates. In addition, we attempted to identify the cell type involved in neurosteroidogenesis in mammalian and non-mammalian vertebrates in order to understand the physiological role of neurosteroids. Glial cells are generally accepted to be the primary site for neurosteroid formation, but the concept of neurosteroidogenesis in brain neurons has up to now been uncertain. We recently demonstrated neuronal neurosteroidogenesis in the brain and indicated that the Purkinje cell, a typical cerebellar neuron, actively synthesizes several neurosteroids de novo from cholesterol in both mammals and non-mammals. This paper summarizes the advances made in our understanding of neurosteroid biosynthesis, including neuronal neurosteroidogenesis, in a variety of vertebrate types.

Age- and region-specific expressions of the messenger RNAs encoding for steroidogenic enzymes p450scc, P450c17 and 3beta-HSD in the postnatal rat brain.

Neurosteroids are now known to be synthesized de novo in the nervous system through mechanisms at least partly independent of peripheral steroidogenic glands. In mammals, the presence of the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and the enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) has been well established in the brain, whereas limited information has been available on the enzyme 17alpha-hydroxylase/c17, 20-lyase (cytochrome P450c17), which converts pregnenolone to dehydroepiandrosterone, one of the most abundant neurosteroids. In addition, little is known regarding developmental changes in these steroidogenic enzymes during postnatal life. Thus, the pathway of neurosteroid formation in the brain is still incomplete. Therefore, we examined expressions of the messenger RNAs (mRNAs) encoding for three key enzymes, P450scc, P450c17 and 3beta-HSD, in the rat brain at different postnatal ages using RT-PCR analysis. The expression of P450scc mRNA was found throughout the brain at the same level, while the 3beta-HSD mRNA expression was higher in the cerebellum and cerebrum than in other brain regions. The P450c17 mRNA was highly expressed in the mesencephalon. On the other hand, higher expressions of the cerebellar and cerebral 3beta-HSD mRNAs were observed only in neonatal life. In contrast, the expression of P450scc mRNA was relatively constant during neonatal life and in adulthood. A similar constant expression of the P450c17 mRNA was evident in the mesencephalon. Serial Southern hybridization in this study confirmed the specific mRNA expression corresponding to each enzyme. These results suggest that in the postnatal rat the expression of 3beta-HSD or P450c17 mRNA may be age- or region-dependent, unlike the P450scc mRNA expression.

Involvement of 26-kDa membrane-bound tumour necrosis factor precursor in bidirectional feedback regulation on 17-kDa tumour necrosis factor production after stimulation by lipopolysaccharide.

The authors have previously shown that 26-kDa membrane-bound tumour necrosis factor precursor (proTNF) on the cell-surface of primed human monocytic cell line THP-1 is involved in positive feedback regulation of lipopolysaccharide (LPS)-dependent TNF-production. Here, we provide direct evidence for modulation of responsiveness of the THP-1 cells against LPS by membrane-bound pro-TNF. When THP-1 cells were cocultivated with a heterogeneous cell line (proTNF/3T3 cells) which constitutively expressed membrane-bound proTNF, LPS-dependent TNF-production by THP-1 cells was significantly suppressed and the normal level was restored by the presence of anti-TNF antibody during cocultivation. The proTNF-3T3-induced decline of TNF-production of THP-1 was observed primarily at the mRNA level, although no difference was observed in the mRNA level of interleukin 1 beta, another LPS-inducible cytokine. These results suggest that proTNF could also be involved in the negative feedback regulation of LPS-dependent TNF-production through cell-to-cell contact. The augmentation of LPS-dependent TNF-production accompanied by the production of endogenous proTNF induced by exogenous agent was inhibited by protein kinase C inhibitor, whereas proTNF/3T3-induced suppression of TNF-production could not be restored to the normal level. It thus seems possible that proTNF might act on macrophages as a bidirectional regulator of its production by THP-1 cells depending on co-induced signals.

Cytochrome P450 side-chain cleavage enzyme in the cerebellar Purkinje neuron and its neonatal change in rats.

Neurosteroids are de novo synthesized in the nervous system through mechanisms at least partly independent of peripheral steroidogenic glands. However, the concept of neurosteroidogenesis in neurons is not clear in mammalian brains. The present study identified the presence of cytochrome P450scc in the rat Purkinje cell, a typical cerebellar neuron. Immunohistochemical analysis with the antibody against the purified bovine adrenal P450scc showed an immunoreaction restricted to somata and dendrites of the Purkinje cells in adult cerebella. Preadsorbing the antibody with P450scc resulted in a complete absence of the immunoreaction. The antibody against inositol triphosphate receptor, a marker of the Purkinje cell, recognized P450scc-immunoreactive cerebellar cells that showed no immunoreaction with glial fibrillary acidic protein, a specific marker of glial cells. Expression of the P450scc-like protein in the cerebellum was verified by Western blot analysis, and cerebellar P450scc messenger RNA, by RT-PCR analysis in adulthood. On the other hand, P450scc-immunoreactive cells were found to scatter throughout the cerebellum at 0 day of age, before the differentiation of the first Purkinje cells, while the site of expression of this protein was localized only in somata of Purkinje cells at 3 days of age. Immunoreactive dendrites of the Purkinje cell spread into the molecular layer during neonatal development concurrently with its maturation. The intensity of the immunoreaction did not change during neonatal life. Expression of the cerebellar P450scc messenger RNA was also detected after birth, and the level was almost constant during neonatal life. A specific RIA indicated that the pregnenolone concentration was unexpectedly high at 0 day and decreased until 7 days. The total amount of pregnenolone in the cerebellum was almost constant from 0-7 days and increased during 7-21 days concurrently with the cerebellar development. In contrast, the pregnenolone sulfate ester level was low and did not significantly change among the developmental stages. These results suggest that steroidogenic enzyme P450scc appears in the rat Purkinje cell immediately after its differentiation. The expression of this enzyme may remain during neonatal development and in adulthood.

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.

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.

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.

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.

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.

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.

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.

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.

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.