Developmental profiles of phosphorylated and unphosphorylated CREBs in murine calvarial MC3T3-E1 cells.

The cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development. We analyzed the changes of the amount, degree of phosphorylation and binding activity of the CREB/ATF family in the course of development of the murine calvarial osteoblastic cell line MC3T3-E1 as an in vitro model system of bone formation. The amount of CREB in the whole-cell extract detectable by Western blot analysis was high through all stages of development and maximal in the proliferation stage. The degree of phosphorylation estimated with anti-phosphorylated CREB antibody changed greatly and reached high levels in the proliferation stage and early mineralization stage. The ratio of phosphorylated CREB to total CREB in the CREB-CRE complex was also examined by gel shift assay. Although the binding to the consensus/CRE probe reached almost equally high levels in the proliferation stage and early mineralization stage, the relative level of phosphorylated CREB in the CREB-CRE complex was different in these two stages. In the early mineralization stage, most CREB bound to consensus/CRE was phosphorylated, while both phosphorylated and unphosphorylated CREB were bound to consensus/CRE in the proliferation stage. ATF-1 was also detected as a minor component bound to the consensus/CRE probe. The alteration of the binding of CREB to consensus/CRE over the course of osteoblast development supports the hypothesis that CREB may regulate the expression of genes defining the developmental sequence of MC3T3-E1 cells.

Transcriptional regulatory elements in the upstream and intron of the fibroin gene bind three specific factors POU-M1, Bm Fkh and FMBP-1.

The transcriptional modulator in the fibroin gene intron is composed of multiple octamer-like AT-rich elements, to which several specific DNA-binding proteins named fibroin-modulator-binding proteins (FMBPs) bind. Three major FMBPs in the silk gland were characterized. Two of them (FMBP-2 and -3) were identified as a Fork head homologue (Bm Fkh) and a POU-domain protein (POU-M1) respectively. These factors were expressed in the silk gland with distinct temporal- and spatial-specificities during late larval development as well as during embryogenesis, and did not correlate directly with fibroin gene expression. The other (FMBP-1) appeared to correlate with the expression of the fibroin gene for temporal- and spatial-specificity. These FMBPs also bind to the elements in the upstream modulator. Transcriptional enhancement by both modulators was inhibited by binding competition for these factors with oligonucleotides. These results suggest that expression of the fibroin gene is controlled by co-ordination of these factors with distinct specificities during silk-gland development.

[A case of recurrent multiple HCC after surgical resection showing regression by two TAEs using 5-FU and zinostatin stimalamer].

A-68-year-old man had hepatocellular carcinoma (HCC) in the area of S6 segment which was resected surgically. Three months after surgery, multiple recurrent lesions were found in both lobes of the liver. A transcatheter arterial embolization (TAE) with 5-FU and zinostatin stimalamer (SMANCS) was done. After TAE, the tumor mass disappeared in the right lobe and reduced to 10% in the left lobe. Although mass lesions remained unchanged for 3 months, the increased of AFP (143.9 ng/ml) was noticed after 4 months. Ten months after the second TAE with 5-FU and SMANCS, the disappearance of tumor masses was confirmed by diagnostic images.

Spatial and temporal expression pattern of Bombyx fork head/SGF-1 gene in embryogenesis.

The embryonic expression of Bombyx fkh/SGF-1 gene has been analysed using in situ hybridization and immunohistochemistry. Both transcripts and protein were first detected in the most anterior and posterior regions at the time of germ anlage formation, and were successively expressed in the foregut and hindgut at later stages. A weaker expression was also detected in the elongated midgut. By the time embryo retraction was finished transcripts and protein were also detectable in the invaginated whole silk glands, and after the blastokinesis stage the products were restricted to the middle and posterior silk glands achieving a state required for the SGF-1 distribution for later stages. Expression could also be detected in the central and peripheral nervous systems. From these observations, we propose that Bombyx Fkh/SGF-1 may play a role in organogenesis processes such as those of the gut, silk glands, and nervous systems, act as a region specific homeotic gene, and in spite of clear embryonic developmental differences between Drosophila and Bombyx, two terminals may be determined by region specific genes such as Bombyx fkh/SGF-1 as opposed to segmental development.

Role of transforming growth factor beta 1 on hepatic regeneration and apoptosis in liver diseases.

AIMS: To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on regeneration and induction of apoptosis of liver cell and bile duct in various liver diseases. METHODS: Formalin fixed paraffin wax sections of 18 liver tissue samples were obtained by needle biopsy, surgery, or necropsy; these included six liver cirrhosis, three obstructive jaundice; five fulminant hepatitis, one subacute hepatitis, and three normal liver. Expression of TGF-beta 1, apoptosis related Le(y) antigen, Fas antigen, a receptor for tumour necrosis factor, and biotin nick end labelling with terminal deoxynucleotidyl transferase mediated dUTP (TUNEL) for locating DNA fragmentation, was investigated histochemically. RESULTS: TGF-beta 1 was expressed in areas of atypical bile duct proliferation, where bile duct continuously proliferated from liver cells. In occlusive jaundice and fulminant hepatitis, TUNEL was positive in nuclei and cytoplasm of metaplastic cells which formed incomplete bile ducts, and these cells appeared to extend from TGF-beta 1 expressing liver cells. Fas antigen was found only on the cell membrane of proliferated bile duct in fulminant hepatitis, which differed from TGF-beta 1 and TUNEL positive areas. Le(y) antigen was expressed in liver cell and bile duct at the areas with atypical bile duct proliferation, but its coexpression with TUNEL was rare. CONCLUSIONS: TGF-beta 1 plays a role in the arrest of liver cell regeneration and atypical bile duct proliferation, and in areas of rapidly progressing atypical bile duct proliferation, such as in fulminant hepatitis or bile retention. Apoptosis appears to be induced by TGF-beta 1. This phenomenon may account for the inadequate hepatic regeneration that occurs with liver disease.

Silk gland factor-1 involved in the regulation of Bombyx sericin-1 gene contains fork head motif.

Silk gland factor-1 (SGF-1) regulates transcription of the Bombyx sericin-1 gene via interaction with the SA site. In this study, two related SGF-1 polypeptides of apparent molecular masses of 40 and 41 kDa were purified. Specific interaction of these proteins with the SA site was demonstrated by electrophoretic mobility shift and dimethyl sulfate methylation interference assays. The SGF-1 40-kDa protein was partially sequenced and characterized as a new member of the fork head/HNF-3 family. Several full-length cDNAs encoding the SGF-1 40-kDa and possibly also the 41-kDa proteins were cloned and sequenced. SGF-1 mRNA is expressed consistently with the presumed role of the SGF-1 protein product in regulating the sericin-1 gene. The SGF-1 protein contains putative transactivation domains. We conclude that the 40- and 41-kDa SGF-1 proteins affect transcription of the sericin-1 gene via binding to the SA site.

Biochemical improvement of chronic hepatitis C after gastrointestinal bleeding.

Although chronic hepatitis C is frequently complicated by iron overload, it remains unclear whether iron cytotoxicity is involved in the disease process. Five patients with chronic hepatitis C showed rapid reduction of serum aminotransferase activity after gastrointestinal bleeding. Posthemorrhagic reduction of liver enzyme levels lasted for more than one week. Anemia was associated with a reduction of serum ferritin concentration. Considering the short half-lives of circulating liver enzymes, reduced release of enzymes, that is inactivation of cell lysis, is the likely cause of the improved biochemical indices. Reactive iron, which is cytotoxic for patients infected by HCV, may be rapidly incorporated into hemoglobin when erythropoiesis is stimulated. Our observation also suggests that intensive iron removal by phlebotomy is a safe, economic treatment for patients with chronic hepatitis C.

Promoter of the POU-M1/SGF-3 gene involved in the expression of Bombyx silk genes.

To characterize the transcription regulation of the POU-M1/SGF-3 gene, we have cloned a genomic DNA fragment encompassing the whole coding region and its flanking sequences. This gene does not contain any intron. The 5'-flanking region of the gene contains several interesting motifs, such as homeodomain-binding motifs, sequences resembling the transcriptional factor Sp1-binding site, and TGTTT motifs, but lacks some of the typical transcriptional regulatory sequences, such as TATA and CCAAT boxes. Transcriptional analysis of a series of deletion mutants of the gene in the nuclear extracts prepared from the middle silk gland of 2-day-old fifth instar larvae revealed the presence of multiple cis-regulatory elements located both upstream and downstream of the initiation site. One of these elements, the homeodomain-binding element, was identified to mediate negative regulation. By mobility shift assay using the POU-M1 specific antibodies, we found that this negative element interacts with the POU-M1/SGF-3. Transcription analysis in vitro using templates mutagenized in the PB region and one of the POU-M1 antibodies indicated that the PB region is an autoregulatory element responsible for SGF-3-dependent transcriptional repression.

Molecular cloning of a POU domain-containing factor involved in the regulation of the Bombyx sericin-1 gene.

The POU domain is a highly conserved region found in a number of transcription factors and products of developmental control genes. We report here the isolation and characterization of a POU domain-containing cDNA (POU-M1) from the middle silk gland of Bombyx mori. It encodes a protein with a POU domain identical to that of the Drosophila Cf1-a protein. By mobility shift and nuclease protection assays, the POU-M1 protein and the putative silk gland factor 3 (SGF-3) were found to interact in an indistinguishable manner with the SC region (positions -204 to -183) of the sericin-1 gene, a key cis-acting element involved in the regulation of the gene through the interaction with SGF-3. Antibodies raised against the synthetic oligopeptide corresponding to the COOH-terminal region of the putative POU-M1 sequence reacted specifically to both the POU-M1 protein and SGF-3. Northern blot hybridization and Western blotting revealed that POU-M1 expression is regulated both temporally and spatially during silk gland development. We conclude that the POU-M1 protein is SGF-3 and propose that the differential expression of the POU-M1 gene is probably involved in the transcriptional regulation of the silk protein genes.

Relationship between superoxide dismutase (SOD) and viral liver diseases.

Immunohistochemical evaluation of Cu, Zn- and Mn-superoxide dismutase (SOD) activity in various viral liver diseases was performed by the peroxidase-conjugated antibody indirect method. Anti-human Cu, Zn-SOD (rabbit) and anti-human Mn-SOD (guinea-pig) derived and purified from SOD of human erythrocytes and placentas were used to determine SOD distribution in liver tissues. SOD in the liver tissues was detected in 68 inpatients of our unit. They consisted of 23 cases with chronic hepatitis caused by hepatitis B virus (13) and hepatitis C virus (10), 24 with liver cirrhosis caused by hepatitis B virus (5) and hepatitis C virus (19) (15: compensatory, 9: decompensatory) and 21 with hepatocellular carcinoma caused by hepatitis B virus (2) and hepatitis C virus (18) complicated of liver cirrhosis. In viral liver diseases, SODs in the liver tissues were distributed to hepatocytes mainly in the pattern of cytoplasmic diffusion. The incidence of immunohistochemical Cu, Zn-SOD and Mn-SOD were 47.8% and 56.5% in chronic hepatitis, 93.3% and 86.7% in compensated liver cirrhosis, 11.1% and 22.2% in decompensated liver cirrhosis, respectively. The aggression of viral liver disease was accompanied with the decrease of SOD concentration in the liver tissues. Hepatocellular carcinoma cells were negative for Mn-SOD in all cases, and weakly positive for Cu, Zn-SOD in 2 out of 21 cases. Comparatively strongly positive SOD findings were obtained from normal regions neighboring carcinomas. A close relationship between the depletion of SOD in liver tissues and carcinogenesis in viral liver diseases was observed.

Purification and characterization of an enhancer-binding protein of the fibroin gene. I. Complete purification of fibroin factor 1.

An enhancer-binding protein of the fibroin gene, fibroin factor 1 (FF1), has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells where this gene is transcribed specifically. There is a multiplicity of FF1; the FF1 activity was eluted as at least three major fractions on column chromatographies. FF1 is able to form a stable complex with the enhancer DNA sequence in the presence of another proteinous factor named FF2, which lacks ability to bind DNA molecules by itself. One of FF1 forms, FF1a, was purified with a combination of classical purification techniques without using a sequence-specific affinity column, and identified as a protein with molecular mass 125 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To obtain homogeneous protein of FF1a, purification of more than 26,000-fold from the starting nuclear extract was necessary.

Purification and characterization of an enhancer-binding protein of the fibroin gene. II. Functional analyses of fibroin factor 1.

Fibroin factor 1 (FF1) when coupled with fibroin factor 2 (FF2) is an enhancer binding protein of the fibroin gene. FF1a, one type of FF1, has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells and identified as a protein with molecular mass 125 kDa (Suzuki, T., Matsuno, K., Takiya, S., Ohno, K., Ueno, K., and Suzuki, Y. (1991) J. Biol. Chem. 266, 16935-16941). FF1 is able to recognize and bind a sequence of -205 to -185 in the enhancer I of the fibroin gene in the presence of FF2. The binding sequence of FF1 with FF2 contains two repeats of the derivative of consensus sequence which is recognized by homeobox-containing proteins. Though FF1 activity to construct the major band complex I is specific to posterior silk gland cells, use of a specific antibody raised against FF1a showed that FF1 protein is ubiquitous in Bombyx cells. These results suggest the possibility that FF1 molecules present as multiple proteins might be specifically modified and activated for the binding to enhancer DNA in posterior silk gland cells. Since the FF1a antibody also inhibited a transcriptional enhancement activity governed by the enhancer sequence, we conclude that FF1 is one of the transcriptional factors of the fibroin gene. The functions of FF1 on fibroin gene transcription are discussed.

Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm Bombyx mori.

In order to establish a transient expression system for genes introduced into early embryos of the silkworm, Bombyx mori, we tested various promoters ligated with CAT reporter genes. The embryos into which we injected supercoiled plasmid DNA of pFb(-860/+10)CAT containing the Bombyx fibroin promoter region ligated to the CAT gene showed a reasonably high CAT activity beginning around 30 h after oviposition. This high activity was observed only when the plasmid was injected before termination of the early nuclear cleavage stage, which was about 8 h after oviposition, but not after this stage. This means that the expression of injected DNA is closely related to the presence of cleavage nuclei in early embryos. Promoters originating from insect genes, like the Bombyx sericin-1 gene, Drosophila hsp70 and Drosophila copia LTR, functioned as strong promoters in the embryos. On the contrary, promoters from mammalian virus genes, such as the SV40 early and Moloney murine leukemia virus LTR genes, functioned as weak promoters. Moreover, linearized DNAs showed no or weak activity of expression in embryos. From these results, we conclude that the silkworm embryo transient expression system is a useful tool for studying the mechanism of regulation of insect genes.

Transcriptional stimulation via SC site of Bombyx sericin-1 gene through an interaction with a DNA binding protein SGF-3.

Three protein binding sites have been identified in the upstream region of the sericin-1 gene. Two of them, SA and SC sites, have been known as putative cis-acting elements. Using synthetic oligonucleotides of these binding sites, it was found that silk gland factor-1 (SGF-1) binds to the SA site, and silk gland factor-3 (SGF-3) binds to the SC site but not to a mutated SC site, SCM. Tissue distribution of the two factors was different. SGF-3 is present abundantly in the middle silk gland (MSG) where the sericin-1 gene is transcribed specifically but is also present in other cell types, though in a much less concentration. SGF-1 is observed very abundantly in the two parts of silk gland, MSG and posterior silk gland (PSG), but not in other cells. Templates containing multimerized SA or SC sites at -39 of the sericin-1 gene promoter were tested in MSG nuclear extracts. The SC multimer strongly activated transcription, while the mutant SCM multimer did not. The SA multimer also gave a slight stimulation of transcription. These results suggest that SGF-3 stimulates transcription through an interaction with the SC site, and SGF-1 does so weakly through the SA site.

A contribution of the core-promoter and its surrounding regions to the preferential transcription of the fibroin gene in posterior silk gland extracts.

Complementation of a posterior silk gland (psg) extract to a HeLa cell extract specifically enhances the transcription of the Bombyx mori fibroin gene. To map the regions responsible for this enhancement, the fibroin promoter was dissected and the transcriptional function of each region was analyzed. Besides the upstream promoter element 5' to the TATA box, two downstream elements were found to be important for the preferential transcription of the fibroin gene in the complementation system as well as in the psg extract. The minimal fibroin promoter from -37 to +10 (core-promoter) was preferentially transcribed in the psg extract, while the transcription efficiencies of other promoters like one of the Bombyx chorion genes and the adenovirus 2 major late promoter (Ad2MLP) were considerably lower. The transcription from the core-promoter was further enhanced when combined with either the intronic element from +156 to +454 or the upstream element. Both the upstream and intronic elements also stimulated the transcription from the Ad2MLP in an orientation independent manner. These results demonstrate that the transcription of the fibroin gene is mediated through an integration of multiple regulatory elements.

Transcription signals and protein binding sites for sericin gene transcription in vitro.

Using nuclear extracts from Bombyx mori middle silk glands (MSG) where the sericin-1 (Ser-1) mRNA is produced specifically, we have studied the in vitro transcription of the Ser-1 gene. To determine the sites required for the promoter function of the Ser-1 gene and characterize the factors binding to these sites, the upstream region was dissected. Footprinting assays revealed the presence of at least three specific protein binding sites, SA (-103 to -85), SB (-149 to -135) and SC (-204 to -183). Removal of SA and SC by 5'-deletions decreased the promoter activity. DNase I footprinting using four extracts of B. mori origin indicated that the factor binding to SA is specific to the silk gland extracts, and the factor binding to SC is very much abundant in the MSG extracts. Stimulation of the promoter activity with the region from -239 to -174 that accommodates the SC site, is stronger in MSG extracts than in the extracts from the posterior silk glands. These results suggest that the SC binding factor abundant in MSG may play an important role in the MSG-specific expression of the Ser-1 gene.

Serum activity and hepatic localization of superoxide dismutase in alcoholics.

A series of investigations was conducted to trace serum superoxide dismutase (SOD) activities by ELISA and localizations of hepatic tissue SOD by the indirect method using peroxidase conjugated antibody, and the diagnostic and physiological significance of SOD in 19 alcoholics was studied. The values increased significantly both in serum Cu, Zn-SOD to 136 +/- 18 ng/ml (normally 33 +/- 9 ng/ml) and in serum Mn-SOD to 859 +/- 686 ng/ml(normally 84 +/- 30 ng/ml) respectively when polyclonal antibody was used (P less than 0.001). The increase in serum Mn-SOD was higher than that in serum Cu, Zn-SOD, fluctuations of these values showed similar tendencies. Meanwhile, serum Cu, Zn-SOD (94 +/- 50 ng/ml) identified by monoclonal antibody, also, showed higher values than that of normal subjects (37 +/- 7 ng/ml) (P less than 0.001). On the other hand, localization of hepatic tissue Cu, Zn-SOD in alcoholics varied, being 63.2% in the cytoplasmic diffuse type, 42.0% in the nuclear diffuse type, 42.1% in the vacuolated membrane type, and 15.8% in the small granular type respectively. Participation of Cu, Zn-SOD in ethanol oxidation, protective roles played by cell membrane, lysosome membrane and nucleic acid of Cu, Zn-SOD to harmful free radicals was presumed. In addition, localization of hepatic tissue Mn-SOD was mostly of the cytoplasmic diffuse type (52.7%) and was extremely variable. From such results, relative or absolute reductions of hepatic tissue SOD in alcoholics was suggested to act on the development of tissue injuries acceleratingly.

cDNA structure and expression of bombyxin, an insulin-like brain secretory peptide of the silkmoth Bombyx mori.

Bombyxin, previously referred to as 4K-prothoracicotropic hormone, is a brain peptide of the silkmoth Bombyx mori, the amino acid sequence of which shows considerable homology with vertebrate insulin family peptides. Two independent clones have been isolated from a Bombyx larval brain cDNA library by using a synthetic oligonucleotide probe, one with the complete coding region for preprobombyxin (lambda Bb360) and the other covering the coding region, possibly for bombyxin, only partially (lambda Bb204). lambda Bb360 encodes preprobombyxin in the order of prepeptide/B-chain/proteolytic cleavage signal/C-peptide/proteolytic cleavage signal/A-chain. This domain organization of preprobombyxin is the same as that of preproinsulins, suggesting that the tertiary structure and posttranslational modification mechanism are conserved through the evolution of bombyxin and insulin. Genomic Southern hybridization analyses using this cDNA as probe suggest that the Bombyx genome contains multiple copies of bombyxin gene. Northern hybridization analyses indicate that the concentration of lambda Bb360-type bombyxin mRNA in the bombyxin-producing cells is remarkably high (2.8 x 10(9) molecules/micrograms of total RNA), without undergoing appreciable change during larval-pupal development.

Complex formation with the fibroin gene enhancer through a protein-protein interaction analyzed by a modified DNA-binding assay.

The distal upstream region of the fibroin gene, -238 to -73, is known to contain the cis-acting element(s) responsible for tissue-specific transcription enhancement in vitro. To identify factor(s) trans-acting on this region, we have developed a modified nitrocellulose blot protein-DNA binding procedure that detects DNA-protein complexes via protein-protein interaction. A protein that does not bind to DNA as a monomer cannot be detected by standard Southwestern blotting. To detect protein-DNA complexes involving both protein-DNA and protein-protein interactions, the protein blot was reacted with the extract protein to form a protein complex on the filter, in the presence of the DNA probe. This new method revealed that the 75-kDa protein binds to the enhancer region of the fibroin gene in the presence of the second protein(s). The 75-kDa protein and the factor(s) mediating DNA binding activity were recovered in two different column fractions. The level of the 75-kDa protein appeared to be much higher in silk gland extracts than in nonsilk gland extracts, whereas the mediating factor(s) distributed evenly. These observations suggest that the distal upstream region of the fibroin gene can be specifically recognized by a protein complex composed of at least two distinct proteins, through protein-protein interaction.

Factors involved in preferential transcription of the fibroin gene.

Transcription factors for class II genes from a tissue source, the posterior silk glands of Bombyx mori, were fractionated by stepwise elution on a phosphocellulose column into five fractions: A (0.04 M KCl flow through), A' (0.1 M KCl eluate), B (0.3 M KCl eluate), C (0.5 M KCl eluate) and D (1 M KCl eluate). The minimal requirement for reconstruction of accurate initiation of transcription of the fibroin gene, as well as of the adenovirus 2 major late promoter, was the combination of fractions A, B and D, suggesting that transcription factors from B. mori can recognize general signals of the promoters for class II genes and that basic transcription factors are conserved even in distantly diverged species of eukaryotes. To detect activities stimulating the transcription governed by the promoter of fibroin gene, each fraction was tested for its function by supplementing a basal amount of HeLa cell extract. When circular templates were used, stimulatory activities specific for the fibroin gene were detected in fraction D. This preferential transcription is composed of at least three activities; the first and the second dependent on the upstream elements and the third dependent on the sequence downstream from the TATA box. However, when linear templates were used the preference for the fibroin gene was apparently lost and transcription activation by fraction D became general.