Spectroscopic characterization of nitrosylheme in nitric oxide complexes of ferric and ferrous cytochrome c' from photosynthetic bacteria.

Reactions of ferric and ferrous cytochromes c' from four photosynthetic bacteria (Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum rubrum ATCC 11170, and Chromatium vinosum ATCC 17899) with nitric oxide have been investigated by electronic absorption and electron paramagnetic resonance spectroscopies. The heme iron(III) of these ferric cytochromes c' has been recently reported to be in a quantum mechanically admixed (S = 5/2, 3/2) state [Fujii, S., Yoshimura, T., Kamada, H., Yamaguchi, K., Suzuki, S., Shidara, S. and Takakuwa, S. (1995) Biochim. Biophys. Acta 1251, 161-169]. The affinity of ferric cytochromes c' for NO among these bacterial species (C. vinosum > Rps. palustris approximately Rb. capsulatus >> R. rubrum) was apparently related to the S = 3/2 content in the or der. In the reaction of ferrous cytochrome c' with NO, six- and five-coordinated nitrosylhemes, which represent species with and without a ligand at the axial position trans to nitrosyl group, have been formed. The content of six-coordinated nitrosylheme in NO-ferrous cytochrome c' has been determined to be Rb. capsulatus approximately Rps. palustris > C. vinosum < R rubrum, suggesting that a stability of iron-to-histidine bond decreases with this order. The NO reactions of ferric and ferrous cytochromes c' from photosynthetic bacteria have been compared with those of cytochromes c' from denitrifying bacteria.

Electron paramagnetic resonance studies of ferric cytochrome c' from photosynthetic bacteria.

Electronic ground nature of ferric cytochromes c' isolated from five photosynthetic bacteria. Chromatium vinosum ATCC 17899, Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum molischianum ATCC 14031, and Rhodospirillum rubrum ATCC 11170 has been investigated by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra indicate that the electronic ground state of five ferric cytochromes c' is a quantum mechanical admixed-spin state of a high spin (S = 5/2) and an intermediate spin (S = 3/2) at pH 7.2 and is high-spin state at pH 11.0. At physiological pH, however, the content of an intermediate spin state differs with the bacterial source of the protein: approximately 50%, Chromatium vinosum; approximately 40%, Rhodobacter capsulatus and Rhodopseudomonas palustris; approximately 10%, Rhodospirillum molischianum and Rhodospirillum rubrum. Computer simulation of the spectra supports this diversity of the contribution of an intermediate spin state. Model studies of the ferric porphyrin complexes suggest that the correlation between content of an intermediate spin state and heme iron displacement from the mean heme plane. Therefore, the variation of the content of an intermediate spin state observed in the present study reflects the subtle difference in the degree of heme iron displacement among the proteins.

Purification and some properties of cytochrome c-552 from a sulfur-oxidizing bacterium, Thiobacillus thiooxidans.

A soluble cytochrome c-552 from Thiobacillus thiooxidans was highly purified and its physico-chemical properteis were studied. The absorption maxima were at 552,523,418 nm in the reduced from and at 412 nm in the oxidized form. The pyridine hemochrome spectrum was the same as that of other cytochromes c. The molecular weight, estimated by the gel filtration method, was found to be 12,600. The isoelectric point was determined to be 9.2-9.3 by the electrofocusing technique. The standard oxidation-reduction potential of this cytochrome was +0.247 V.

Multicentric infantile myofibromatosis in the cranium: case report.

Infantile myofibromatosis is a rare clinical entity characterized by multiple mesenchymal tumors in the neonatal period. We describe a 15-month-old girl with multicentric cranial lesions involving the parietal and occipital bones associated with a single small subcutaneous lesion in the back. Magnetic resonance imaging clearly demonstrated the isointense lesions on T1-, T2-, and proton density-weighted images, which showed marked gadolinium enhancement of the tumors and adjacent dura mater. A histological examination of the resected temporal lesion revealed the myofibroblastic nature of the tumor cells. This is the first description of magnetic resonance features of multicentric infantile myofibromatosis in the cranium, and gadolinium-enhanced magnetic resonance images were useful in showing dural involvement. The importance of recognizing this disorder is emphasized because of its special clinical behavior.

Plasma direct injection high-performance liquid chromatographic method for simultaneously determining E3810 enantiomers and their metabolites by using flavoprotein-conjugated column.

A high-performance liquid chromatographic method using the column-switching technique was developed for the simultaneous determination of E3810 and its metabolites (M1-M4). An avidin column was used for in-line pretreatment to exclude plasma proteins, allowing direct injection of a large volume of plasma. A flavoprotein-conjugated chiral stationary phase in a gradient elution mode then gave baseline separation of E3810 and the four metabolites. The enantiomers of E3810 and M3 were also separated. The method is simple, rapid, accurate, and precise. Since no extraction procedure is employed, which might involve different recoveries of different metabolites, no internal standard is necessary. The method was applied to analyze E3810 and its metabolites in plasma after intravenous injection of the drug in beagle dogs.

Light intensity distribution in the externally illuminated cylindrical photo-bioreactor and its application to hydrogen production by Rhodobacter capsulatus.

The light distribution in the externally illuminated cylindrical photo-bioreactor for production of hydrogen by a photosynthetic bacterium Rhodobacter capsulatus ST-410 was estimated. The estimation was performed on the basis of the Matsuura and Smith's diffuse model [1]. In the diffuse model, the incident light rays are assumed to proceed in every direction and the local intensity is calculated as the sum of the intensities of light. Since Lambert-Beer's law, extensively used in photometry, was not useful for explaining the decrease in the intensity of light by the biomass, an empirical expression was used. The measurement of the intensities from every direction was conducted in an externally illuminated cylindrical photo-bioreactor having an inner diameter of 60mm and a working volume of 550ml. The obtained results confirmed our estimation. The light distribution was applied to estimate the hydrogen production by R. capsulatus ST-410 using the same photo-bioreactor. The overall hydrogen-production rate was successfully estimated.

Transport of monocarboxylic acids at the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells.

The kinetics and mechanism of the transport of monocarboxylic acids (MCAs) were studied by using primary cultured bovine brain capillary endothelial cells. Concentration-dependent uptake of acetic acid was observed, and the kinetic parameters were estimated as follows: the Michaelis constant, Kt, was 3.41 +/- 1.87 mM, the maximum uptake rate, Jmax, was 144.7 +/- 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 +/- 1.98 microliters/mg of protein/min. At medium pH below 7.0, the uptake rate of [3H]acetic acid increased markedly with decreasing medium pH, whereas pH-independent uptake was observed in the presence of 10 mM acetic acid. An energy requirement for [3H]acetic acid uptake was also demonstrated, because metabolic inhibitors (2,4-dinitrophenol and rotenone) reduced significantly the uptake rate (P less than .05). Carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, a protonophore, inhibited significantly the uptake of [3H]acetic acid at medium pH of 5.0 and 6.0, whereas 4,4'-diisothiocyanostilben-2,2'-disulfonic acid did not. Several MCAs inhibited significantly the uptake rate of [3H]acetic acid, whereas di- and tricarboxylic acids did not. The uptake of [3H]acetic acid was competitively inhibited by salicylic acid, with an inhibition constant, Ki, of 3.60 mM, suggesting a common transport system between acetic acid and salicylic acid. Moreover, at the medium pH of 7.4, salicylic acid and valproic acid inhibited significantly the uptake of [3H]acetic acid, demonstrating that the transport of MCA drugs could also be ascribed to the MCA transport system at the physiologic pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectral properties of nitric oxide complex of cytochrome c' from Rhodopseudomonas capsulata B100.

The spectral properties for NO complexes of ferric and ferrous cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata B100 are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the NO complexes of the other cytochromes c' and horse heart cytochrome c. The NO-ferrous cytochrome c' would be a mixture of NO complexes with six- and five-coordinate nitrosylheme, suggesting that the heme-iron to histidine bond in the ferrous cytochrome c' is more stable than that from chemoheterotrophic bacteria. The reaction product of ferric cytochrome c' with NO exhibited the spectra similar to NO-ferric derivatives of the other hemoproteins, which indicates the formation of NO-ferric cytochrome c'.

Spectral properties of cytochrome c' from Rhodopseudomonas capsulata B100 and its CO complex.

The spectral properties of cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata (= Rhodobacter capsulatus) B100 and its CO complex are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the other cytochromes c' and horse heart cytochrome c. EPR and electronic spectral results for the ferric cytochrome c' suggest that the ground state of heme-iron(III) at neutral pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state and that at pH 11.0 is in a high-spin state. In the MCD spectrum of the CO-ferrous cytochrome c', the MCD intensity in the Soret band region was much higher than that of CO complexes of hemoproteins with a protoheme. The differences in a stereochemistry of the sixth-coordination position is discussed.

Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells.

The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [125I]ebiratide (H-Met(O2)-Glu[125I]His-Phe-D-Lys-Phe-NH(CH2)2NH2), was examined in cultured monolayers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [125I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [125I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [125I]ebiratide. The acid-resistant binding of [125I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM-1 mM). The maximal internalization capacity (Bmax) at 30 min was 7.96 +/- 3.27 pmol/mg of protein with a half-saturation constant (Kd) of 15.9 +/- 6.4 microM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.

Enantioselective high-performance liquid chromatographic assay for determination of the enantiomers of a new anti-ulcer agent, E3810, in beagle dog plasma and rat plasma.

An enantioselective high-performance liquid chromatographic method for the determination of E3810, a new anti-ulcer agent, in Beagle dog plasma and rat plasma has been developed. After extraction from plasma with ethyl acetate, E3810 enantiomers were measured by reversed-phase high-performance liquid chromatography on a Chiralcel OD-R column. The enantiomers were detected by ultraviolet absorbance detection at 290 nm. The recoveries of E3810 enantiomers and internal standard were greater than 91%. The calibration curves were linear from 0.03 to 20 micrograms/ml for Beagle dog plasma and from 0.1 to 100 micrograms/ml for rat plasma. The limits of quantification of both enantiomers were 0.03 micrograms/ml for Beagle dog plasma and 0.1 micrograms/ml for rat plasma. The intra- and inter-day accuracy and precision data showed good reproducibility of the method. The assay was applied for the analysis of E3810 enantiomers in plasma after intravenous administration of racemic E3810 to Beagle dogs and rats. This method should be very useful for enantioselective pharmacokinetic studies of E3810.

Effects of Ethanolamine as a nitrogen source on hydrogen production by Rhodobacter capsulatus.

Ethanolamine was examined as a nitrogen source in the production of hydrogen by Rhodobacter capsulatus ST-410, a hydrogenase-deficient mutant of the strain B-100. It was found that ethanolamine supports cell growth as the sole nitrogen source and permits a large amount of hydrogen evolution, detected at 138 micromol/ml-culture from 3.5 mM ethanolamine and 30 mM DL-malate. The amount corresponded to a stoichiometric yield of 77% and was close to that obtained from 7.0 mM L-glutamate and 30 mM DL-malate. The hydrogen evolution rate per unit biomass (cells) was higher than that with L-glutamate, and the cells grown with ethanolamine had higher nitrogenase activity than the cells grown with L-glutamate. In terms of bioconversion of cellulosic and hemicellulosic biomass to hydrogen, D-glucose, D-xylose, and D-cellobiose were tested as substrates. The results indicated that those sugars permit a large evolution of hydrogen through cultivation with ethanolamine as a nitrogen source. For instance, the cells grown with 3.5 mM ethanolamine evolved hydrogen of 289 micromol/ml-culture (80% yield) from 30 mM D-glucose under a controlled pH of 6.4 to 6.9.