Regulation of primary erythropoiesis in the ventral mesoderm of Xenopus gastrula embryo: evidence for the expression of a stimulatory factor(s) in animal pole tissue.

We have previously shown that the animal pole tissue from a st.10+ early gastrula Xenopus embryo stimulates the primary differentiation of erythrocytes in the ventral mesoderm in combination culture. To characterize the nature of this stimulation, various sizes and different portions of animal pole tissue were combined with the ventral mesoderm explants.

Cloning and expression of cDNA encoding Xenopus laevis bone morphogenetic protein-1 during early embryonic development.

The Xenopus laevis DNA fragment encoding a protein homologous with human bone morphogenetic protein-1 (BMP-1) was amplified by polymerase chain reaction (PCR) from cDNA derived from stage 26 (st.26) embryonic RNA. Subsequently this fragment was used as a probe to isolate cDNA clones by screening of a X.

Characteristics of a 28-kDa collagenous protein extracted with guanidine from EDTA-demineralized rabbit alveolar bone.

Bone proteins in alveolar bone of mandibles from young adult rabbits (3-month-old) were extracted with 4.0 M guanidine hydrochloride (GuHCl), followed by 0.5 M ethylenediaminetetraacetate, and again with 4.

[The significance of 201Tl/123I MIBG (metaiodobenzylguanidine) mismatched myocardial regions for predicting ventricular tachycardia in patients with idiopathic dilated cardiomyopathy].

123I-MIBG (MIBG) regional defects in myocardial regions with preserved 201Tl (Tl) uptake have been observed in patients with idiopathic dilated cardiomyopathy (DCM). To evaluate whether the presence of Tl/MIBG mismatched regions is related to the occurrence of ventricular tachycardia (VT), we performed myocardial dual SPECT imaging with Tl (111 MBq) and MIBG (111 MBq) in 17 patients with DCM, 11 (Gp A) with and 6 (Gp B) without VT. Myocardial dual SPECT imaging was performed at 15 minutes after and 4 hours after the tracer injection.

Murine stem cell factor stimulates erythropoietic differentiation of ventral mesoderm in Xenopus gastrula embryo.

We have reported that the animal pole cells stimulate the ventral mesoderm of early gastrula Xenopus embryo (stage 10) to differentiate into erythrocytes. To determine the molecular mechanism(s) involved in the stimulatory effect of the animal pole, ventral mesoderm explants were cultured in the presence of various defined cellular factors. In this study, we report that murine stem cell factor (SCF) stimulates globin expression at the optimum dose of 10 ng/ml.

Nature and distribution of mineral-binding, keratan sulfate-containing glycoconjugates in rat and rabbit bone.

The presence of keratan sulfate (KS) and KS proteoglycans in bone has been demonstrated in birds and rabbits but comparison with other animal species has not been investigated. The nature and distribution of mineral-binding, KS-containing glycoconjugates in rat and rabbit bone were investigated with a monoclonal antibody (MAb 5D4) specific for KS. Mineral-binding proteins were extracted from the mineralized bone with 0.

Positive and Negative Regulation of the Differentiation of Ventral Mesoderm for Erythrocytes in Xenopus laevis: (Xenopus laevis/erythropoiesis/embryonic blood island/explant/regulation).

To elucidate the mechanism of determination and regulation of hemopoiesis in the early Xenopus embryo, explants of dorsal and ventral mesoderm from various stage embryos were cultured alone or combined with various tissues derived from the same stage embryo. Western blot analysis of larvae-specific globin expression using monoclonal antibody L5.41 revealed that extensive erythropoiesis occurred in the explants of ventral mesoderm from st.

Characterization of mineral-binding 40-kDa glycoprotein extracted from young adult rabbit alveolar bone.

A forty-kilodalton (40-kDa) protein was extracted from alveolar bone of young adult rabbit with 0.5 M EDTA after extraction with 4 M GuHCl, and purified by gel-filtration, anion-exchange and hydroxyapatite columns using a high-pressure liquid chromatography system under denaturing conditions. The purified 40-kDa protein was not susceptible to bacterial collagenase and thrombin, but was cleaved by cyanogen bromide.

Extracellular production system of heterologous peptide driven by a secretory protease inhibitor of Streptomyces.

The value of a heterologous peptide extracellular production system in Streptomyces using a secretory protease inhibitor, was examined. DNA was synthesized encoding apidaecin 1b (AP1), an interesting antibacterial peptide discovered in lymph fluid of the honeybee, and was joined to the Streptomyces subtilisin inhibitor (SSI) gene via a 12-bp nucleotide sequence corresponding to the amino acid sequence specific for cleavage by blood coagulation factor Xa. The fusion protein (SSI-AP1) could be expressed and excreted efficiently into the medium by culturing S.

Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization.

The present study utilized biochemical and immuno- and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific for N-acetyl-D-glucosamine, N-acetylneuraminic acid, and N-acetyl-D-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl.

Transplantation studies on human and duck hepatocytes in athymic nude mice.

For determination of the most suitable tissue for heterotopic transplantation of exogenous hepatocytes, dissociated hepatocytes or small pieces of liver tissue were transplanted into the spleen, adipose tissue and inside the capsule of the kidney of BALB/c mice. Survival of syngeneic grafts of dissociated hepatocytes was highest in the spleen and that of pieces of liver tissue in the adipose tissue, but only the latter system was suitable for xenogeneic transplantation. Histological examination showed that a total of 50% of the human or duck liver tissue implants survived in the inguinal fat pad of athymic nude mice (BALB/c-nu).

In vitro transcription of the hepatitis B virus gene by nuclear extracts of human hepatoma cells.

In vitro transcription of hepatitis B virus DNA (HBV DNA) was studied using nuclear extracts of human hepatoma cell lines. RNA polymerase II-dependent run-off transcription of pre-S mRNA under the control of pre-S1 promoter was observed in nuclear extracts obtained from HepG2 and PLC/PRF/5 cells, and the efficiencies in these extracts were significantly higher than those in nuclear extracts of non-liver cells such as HeLa, Molt-4, and Ehrlich. Analysis of run-off transcripts by the pre-S1 promoter, using deletion mutants of HBV DNA as templates and synthetic oligonucleotides as competitors, showed that hepatocyte nuclear factor 1 was necessary for initiation of in vitro transcription of pre-S mRNA.

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone.

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3.

A cDNA clone closely associated with non-A, non-B hepatitis.

A lambda gt11 cDNA library was constructed from RNA purified from hepatitis B viral surface antigen-negative human plasma with high alanine aminotransferase activity. A cDNA clone, designated as C8-2, was isolated by immunoscreening with mixed sera from non-A, non-B hepatitis (NANBH) carrier and convalescent chimpanzees. The recombinant protein produced by C8-2 reacted specifically with sera of patients in the chronic phase of NANBH.

Characterization of multiple forms of small collagenous apatite-binding proteins in bone.

A number of small (Mrs 25-28 kDa) collagenous apatite-binding (SCAB) proteins that stain blue with 'Stains-All' have been isolated from fetal porcine bone by sequential extractions with 4M GuHCl (G1), followed by 0.5M EDTA (E), and again with 4M GuHCl (G2). Following purification under dissociative conditions, two types of SCAB proteins both with approximately one-third of their structure being collagenous, were identified in the EDTA extract.

Identification of small collagenous proteins with properties of procollagen alpha 1 (I) pN-propeptide in fetal porcine calvarial bone.

Several small collagenous apatite binding (SCAB) proteins have been extracted from the mineralized matrix of fetal porcine calvarial bone. One protein (SCAB 3), released on demineralization of bone with 0.5 M EDTA, appears to represent the alpha 1 pN-propeptide that is normally released during proteolytic processing of type I procollagen.

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations.

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.

Effective production of mouse monoclonal antibodies against influenza virus by repeated intrasplenic immunization.

Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50 micrograms of antigens was used for the in vitro immunization method, and at least 5 micrograms was necessary for a single intrasplenic immunization.

Analysis of allotolerance in thymectomized Xenopus restored with semiallogeneic thymus grafts.

The triploid J strain Xenopus laevis (MHC haplotype, j/j/j) were thymectomized as early larvae, and in the adult stage each animal was given a pair of thymuses or lymphocytes from semiallogeneic diploid donor frogs (j/k). The alloreactivity of host frogs was restored to third-party donors in terms of skin graft rejection and mixed leukocyte reaction, but there was specific unresponsiveness against the k haplotype. Grafting of heavily irradiated (10,000 rads) thymuses also restored host reactivity with induction of tolerance to the k haplotype.