Amino acid sequence and function of rebredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-methionyl residue is partially formalated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 are 8.1 and 18.5, respectively, and the standard redox potential is +5 mB, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the rection was stimulated with 2-methyl-1, 4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membraous quinone, functions as natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.     

Transfilter analysis of the inductive influence of proventricular mesenchyme on stomach epithelial differentiation of chick embryos

Summary To clarify the precise conditions under which chick embryonic proventricular mesenchyme can induce proventricular epithelial differentiation, transfilter experiments were carried out. Six-day proventricular epithelium formed glands and expressed pepsinogen when a Nucleopore filter with a pore size of more than 0.6 m, but not 0.2 m, was inserted between the epithelium and the proventricular mesenchyme. The larger the pore size of the filter, the more elongated the glands and the more pepsinogen was induced in the explants. The quail nuclear marker and scanning electron microscopy were used to examine penetration of mesenchymal cells through the Nuclepore filter. The filter of more than 0.2 m pore size allowed cell processes of mesenchymal cells to pass through. However, only the filter with a pore size of more than 0.6 m allowed actual migration of mesenchymal cells through the filter, and the larger the pore size of the filter, the more mesenchymal cells passed through. Under the same conditions 6-day and 4.5-day gizzard epithelium formed glands and expressed pepsinogen. These results indicate that a flow of diffusible substances through a Nuclepore filter and even direct contact of a few short cell processes of mesenchymal cells with epithelial cells are not sufficient for induction, and that direct contact of mesenchymal cell processes and/or mesenchymal cells with epithelial cells over a considerably wide area may be prerequisite for the induction.     

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.     

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.     

(6R)-Tetrahydrobiopterin Increases the Activity of Tryptophan Hydroxylase in Rat Raphe Slices

The effects of (6R)- and (6S)-tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6-methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5-hydroxytryptophan (5-HTP) formation under inhibition of aromatic L-amino acid decarboxylase with use of HPLC-fluorometric detection. (6R)-BPH4 (the naturally occurring form) at 42 microM, tetrahydroneopterin at 50 microM, and 6-methyltetrahydropterin at 100 microM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)-BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)-BPH4, and that the concentration of (6R)-BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.     

Combined effects of gamma rays, cis-diamminedichloroplatinum (CDDP) and systemic hyperthermia mastocytoma transplanted into muscle in the mouse

Comparison was made between the effects of local irradiation of gamma rays, s. c. injection of cis-diamminedichloroplatinum (II) (CDDP), systemic hyperthermia and their combinations on the i. m. transplanted murine mastocytoma. Increase of the mean survival time (M. S. T.) by a factor of 1.72 and of 1.68 was achieved by a single irradiation at 20 Gy, given on day 5 after transplantation, and by injections of CDDP at 2 mg/kg, given s. c. on days 5 and 12 after transplantation, respectively. Increase of M. S. T. by a factor of 1.10 which was achieved with systemic hyperthermia of 41.8 degrees C of the core body temperature for 5 min, given twice, on days 5 and 12 after transplantation, was not statistically significant. The most effective one among all possible combinations within the 3 modalities was that of radiation and CDDP. Increase of M. S. T. was by a factor of 4.01.     

Gland formation induced in the allantoic and small-intestinal endoderm by the proventricular mesenchyme is not coupled with pepsinogen expression

Abstract. Allantoic and small-intestinal endoderms of chick and quail embryos were associated with the proventricular mesenchyme of chick embryos and then cultivated on chorioallantoic membrane. This resulted in the induction of complex glands, but the recombinates never produced embryo-specific pepsinogens; also, glandular cells developed a brush border, expressed sucrase antigen on their apical surface, and sometimes differentiated into goblet cells, thus indicating that both endoderms have the tendency to differentiate into an intestinal epithelium. In the recombinates composed of allantoic endoderm and proventricular mesenchyme, acid-protease activity was detected, but biochemical analysis revealed that this activity was not due topepsinogens. These results indicate that the gland formation induced in allantoic and small-intestinal endoderms by the proventricular mesenchyme is not accompanied by the expression of pepsinogens, suggesting that independent mechanisms are responsible for the morphogenesis and cyto chemical differentiation of the endoderm.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.