An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates.

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at -130 degrees C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.

Kyotorphin synthetase activity in rat adrenal glands and spinal cord.

Kyotorphin, an endogenous [Met5]enkephalin-releasing antinociceptive dipeptide (L-Tyr-L-Arg), is formed by kyotorphin synthetase from its constituent amino acids, L-Tyr and L-Arg, in the brain in an ATP-Mg(2+)-dependent manner. To elucidate the physiological role of kyotorphin in organs other than the brain, we examined the activity of kyotorphin synthetase in the rat adrenal glands and spinal cord. By Sephacryl S-300 gel-filtration chromatography of the soluble extracts from both the organs, the enzyme activity forming immunoreactive kyotorphin from L-Tyr and L-Arg in the presence of ATP and MgCl2 was detected in the fractions with the molecular mass of 200-300 kDa, being drastically reduced by the omission of ATP and MgCl2 from the reaction medium. The Km values of the partially purified adrenal and spinal kyotorphin synthetase for L-Tyr, L-Arg, ATP, and MgCl2 were close to those of the brain enzyme. The activity of adrenal kyotorphin synthetase was inhibited by some L-Arg analogues. NG-nitro-L-arginine methyl ester, alpha-methyl-L-ornithine and D-Arg, but not by NG-nitro-L-arginine and N-iminoethyl-L-ornithine. In the crude soluble extracts from the adrenal glands and spinal cord, kyotorphin was formed by kyotorphin synthetase, and also by the enzymatic processing of the precursor proteins, in the presence of physiological concentrations of L-Tyr and L-Arg in addition to ATP and MgCl2. Thus, kyotorphin synthetase resembling that in the brain is present in the rat adrenal glands and spinal cord. The present findings may predict a functional role of the L-Arg-kyotorphin pathway in these organs.

NG-nitro-L-arginine methyl ester and alpha-methyl-L-ornithine inhibit kyotorphin synthetase from rat brain.

Kyotorphin (KTP), an antinociceptive dipeptide (Tyr-Arg), is formed by KTP synthetase from L-Tyr and L-Arg in the brain. We examined the effects of various L-Arg analogues on immunoreactive KTP (iKTP) formation by KTP synthetase purified partially from rat brain. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), but not NG-nitro-L-arginine and N-iminoethyl-L-ornithine, suppressed iKTP formation by KTP synthetase from 1 mM of L-Arg and L-Tyr, the IC50 value being 2.33 mM. Similarly, alpha-methyl-L-ornithine (alpha-MO) inhibited KTP synthetase, the IC50 value being 2.51 mM. D-Arg at high concentrations also exhibited a weak inhibitory effect. Kinetic experiments indicated that the inhibition by L-NAME and alpha-MO of KTP synthetase is competitive. Thus, these L-Arg analogues appear to act as the competitive inhibitor of KTP synthetase.

Light dose and time dependency of photodynamic cell membrane damage.

We have investigated the light dose and time dependency of photodynamic cell membrane damage using electrophysiological methods. This study controls the level of cell membrane damage by precisely administration of the light dose. The photosensitizer used was 5',5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride (BAT). A confocal laser scanning microscope was used to provide rapid light activation (< 1 s) and the subsequent membrane damage was monitored using standard patch clamp techniques. In the presence of 49 microM BAT, light levels less than 0.94 J/cm2 led to a reversible depolarization (approximately 20 mV) and reduction of resistance (approximately 10%) within 3 s of illumination. Higher intensities of illumination (> 1.57 J/cm2) caused a complete and irreversible loss of membrane potential and cell membrane resistance within 8 s illumination. The threshold dose of light required to induce cell death by illumination in the presence of BAT was increased in the presence of the antioxidant Trolox-C.

Phototoxic process after rapid photosensitive membrane damage of 5,5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride.

We report a new aspect of rapid (<30 s) light-induced cell membrane damage photosensitized by 5,5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride (BAT), which is a water-soluble alpha-terthienyl analogue, using a high-power laser (light intensity 1.6 W cm(-2)). In this paper, we will discuss the relationship between the exposure time of the cells to the photosensitizer and the phototoxic process. Three toxic processes can be identified: first, a non-light-mediated toxicity dependent on BAT-cell incubation; second, a phototoxicity independent of BAT exposure time when the BAT concentration is in the 2-10-microM range; third, a phototoxicity dependent on BAT exposure time when BAT concentration becomes 20 microM. The cytotoxicity decreases when alpha-tocopherol, an antioxidant, is added to a cell membrane. This pattern of phototoxicity is the typical of a phospholipid peroxidation chain reaction and oxidative damage of membrane proteins triggered by a reactive oxygen species generated by a triplet state of BAT. The BAT exposure time is clearly correlated with the partition of the photosensitizer in the cell membrane and inside the cell.

[Pulmonary infarction diagnosed by transbronchial lung biopsy].

A 43-year-old man complained of chest pain, fever, and hemosputum in February 1997. Chest X-ray films revealed small opacity in the right lower lung field. The patient received therapy for pneumonia and his condition gradually improved. However, on March 21 he was admitted to our hospital with worsened chest pain and radiographic findings. A transbronchial lung biopsy revealed thrombus and necrosis, thus yielding a diagnosis of pulmonary infarction. The patient did not exhibit any underlying disease or coagulation abnormalities. Treatment with ticlopidine resulted in a favorable course. This was a rare case of pulmonary infarction in which TBLB findings led to the diagnosis.

[Four cases of cancer of unknown primary site that manifested as mediastinal metastatic lesions].

We encountered 4 patients with cancers of unknown primary sites that were manifested by mediastinal lesions. Patient 1 was a 58-year-old man with enlarged superior mediastinal lymph nodes. An exploratory thoracotomy yielded a diagnosis of lymph node metastasis of poorly differentiated adenocarcinoma. The patient was treated with chemotherapy and radiation therapy. Patient 2 was a 68-year-old man with a tumor in the right superior mediastinum. A total resection of the tumor was performed through a thoracotomy. The diagnosis was lymph node metastasis of squamous cell carcinoma, and treatment consisted of irradiation. A tumor shadow in the right upper lobe appeared 14 months after the thoracotomy, and was considered to be a primary lesion requiring a right pneumonectomy. The patient died of hepatic metastasis 6 months after the second operation. Patient 3 was a 59-year-old man with mediastinal and hilar lymph node swelling. Mediastinoscopic findings resulted in a diagnosis of squamous cell carcinoma. Because of the patient's insistence, only radiation therapy was performed. Patient 4 was a 65-year-old woman with a tumor in the right superior mediastinum who underwent a median sternotomy for total resection of the tumor. The pathological findings were strongly suggestive of metastasis of clear cell carcinoma. Patients 1, 3, and 4 were alive 33, 24, and 51 months, respectively, after their initial operation, without detectable primary sites. Patient 2 was considered to have had T 0 N 2 lung cancer.

Thin-film glucose biosensor based on plasma-polymerized film: simple design for mass production.

We propose a simple thin-film glucose biosensor based on a plasma-polymerized film. The film is deposited directly onto the substrate under dry conditions. The resulting films are extreme thin, adhere well onto the substrate (electrode), and have a highly cross-linked network structure and functional groups, such as amino groups, which enable a large amount of enzyme to be immobilized. Since this design allows fabrication through a dry process, with the exception of the enzyme immobilization, which is the last stage of the process, the chip fabrication can be designed as a full-wafer process to achieve mass production compatibility. The resulting sensors produced using this film are more reproducible, exhibit lower noise, and reduce the effect of interference to a greater degree than sensors made using conventional immobilization methods, e.g., via 3-(aminopropyl)triethoxysilane. The obtained film is a good interfacial design between enzyme and electrode; enzyme two-dimensionally locates very close to the electrode in a manner that is quite reproducible. Therefore, a wide dynamic range (up to 60 mM) and rapid response time (11.5+/-0.8 s) were obtained. Because of its highly cross-linking network structure, the amperometric response due to interferences such as ascorbic acid and acetaminophen was reduced by size discrimination of plasma-polymerized films.

A novel method of immobilizing antibodies on a quartz crystal microbalance using plasma-polymerized films for immunosensors.

A novel method of immobilizing antibodies on quartz crystals for use in immunosensors was developed using an ethylenediamine plasma-polymerized film matrix. The films formed on the quartz crystals are extremely thin and homogeneous, and they incorporate amino groups. Sensors produced using this method are more reproducible from sample to sample and exhibit lower noise and higher sensitivity than sensors made using conventional immobilization methods, e.g., via polyethylenimine and (gamma-aminopropyl)trimethoxysilane. This is, to our knowledge, the first reported application of plasma-polymerized films to quartz crystal microbalance immunosensors. Results on orientation-controlled immobilization of anti-bodies, reusability and calibration tests are also presented.

Application of polymer-embedded proteins to fabrication of DNA array.

A plasma-polymerized film (PPF) of hexamethyldisiloxane [HMDS; (CH(3))(3)SiOSi(CH(3))(3)] was used to immobilize streptavidin on a glass substrate. Another layer of HMDS-PPF was also applied to the protein, which was first adsorbed to an underlayer of the same kind of film. As the result, the streptavidin was "embedded" between the two layers of HMDS, whereby biotinylated molecules could be efficiently captured. The second layer of approximately 30 to 45 A PPF was sufficient to allow the binding of biotinylated molecules, whereas thicknesses of >90 A significantly hindered the streptavidin-biotin interactions. Fluorescence analysis revealed that the absence of an HMDS plasma-polymer (HMDS-PP) layer on either side of the streptavidin film resulted in a decrease in biotin binding. This immobilization technique was used to bind biotinylated oligonucleotides in sequence-specific DNA-DNA interactions. The hydrophobic properties of the plasma-polymerized HMDS thin film acted to minimize nonspecific DNA binding to the glass substrate. A DNA array was fabricated using this procedure and showed greatly decreased nonspecific DNA binding compared with a poly-L-lysine coated substrate.

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques.

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.

Evidence for axial coordination of 5,6-dimethylbenzimidazole to the cobalt atom of adenosylcobalamin bound to diol dehydratase.

It was demonstrated by electron paramagnetic resonance (EPR) spectroscopy that organic radical intermediates disappeared and cob(II)alamin accumulated upon suicide inactivation of diol dehydratase by 2-methyl-1,2-propanediol. The resulting EPR spectra showed that the eight hyperfine lines due to the divalent cobalt atom of cob(II)alamin further split into triplets by the superhyperfine coupling to the 14N nucleus. Essentially the same superhyperfine splitting of the octet into triplets was observed with [14N]- and [15N]apoenzyme. When the adenosyl form of [14N2]- and [15N2]imidazolyl analogues of the coenzyme [Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437] was used with unlabeled apoenzyme, the octet showed superhyperfine splitting into triplets and doublets, respectively. Therefore, it was concluded that cobalamin is bound to this enzyme with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. This conclusion is consistent with the fact that the consensus sequence forming part of a cobalamin-binding motif, conserved in methionine synthase and some of the other cobalamin enzymes, was not found in the deduced amino acid sequences of the subunits of diol dehydratase. Adenosylcobinamide methyl phosphate, a coenzyme analogue lacking the nucleotide moiety, underwent cleavage of the cobalt-carbon bond upon binding to the enzyme in the presence of substrate, forming a cob(II)inamide derivative without nitrogenous base coordination, as judged by EPR and optical spectroscopy. Therefore, this analogue may be a useful probe for determining whether the replacement of the 5, 6-dimethylbenzimidazole ligand by a histidine residue takes place upon binding of cobalamin to proteins.