[Nifekalant hydrochloride as an effective treatment for postoperative ischemic heart disease].

We experienced 2 effective cases of nifekalant hydrochloride. One patient was 76-year-old female who underwent emergent coronary artery bypass grafting (CABG) because of unstable angina pectoris (AP) and ventricular fibrillation (Vf). Her cardiac function had been decreased preoperatively due to old myocardial infarction (OMI). One day after CABG, she revealed sustained ventricular tachycardia (VT) and Vf. Although administrations of neither lidocaine hydrochloride nor magnesium sulfate were effective, nifekalant hydrochloride finally stopped the life-threatening arrhythmia without hypotension. Another patient was 77-year-old male who underwent CABG and Dor operation. His cardiac function also had been decreased due to OMI. He revealed VT attack at midnight 3 days after operation. VT attack still appeared at next 2 midnight under lidocaine hydrochloride infusion, but finally it has disappeared after starting a drip infusion of nifekalant hydrochloride. Nifekalant hydrochloride is quite useful as a new therapeutic strategy for uncontrollable VT and Vf and for the patient who has a reduced left ventricular function because it has an inotropic effect.

[Observation of the right atrial wall suture line after tricuspid valve supra-annular implantation].

Strength of the right atrial wall suture line after tricuspid valve supra-annular implantation (TVSI) is controversial. We observed the right atrial supra-annular position of a 63-year-old male during his third mitral operation who underwent mitral valve replacement (MVR) and TVSI 15 years ago. Eight years later, he received the second MVR and removal of the bioprosthetic valve from the tricuspid position due to primary tissue failures. The annular size of the tricuspid valve had decreased enough to be fixed by tricuspid annuloplasty (TAP) and re-TVSI was not needed at that time. In this operation, 7 years following bioprosthetic valve removal, the circularly bulging atrial wall still remained and seemed to have enough strength for holding the prosthetic valve. This finding may support the conclusion that the right atrial wall has enouth strength for holding a prosthetic valve in position.

[Surgical treatment for left ventricular false aneurysm caused by infective endocarditis; report of a case].

A 24-year-old man who had left ventricular (LV) false aneurysm, which is caused by mitral valve infective endocarditis, underwent aneurysmectomy, direct closure of aneurysmal mouth and concomitant mitral valve replacement. Post-operative course was uneventful. It has been reported that the etiology of this false aneurysm was due to the vegetations' friction, which could have caused an initial endocardial ulceration that progressively expanded into the myocardium. The false aneurysmal wall should be completely removed in order to eliminate the possibility of recurrence of the infective endocarditis. We believe that the surgical treatment should be carried out as soon as possible after completion of diagnosis because the aneurysmal wall is usually quite thin and could rupture easily.

[Prosthetic valve endocarditis after aortic valve replacement with freestyle stentless xenograft].

A 52-year-old man underwent aortic valve replacement with freestyle stentless xenograft, using subcoronary technique for active infective endocarditis in June, 2001. Eighteen month later he had late prosthetic valve endocarditis associated with aortic annular abscess due to Staphylococcus epidermidis infection. The abscess was debrided and gelatin-resorcin-formalin glue (GRF glue) was injected into the abscess cavity. Abscess cavity was closed with continuous running suture of 3-0 polypropylene stitches. Finally the aortic valve was replaced with ATS mechanical valve (20 mmAP). After administration of vancomycin and gentamicin for 4 weeks, he discharged on 57th postoperative day in good condition. We strongly suggest that GRF glue is essential to close the aortic annular abscess of combined with aortic regurgitation due to active infective endocarditis.

Ascorbate-induced high-affinity binding of copper to cytosolic proteins.

Copper chaperones are necessary for intracellular trafficking of copper to target proteins. This is probably because the milieu inside the cell has a large capacity for sequestering this metal. By fluorometry using a fluorescent Cu(II) chelator and by centrifugal ultrafiltration, we have studied copper binding of the whole cytosolic proteins from mouse brain and liver, and found that their binding capacity and affinity for copper were markedly increased by ascorbate. Brain cytosolic protein bound, with high affinity, 63 nmol of copper/mg, more than half of which was redox-inactive, as indicated by its inability to catalyze oxidation of ascorbate. Most of the bound copper was in the Cu(I) state, coordinating to thiol groups of protein. Cytosolic protein competed for copper more strongly than GSH when compared at their relative concentrations in tissues. The results taken together suggest that protein thiols of cytosol can strongly sequester copper.

A helical arrangement of beta-substituents of dehydropeptides: synthesis and conformational study of sequential nona- and dodecapeptides possessing (Z)-beta-(1-naphthyl)dehydroalanine residues.

Sequential nona- and dodecapeptides possessing three and four (Z)-beta -(1-naphthyl)dehydroalanine (Delta(Z)Nap) residues, Boc-(L-Ala-Delta(Z)Nap-L-Leu)(n)-OCH(3) (n = 3 and 4; Boc = t-butoxycarbonyl), were synthesized to design a rigid 3(10)-helical backbone for a regular arrangement of functional groups using dehydropeptides. Their solution conformations were investigated by NMR and CD analyses, and theoretical energy calculations. Both peptides were found to adopt a 3(10)-helical conformation in CDCl(3) from their nuclear Overhauser effect spectroscopy (NOESY) spectra, which showed intense cross peaks for N(i)H-N(i+1)H proton pairs, but no cross peaks for C(alpha)(i)H-N(i+4)H pairs. The predominance of a 3(10)-helix was also supported by solvent accessibility of NH resonances. CD spectra of both peptides in tetrahydrofuran showed strong exciton couplets at around 228 nm assignable to naphthyl side chains, which are regularly arranged along a right-handed helical backbone. Chain-length effects on conformational preference in sequential peptide -(Ala-Delta(Z)Nap-Leu)(n)- were discussed based on spectroscopic analysis, energy minimization, and molecular dynamics simulations. Consequently, the repeating number n > or = 3 forms predominantly a right-handed 3(10)-helical conformation. The energy calculation also revealed that the midpoint naphthyl groups of peptide n = 4 are highly restricted to one stable orientation. In conclusion, beta-substituted alpha,beta-dehydroalanine is expected to be a unique tool for designing a rigid molecular frame of 3(10)-helix along which beta-functional groups are regularly arranged in a specific manner.

Structural and conformational properties of (Z)-beta-(1-naphthyl)- dehydroalanine residue.

To understand how chemical structure of beta-substituted alpha, beta-dehydroalanine (particularly size and pi conjugation of beta substituent) affects conformational property, x-ray crystallographic analysis was performed on Boc-Ala-Delta(Z) Nap-Val-OMe [Boc: t-butoxycarbonyl; Delta(Z) Nap: (Z)-beta-(1-naphthyl)dehydroalanine; OMe: methoxy] having the naphthyl group as a bulky beta substituent. Single crystals were grown by slow evaporation from an ethanol solution in the triclinic space group P1 with a = 9.528 (3) A, b = 12.410(4) A, c = 5.975(2) A, alpha = 96.77(3) degrees, beta = 102. 81(2) degrees, gamma = 88.74(3) degrees, V = 684.1(4) A3, and Z = 1. Phase determination was carried out by a direct method (SHELEXS), and the final structure was refined to R = 8.1% and R(w) = 9.0% for 1964 observed reflections. The bond lengths and bond angles of the Delta(Z)Nap residue, characterized by a sp(2) hybridized C(alpha) atom, did not differ from those of other dehydroresidues such as Delta(Z) Phe, Delta(Z) Leu, and DeltaVal essentially. The peptide backbone took a type II beta-turn conformation involving an intramolecular hydrogen bond between CO(Boc) and NH(Val), similar to di- or tripeptides containing a Delta(Z) Phe or Delta(Z) Leu residue in the second positions. Here the naphthyl group was found to be nonplanar [chi(2) = 55(1) degrees ] relative to the C(alpha)==C(beta)==C(gamma) plane. The nonplanarity was supported by conformational energy calculation. The molecular packing was stabilized by two kinds of intermolecular hydrogen bonds and van der Waals interactions. Naphthyl groups were arranged in a partially overlapped face-to-face orientation with a center-to-center distance of 5.97 A. For additional information, peptide Boc-(Ala-Delta(Z) Nap-Leu)(2)-OMe was synthesized and its solution conformation was investigated by (1)H-NMR spectroscopy. The hexapeptide showed the tendency to form a 3(10)-helical conformation in solution essentially. Conformational properties of Delta(Z) Nap residue, characterized by a type II beta-turn and 3(10)-helix, were supported by a conformational energy contour map of the Delta(Z)Nap residue.

Effects of a magnetic fields on the various functions of subcellular organelles and cells.

Magnetic fields (MF) are widely distributed in environment and their effects are increasing by the development of electrical machines. Several investigators reported that the MF might affect various functions of cells. However, an acceptable hypothesis has not yet been proposed. Thus, we studied the effects of weak MFs on various biological functions of cells, such as mitochondrial functions, stimulation dependent signal transduction of neutrophils, cell growth and transformation of HL-60 cells, H(2)O(2)-induced apoptosis and the expression of apoptotic genes in HL-60 cells. As a result of the study, a weak MF has scarcely any effects on various biological functions of cells. We also studied the direct effect of a static strong MF (SSMF, 600-2000 G) on the functions of cells or on Fe(2+)-induced lipid peroxidation and on reactive oxygen species (ROS) generation in oral polymorphonuclear leukocytes (OPMN) without stimulation using Ferrite magnets. The generation of ROS from OPMN was slightly inhibited but Fe(2+)-induced lipid peroxidation of biological membrane was slightly stimulated by exposure to the SSMF. At present, however, conclusive results have been neither obtained experimentally nor any acceptable idea proposed.

Direct temperature-controlled trapping system and its use for the gas chromatographic determination of organic vapor released from human skin.

For controlling of trap temperature, the relationship between electric resistance of the trap tube and temperature is used. As the electric resistance of the trap tube (20 cm long stainless steel tubing) was very small, such as ca. 0.040 ohm for -70 degrees C and ca. 0.064 ohm for +90 degrees C, it was estimated by using the value of voltage output at both ends of the trap tube when a direct current (5 A) was applied for 6.5 ms at every 100 ms on the trap. By using this temperature measurement, a cycle of trapping is shortened, especially at the process of desorption, because it is possible to set a large increasing rate of temperature, such as 20 degrees C/s. The present trapping system has faster temperature response compared to that with a thermocouple. This system was applied for the study of the releasing of ethanol and water vapors from the human finger, which was treated as follows: dipping in 10% ethanol aqueous solution for 1 min, followed by washing with water and then drying in the air. In this case, a cycle of trapping took 53 s, and the period of total analysis was only 3 min. The present system is an efficient tool for the study of the exhalation of organic vapors from human skin.

Inactivation of wild-type BCR/ABL tyrosine kinase in hematopoietic cells by mild hyperthermia.

Temperature-sensitive mutants of BCR/ABL tyrosine kinase have been extensively used to study the mechanisms of cell transformation and signal transduction. However, little is known about the effect of temperature on the activity of wild-type BCR/ABL gene product. In this study, we demonstrate that in vivo tyrosine kinase activity of p210, p190 BCR/ABL and v-abl are temperature-sensitive when expressed in hematopoietic cells and decline when temperature is raised 2 degrees C above normal range. In vitro tyrosine kinase activities of purified recombinant Abl and immunoprecipitated p210 BCR/ABL were also sensitive to increased temperature. Tyrosine phosphorylation of cellular proteins was markedly reduced in BCR/ABL transformed cells after 16 h at 39 degrees C, whereas the expression of BCR/ABL was unchanged. Temperature-induced downregulation of BCR/ABL kinase activity was reversible when cells were shifted back to 37 degrees C. The downregulation of Abl tyrosine kinase activity was not influenced by mutation or deletion of SH2 or SH3 domains or mutation of the GRB2 binding site. No increase in functional activity or expression of protein-tyrosine phosphatases, PTP-1B, SH-PTP1 or SH-PTP2 was detected in cells grown at 39 degrees C. Temperature-induced downregulation in tyrosine kinase activity correlated with decline in phosphotyrosine-associated PI 3-kinase whereas there was no change in growth factor independence of transformed hematopoietic cells. In conclusion, Abl tyrosine kinase has intrinsic sensitivity to temperature and BCR/ABL expressed in hematopoietic cells is downregulated by increasing temperature 2 degrees C. These observations provide a unique opportunity to identify cellular factor(s) which regulate BCR/ABL kinase in vivo and suggests possible novel treatment of CML by a mild hyperthermia.

Synthesis of delta(E)Phe-containing tripeptide via photoisomerization and its conformation in solution.

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.

Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius.

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus. Thus, the intermedilysin gene is useful as a marker gene of S. intermedius. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius.

[Progressive mitral regurgitation which is necessitated the mitral valve replacement after partial left ventriculectomy (Batista procedure): a case report].

The patient is 61-year-old woman who underwent partial left ventriculectomy, (Batista procedure) due to dilated cardiomyopathy and multiple thromboembolism. Although postoperative course was uneventful, she has had clinical symptoms of the left heart failure due to the increased mitral valve regurgitation at the early postoperative period, gradually. Even though mitral valve regurgitation was severe, it was not apt to re-dilatate the left ventricular capacity evaluated by echocardiography. She underwent the mitral valve replacement on the 92nd postoperative day, and was once possible for weaning from cardiopulmonary bypass under the support of IABP. However, she died on the 19th postoperative day caused by sepsis. It is important to evaluate the accurate mitral valve regurgitation preoperatively for Batista procedure. Although there was the mild mitral valve regurgitation, it is essential to repair or replace the mitral valve for Batista procedure.

VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.

Vascular endothelial growth factor (VEGF) has been shown to promote neovascularization in animal models and, more recently, in human subjects. This feature has been assumed to result exclusively from its direct effects on fully differentiated endothelial cells, i.e. angiogenesis. Given its regulatory role in both angiogenesis and vasculogenesis during fetal development, we investigated the hypothesis that VEGF may modulate endothelial progenitor cell (EPC) kinetics for postnatal neovascularization. Indeed, we observed an increase in circulating EPCs following VEGF administration in vivo. VEGF-induced mobilization of bone marrow-derived EPCs resulted in increased differentiated EPCs in vitro and augmented corneal neovascularization in vivo. These findings thus establish a novel role for VEGF in postnatal neovascularization which complements its known impact on angiogenesis.

Expression and activity of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase in different regions of the avian brain.

Recently, we have demonstrated, using biochemical and immunochemical methods, that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and produces pregnenolone and its sulfate ester. To clarify progesterone biosynthesis in the avian brain, therefore, we examined the expression of messenger RNA (mRNA) encoding for the enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) and its enzymatic activity using the quail. RT-PCR analysis together with Southern hybridization indicated the expression of 3beta-HSD mRNA in the brain of sexually mature birds but with no clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of pregnenolone to progesterone was found in brain slices of mature males. Progesterone biosynthesis was increased in a time dependent manner and completely abolished by trilostane, a specific inhibitor of 3beta-HSD. The enzymatic activity of 3beta-HSD was greatest in the cerebrum and lowest in the mesencephalon. A specific RIA indicated that progesterone concentrations in the different brain regions closely followed the level of 3beta-HSD activity. High levels of progesterone concentration were observed in the diencephalon and cerebrum with lowest values in the mesencephalon. Progesterone levels in the brain regions were significantly higher than those in the plasma. These results suggest that the avian brain possesses not only cytochrome P450scc but also 3beta-HSD and produces progesterone. It is also indicated that progesterone biosynthesis in the avian brain may be region-dependent.

Mitochondrial dysfunction in the senescence accelerated mouse (SAM).

Oxidative damage to DNA, proteins, and lipids in mitochondria caused by free radicals may be one factor in aging. Oxidative phosphorylation was estimated in liver mitochondria from senescence accelerated mice (SAMP8) and a senescence resistant substrain (SAMR1). The respiratory control ratio decreased in liver mitochondria of SAMP8 during aging, and it was estimated that at 18 months of age this respiratory control value suggested that it might be insufficient to provide ATP synthesis necessary for normal cell metabolism. In addition, the ADP/O, an index of efficiency of ATP synthesis, was depressed at 18 months of age. Dinitrophenol-dependent uncoupled respiration in liver mitochondria of SAMP8 mice was markedly decreased with aging, suggesting a dysfunctional energy transfer mechanism in mitochondria of aged SAMP8 mice. Active uptake of calcium in liver mitochondria was markedly dysfunctional in SAMP8 mice with aging, and uncoupling of respiration was induced more easily in aged mitochondria. Milder effects on these functional parameters were observed in SAMR1 mice. A similar dysfunction was also observed in heart mitochondria of SAMP8 mice at 12 months of age. The amount of Bcl-x in liver mitochondria was slightly decreased in SAMP8. We suggest that these changes in mitochondrial function may be related to the shorter life span of the senescence accelerated mouse.

Molecular mechanisms of apoptosis in HL-60 cells induced by a nitric oxide-releasing compound.

Nitric oxide (NO) generated from 1-hydroxy-2-oxo-3, 3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-1 beta converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.

Oxygen-dependent fragmentation of cellular DNA by nitric oxide.

Although active oxygen species and related metabolites, such as nitric oxide (NO), have been postulated to play important roles in the apoptosis of various cells, a precise mechanism leading to cell death remains to be elucidated. Recently we found that the lifetime of NO depends greatly on the concentration of environmental oxygen and that NO reversibly inhibits mitochondrial respiration and ATP synthesis; the inhibitory effect is stronger at physiologically low oxygen tension than under atmospheric conditions (Arch. Biochem. Biophys. 323, 27-32, 1995). The present work describes the effects of the NO-generating agent, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18) and oxygen tension on the respiration, ATP synthesis and apoptosis of HL-60 cells. When respiration was inhibited by NOC 18, cellular ATP levels decreased significantly and DNA fragmentation was elicited. Both events were enhanced by decreasing oxygen tension and suppressed by adding NO-trapping agents, such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) and oxyhemoglobin. The fragmentation of cellular DNA was inhibited in a dose dependent manner by herbimycin A, a tyrosine kinase inhibitor. Fragmentation of the DNA of HL-60 cells was also induced either by peroxynitrite, superoxide or hydroxyl radical by some mechanism which was diminished by lowering the oxygen tension. These results indicated that the decrease in cellular ATP and activation of tyrosine kinase might play important roles in NO-induced apoptosis particularly under physiologically low oxygen tensions.

Valinomycin induces apoptosis of ascites hepatoma cells (AH-130) in relation to mitochondrial membrane potential.

Valinomycin is a potassium ionophore, and is well known to cause the collapse of the mitochondrial membrane potential. It has been reported that loss of mitochondrial membrane potential is observed in the early stages of apoptosis induced by various agents. Thus, the effects of valinomycin on tumor cells were examined. Valinomycin induced uncoupling of respiration and depolarization of isolated mitochondria. Depolarization of intact mitochondria in AH-130 rat ascites hepatoma cells was also induced by valinomycin. Valinomycin induced apoptosis revealing the typical apoptotic characteristics such as fragmentation and ladder formation of DNA, shrinkage of cells, and formation of pycnotic nucleus. There was a correlation between the depolarization of mitochondria and DNA fragmentation. After depolarization of mitochondria, the activity of caspase-3-like protease but not caspase-1-like protease increased markedly. In contrast, this apoptosis did not involve the release of reactive oxygen species from mitochondria, increase in intracellular calcium concentration, or protein synthesis. In addition, anti-apoptotic members of the Bcl-2 family (Bcl-xL and Bcl-2) were not correlated with apoptosis. These results indicate that valinomycin might induce apoptosis through degradation of the mitochondrial membrane potential. Taken together, these observations suggest that there may be a mechanism that transmits the signal from mitochondrial depolarization to subsequent apoptosis execution steps.

Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide.

Effects of nitric oxide (NO) and NO generating agents, on the electron transport system of mitochondria were examined in a study of the mechanism and physiological importance of NO in energy metabolism. In the presence of various substrates, uncoupled respiration was inhibited by NO in manner which was both dose- and oxygen tension-dependent. Simultaneously measuring changes in cytochrome absorption spectra and respiration showed that the site of action of NO is cytochrome oxidase. Similar inhibition was also brought about by 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO donor. Electron paramagnetic resonance (EPR) analysis revealed that inhibition of uncoupled respiration occurred only during the presence of NO in the reaction mixture. The inhibitory effect of NO was increased significantly by lowering the concentration of mitochondrial protein. No appreciable inhibition of respiration was observed in the presence of 3-morpholinosydnonimine (SIN-1), a peroxynitrite anion (ONOO-) generating reagent, but inhibition did occur in the presence of superoxide dismutase (SOD). These results indicate that NO reversibly interacts with mitochondria at complex IV thereby inhibiting respiration particularly under physiologically low oxygen tension and that de novo generated ONOO may have no significant effect under the present experimental conditions.