Constitutive expression of FosB and its short form, FosB/SF, induces malignant cell transformation in rat-1A cells

We have established Rat-1A cell lines constitutively expressing c-Fos and the two products of the fosB gene, FosB and its short form, FosB/SF. The expressed proteins in the different stable transfectants have been characterized by immunofluorescence and immunoprecipitation analysis. Our results demonstrate that constitutive expression of FosB, like the constitutive expression of c-Fos and, to a lesser extent, FosB/SF, results in cells that grow to increased saturation densities and have the ability to grow in an anchorage-independent manner. Most important is the finding that expression of these proteins augments the tumorigenic potential of Rat-1A cells. These results show that both forms of FosB have a stimulatory effect on cell proliferation.     

Stimulatory Effects of Exogenous Thyroid Hormone and Growth Hormone on sn-Glycerol-3-Phosphate Dehydrogenase Activity in the Genetic-Hypothyroid Mutant Mouse Cerebellum: Restricted to the Second 20 Days of Postnatal Life

We recently reported that by postnatal day 40 the activity of sn-glycerol-3-phosphate dehydrogenase (GPDH) was significantly depressed in the cerebellum of genetic-hypothyroid mutant mice. This mutant mouse-GPDH combination was used in the present study to define the critical time period during which thyroid hormone (T4) and growth hormone (GH) are essential for maturation of Bergmann glial cells. Our findings are that (a) induction of GPDH activity in the Bergmann glial cell is dependent on T4, (b) T4 is most effective when administered during the second 20 days of postnatal life, (c) the effect of GH on GPDH activity is complementary to or synergistic with that of T4, and (d) Bergmann glial cells and radial glial fibers of the mutant mice contain immunoreactive GPDH following various hormonal treatments. These results suggest that T4 is indispensable for the maturation of Bergmann glial cells.     

Mouse Offspring Derived from Fetal Ovaries or Reaggregates Which were Cultured and Transplanted into Adult Females

Primordial germ cells (PGCs) and gonia could be promising novel targets and vehicles for manipulation of the mammalian germ line. To make such manipulation a practical possibility, PGCs or gonia must be allowed to produce gametes and offspring after they were isolated from embryos and manipulated in culture. As the first step to develop such research strategy, we obtained offspring from mouse oogonia which were isolated from embryonic ovaries and cultured as dispersed cells before transplantation into female mice as reaggregates.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

Local formation and degradation of endothelin-1 in guinea pig airway tissues

Endothelin(ET)-1 and big ET-1 caused potent and sustained constriction of isolated guinea pig bronchus. The response to ET-1 was enhanced by phosphoramidon in a simple dose-related manner (0.01-1000 microM), while the response to big ET-1 was enhanced at lower doses (0.01-0.1 microM) but was suppressed at higher doses (100-1000 microM) of phosphoramidon. Big ET-1, when given intravenously (i.v.) to anesthetized guinea pigs, increased both bronchopulmonary inflation pressure and mean arterial blood pressure (2.5, 5, 10 nmol/kg i.v.). The pressor response to big ET-1 was attenuated by phosphoramidon dose-relatedly, while the pulmonary response was modified in a complex fashion composed of delayed onset and prolonged duration of action. These results suggest that ET converting as well as degrading enzymes coexist in the airway tissue and both enzymes are sensitive to phosphoramidon, so that phosphoramidon acts bifunctionally to reduce and stimulate the airway responses to big ET-1.     

Further fractionation of the glycoprotein families of porcine zona pellucida by anion-exchange HPLC and some characterization of the separated fractions

The glycoproteins of porcine zonae pellucidae have been fractionated into three families (PZP1-3) by gel filtration HPLC [Nakano et al. (1987) Biochem. Int. 14, 417-423]. However, they still comprise heterogeneous molecular species differing in electric charge. We found that sulfate, but not phosphate, is contained in PZP1-3 by a simple and rapid method for microanalysis of the anionic groups. These families were efficiently separated into many fractions by anion-exchange HPLC. When elution was performed by stepwise increase in NaCl concentration in 8 M urea/20 mM Tris-HCl, pH 8.0, a single distinctive peak emerged for each step. The analyses of amino acids, monosaccharides, and anions of the eight separated fractions of the major family, PZP3, showed that larger amounts of sulfated lactosamine linked to the constituent proteins are present in the fractions that are eluted later: the chain length and/or the chain number of these polylactosamines and the sulfate content increased with stepwise increase in NaCl concentration. Composition analyses also revealed that twice as much N-glycolylneuraminic acid is present as N-acetylneuraminic acid in all fractions. The contents of these sialic acids in the fractions slightly increased in the order of elution. These results together with those of the analyses of endo-beta-galactosidase digests showed that the charge heterogeneity of the porcine zona proteins is due mainly to differences in the amount of sulfated lactosamine, which is predominantly distributed in the non-reducing regions of the sugar chains.     

Alanine: glyoxylate aminotransferase 1 is present in the peroxisomes of guinea pig kidney

The subcellular distribution of alanine: glyoxylate aminotransferase 1 in guinea pig and rabbit kidneys was examined by centrifugation in a sucrose density gradient. The enzyme was located in the peroxisomes of guinea pig kidney and cross-reactive with the antibody against rat liver alanine: glyoxylate aminotransferase 1. This is the first report on the presence of the enzyme in the peroxisomes of mammalian kidney. The enzyme was found to be located in the mitochondria but not in the peroxisomes in rabbit kidney.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

Inhibition of biological actions of big endothelin-1 by phosphoramidon

Endothelin (ET)-1 and big ET-1 both caused contraction of isolated porcine coronary arteries, but the potency of big ET-1 was 1/100-1/200 that of ET-1. These responses were independent of the vascular endothelium. Phosphoramidon blocked the vasoconstriction caused by 30 nM big ET-1, but was ineffective on the action of 0.3 nM ET-1. Also in vivo, phosphoramidon had no effect on the ET-1-induced pressor actions, but blocked the pressor and airway-contractile responses to big ET-1 in rats and/or guinea pigs. Thus, it is likely that the vascular responses to exogenous big ET-1 are at least in part due to its conversion to ET-1 by a phosphoramidon-sensitive ET converting enzyme(s) in the vascular smooth muscle in vitro and in vivo.     

Slalom chromatography: size-dependent separation of DNA molecules by a hydrodynamic phenomenon

Slalom chromatography, a size-dependent DNA fractionation method based on a new principle [Hirabayashi, J., & Kasai, K. (1989) Anal. Biochem. 178, 336-341], was systematically studied in detail. In this method, larger DNA fragments are eluted much later than smaller ones from columns packed with spherical microbeads. Elution of a series of DNA fragments was systematically examined by using columns packed with polymer-based packings of different diameter and different pore size for high-performance gel permeation chromatography. Packings of smaller diameter proved to be superior for resolving the smaller size range of DNA, while the reverse was the case for larger DNAs. Application of a faster flow rate led to larger retardation of every DNA fragment, while at the lowest flow rate applied (0.067 cm/min), all the fragments were eluted almost at the void volume. When the column temperature was lowered, retardation of DNA became larger. On the other hand, differences in the chemical nature and the pore size of packings, or in the hydrophobicity of the eluting solvent, had little effect on DNA retardation. Size-dependent fractionation of DNA was also achieved even on columns packed with nonporous packings having anionic groups (cation exchangers). In conclusion, these results confirmed the previous conclusion that slalom chromatography is not based on an adsorption or equilibrium phenomenon but should be attributed to a hydrodynamic phenomenon.