cis-4-Hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate prevent myogenesis of C2C12 muscle cells and block MyoD1 and myogenin expression.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.     

Parathyroid hormone stimulates ATP-dependent calcium pump activity by a different mode in proximal and distal tubules of the rat.

A new technique was developed to isolate basolateral membrane vesicles individually from proximal and distal tubules of the rat cortex. This new technique enabled us to study differences in their kinetics and mechanisms of hormonal regulation of Ca pump between proximal and distal tubules. The Ca pump in distal tubule has very high affinity (42.6 nM Ca2+) and the one in proximal tubule has relatively low affinity (75.6 nM Ca2+). Parathyroidectomy (PTX) decreased the Vmax of Ca pump activity in proximal tubule (4.68 +/- 0.99 vs. 9.08 +/- 2.21 nmol 45Ca2+/min per mg protein BLMV, P less than 0.05), while it increased Km in distal tubule (93.1 +/- 11.0 vs. 35.1 +/- 16.1 nM Ca2+, P less than 0.05). Restoration of serum Ca2+ concentration by 1,25(OH)2D3 supplement could not reverse these changes by PTX in Ca pump activity in either the proximal or the distal tubule. In conclusion, this study strongly suggested that parathyroid hormone stimulated Ca pump activity by increasing the Vmax in proximal tubule and by increasing the affinity in distal tubule. 1,25(OH)2D3 does not have a direct effect on the basolateral membrane Ca pump activity.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.     

Developmental analysis of the eye lens obsolescence (Elo) gene in the mouse: cell proliferation and Elo gene expression in the aggregation chimera.

This study investigates the primary effect of the eye lens obsolescence (Elo) gene of the mouse. Morphological features of the Elo lens were defined as follows: (1) deficient elongation of lens fiber cells, (2) morphological abnormality of nuclei of lens fiber cells, (3) lack of eosinophilic granules in the central fiber cells and (4) rupture of lens capsule in the posterior region. We have immunohistologically examined, by means of an in vivo BrdU incorporation system, whether or not the Elo gene regulates cell proliferation during lens development. The lens fiber cells were morphologically abnormal in day 13 embryonic Elo lens. However, there were no significant differences in morphology or cell proliferation between normal and Elo lens epithelium until day 14 of gestation. After day 15, the total cell number in the Elo lens epithelium was significantly less than that in the normal, but the total numbers of S-phase cells in the two genotypes were not significantly different. The ratio of the total S-phase cell number to the total number of lens epithelial cells may be affected by the developmental stage, but not directly by the genotype. The genotype, however, may be having a direct influence at later ages because malformation of Elo lens fiber cells must cause reduction of the total number of lens epithelial cells in older embryos. Although, at 30 days old, Elo lens cells were externally extruded through the ruptured capsule into the vitreous cavity, BrdU-labelled lens epithelial cells were detectable. To investigate whether the Elo lens phenotype is determined by its own genotype or by its cellular environment, we produced aggregation chimeras between C3H-Elo/+(C/C) and BALB/c(c/c). Most lenses of BALB/c dominant chimeras were oval in shape without the ruptured lens capsule. However, they were opaque in the center and slightly smaller in size than normal. The lenses of C3H-Elo/+ dominant chimeras were morphologically similar to the Elo lens. Although normal nuclei were regularly arranged in the anterior region, Elo-type nuclei were located in the posterior region. Immunohistological staining by using anti-C3H strain-specific antibody demonstrated that the lens fiber cells with abnormal nuclei were derived only from C3H-Elo/+, not from BALB/c. These observations suggest that the primary effect of the Elo gene in the developing lens may be specific to the fiber cell differentiation rather than to the cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

An antigen present in the Drosophila central nervous system only during embryonic and metamorphic stages

We report here about an antigen that is expressed in the central nervous system (CNS) of Drosophila only during the embryonic and metamorphic stages. In Drosophila, axonogenesis and synaptogenesis occur twice during the development: first in the embryonic and second in the metamorphic stages. We generated monoclonal antibodies (MAbs) in order to obtain molecular probes for analyzing axonogenesis or synaptogenesis in the CNS on the assumption that good candidates for molecules responsible for such phenomena must be present in the neuropil during those stages exclusively. As a result, we found MAb 66B2 whose intense immunoreactivity in the neuropil of the CNS was observed exclusively in the embryo and pupa, and not in the larva and adult. Immunoblot analyses showed that MAb 66B2 binds specifically to a protein with an apparent molecular weight of 350 K and neutral pl in the prepupal CNS. A significant amount of the antigen was isolated in forms that were soluble without detergent. Results of immunohistochemistry with MAb 66B2 in a primary culture of embryos showed that some live cells in the ganglion-like cluster were stained, and that neuronal cell bodies and neurites emanating from there were negative. These results strongly suggest that the 66B2 antigen observed in the CNS is an extracellular matrix component secreted from nonneuronal cells. These developmental changes in the 66B2 immuno-reactivity in the CNS presumably reflect dynamic changes of an extracellular matrix in the CNS that are accompanied by axonogenesis or synaptogenesis. © 1992 John Wiley & Sons, Inc.     

Purification and characterization of a novel GTP-binding protein with a Mr value of 24,000 from rat liver

About 15% of the total GTP-binding proteins (G proteins) of rat liver homogenate was found in the microsomes-Golgi complex fraction. From this fraction, we purified to near homogeneity and characterized a G protein with a Mr value of 24,000 (24K G). 24K G specifically bound guanosine 5'-(3-Q-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 30 nM. 24K G bound maximally about 0.7 mol of GTP gamma S/mol of protein. 24K G hydrolyzed GTP to liberate Pi with a turnover number of about 0.008 min-1. 24K G was not copurified with the beta gamma subunit of heterotrimeric G proteins. The partial amino acid sequences of 24K G revealed that this protein was a novel small G protein.     

Studies on the Pectic Depolymerases I. Exopolygalacturonase from Root Tissues and Cell Suspension Cultures of Carrots

Three polygalacturonases (PG) have been isolated from carrots(Daucus carota L. cv. Kintoki). Two were isolated from roottissues (PG-I and PG-II) and one from cell suspension cultures(PG-III). PG-I and PG-III were readily solubilized in a lowionic strength buffer, whereas PG-II required additional NaClto be solubilized. These seems to be a change in the propertiesof PG between the original tissue and carrot cell cultures.The three PGs were partially purified by chromatography on SephadexG-150, and characterized. Elution from a Sephadex G-150 column indicated a molecular weightof about 48,000 for all three PGs. PG-III, studied in detail,hydrolyzed the galacturonan chain in an exo-fashion, and wasnot activated by a variety of cations at concentrations of 0.5or 1.0 mM. The pH optimum, and pH and heat stability of PG-Iand PG-III were slightly different from those of PG-II. PG-Iwas also different from PG-II and PG-III in its pectin hydrolyzingactivity. These results indicate that the enzymatic properties of PG-IIIfrom cell cultures are very similar to those of PG-I or PG-IIfrom root tissues; the only significant difference seems tobe the binding properties of the PGs to the cell wall materials. (Received March 23, 1981; Accepted June 11, 1981)     

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide by 3-chloro-D-alanine hydrogen chloride-lyase (deaminating) of Pseudomonasputida

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide is catalyzed by highly purified 3-chloro-D-alanine hydrogen chloride-lyase from $\text{Pseudomonas}\text{putida}$. The synthetic reaction proceeds optimally at pH 8.5, as a function of enzyme concentration and incubation time. The enzymatically synthesized D-cysteine was isolated from the large scale reaction mixture and identified by physicochemical means.