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Summary Background We evaluated changes in the levels of urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) in patients undergoing mild hypothermia therapy and compared 8-OHdG expressions in those receiving whole-body cooling or selective head cooling. Material/Methods The subjects were 15 patients undergoing mild hypothermia therapy following resuscitation after cardiac arrest in our intensive care unit. We divided the patients into 2 groups receiving either whole-body cooling or selective head cooling, according to their circulatory stability. We examined urinary 8-OHdG level for 1 week and neurological outcomes 28 days after admission. Results We observed significant decreases in urinary 8-OHdG levels on days 6 and 7 compared with that on day 1 in the whole-body cooling group. Furthermore, we noted significantly lower urinary 8-OHdG levels after days 5, 6 and 7 in the whole-body cooling group than in the selective head-cooling group. Neurological outcomes were similar in both groups. Conclusions Mild hypothermia therapy with whole-body cooling had a greater effect on the suppression of free radical production than selective head cooling. However, selective head cooling might be an appropriate indication for patients with circulatory instability after resuscitation, because it provides neuroprotection similar to that of whole-body cooling.

Sepsisのなかで臓器障害または重要臓器の灌流障害を伴うsevere sepsisの患者に対し持続的血液濾過透析(continuous hemodiafiltration: CHDF)を行い,補体系に及ぼす影響からその有効性について検討した。対象は当センターICUにおいて3日以上CHDFを施行したsevere sepsisの患者9例で,hemofilterはpolymethyl methacrylate (PMMA)膜を用い,抗凝固剤はメシル酸ナファモスタットを投与した。測定項目は,CH50, C1q, C3プロアクチベーター,C3, C4, C3a, CRP,アルブミンおよびcolloid osmotic pressure (COP)であり,測定はCHDF開始前,開始2, 6, 12時間後と3日後に行った。CHDF開始2週間後の転帰で生存群と死亡群に分け,群内,群間とhemofilterへの脱血側と送血側での変化について各々有意差検定を行った。転帰は生存5例,死亡4例であり,CHDF施行時間は134±36時間(mean±SD)であった。両群ともCHDF開始前C3aは高値を示し,CH50, C3は低値を示した。C3aはCHDF施行中に生存群では有意に低下し死亡群では増加傾向にはあったが,CH50, C3に有意の変化はなかった。CHDFの濾液中にCH50, C3aが検出され,C3aは脱血側に比し送血側では2および6時間後には増加したが12時間後には低下した。CH50には有意の変化はなかった。severe sepsisの患者では補体系のalternative pathwayが活性化されC3aが高値を示した。生存群ではCHDF施行によるC3aの除去が転帰の改善に寄与したと思われ,CHDFは敗血症の治療に有利に作用すると思われた。しかしhemofilter自体が補体を活性化する可能性も示唆された。

This paper describes a fully integrated single-chip CMOS mixed-signal system on a chip (SoC) for DVD player applications. It integrates one digital signal processor (DSP), two 32-bit CPUs, three dedicated processing units, a partial response maximum likelihood (PRML) read channel with an analog front end (AFE), and many other subsystems on the same die. The AFE includes a fifth-order G/sub m/-C filter and attains over 66 dB C/N overall. PR(3,4,4,3) structure is employed in the PRML read channel. Owing to the PRML signal processing and the mixed-signal system level optimization in the PRML read channel, less than 10/sup -6/ of bit-error rate (BER) is obtained for the focus offset margins over /spl plusmn/0.5 /spl mu/m. This SoC is fabricated in 0.13-/spl mu/m one-poly six-Cu CMOS technology. It contains 24 million transistors in a 63.87 mm/sup 2/ die and consumes 1.5 W at 40 MSample/s data rate, which corresponds to DVD 1.5 times playback operation mode.

This paper shows our experimental results for the heating characteristics of magnetic wood. We made a separate heating board and a multiple-ply heating board, to both of which were adhered magnetic wood and an excitation coil. The results showed that the multiple-ply heating board, which was combined with coated magnetic wood and a spiral coil, had superior heating characteristics and heating efficiency.

IAEA transport regulations require the 9 m drop test for type B packages. The target used in this drop test must be unyielding. However, the real target that a transport package might encounter at an accident during transport is a yielding target, such as concrete, asphalt, or soil. To compare the impact acceleration between a real target and an unyielding target, analyses of the drop test onto a real target and an unyielding target were performed by using a LS-DYNA-3D computer code. A road surface is usually concrete or asphalt, and such surfaces are very hard; but there are soil, sand, etc. under these materials, so the impact caused by a drop accident is relatively small. It becomes clear that the drop height onto a road, corresponding to the 9 m drop height required by IAEA regulations is about 50 m, and the impact velocity is about 110 km.s−1

It is important to determine the best way to arrange the fuel assemblies in the casks to reduce the effects of exposure on the operators that work with them. A system for arranging the fuel assemblies in TN-12/2 casks and predicting the dose rates around a cask using a personal computer is described here. For this system a new code, named SOURCE, has been developed to calculate the radiation source intensity precisely and quickly. In advance, the detailed shielding calculation for the cask is performed and the data which show the contribution from each fuel assembly location to a certain point around a cask is stored in the prediction system. To verify the accuracy of this prediction system, the measurement data of TN-12/2 casks are compared with the results of the system; fairly good agreements are obtained.

The study of highly effective neutron shields for packaging, which maintain integrity under severe thermal conditions, such as extended use under high temperatures or hypothetical fire conditions, is described. Such neutron shields are of the following four types. (i) Silicone rubber type—a good heat resistant type; because of its good fluidity, it can be poured into complex cases. (ii) Polypropylene type—the lightest type with a higher atomic number density of hydrogen. (iii) Ethylenepropylene rubber type—an intermediate type between the above two types in shielding ability and heat resistance. (iv) Titanium hydride type—the most hydrogenous type. The shielding ability of the above four neutron shields is evaluated. In addition, an extended heat resistance test was carried out, and the maximum allowable temperatures of these shields is evaluated. In conclusion, the neutron shields presented here are appropriate for transport/storage packaging for high burnup fuels.

The relationship between wood construction and DC magnetic characteristics for three types of magnetic wood was experimentally investigated. The results show that the magnetic characteristics of each type of magnetic wood are dependent on the magnetic materials, the density of the magnetic material and the construction of the wood. Furthermore, it was determined that the relationship between the fiber direction and the magnetic path direction of the magnetic wood influenced the wood's magnetic characteristics.

High mobility group box 1 (HMGB-1) has recently received attention as a late mediator of lipopolysaccharide-induced shock, and is thought to function as a mediator in such a disorder as multi-organ failure (MOF). In Japan, we have access to an immobilized polymyxin B fiber column using a direct hemoperfusion (PMX-DHP) for patients with septic shock to improve hemodynamics and organ dysfunction. In this study, we looked at HMGB-1 levels in each category based on the sequential organ failure assessment (SOFA) scores to further dissect its importance in specific aspects of organ failure in patients undergoing PMX-DHP.Sixty patients with septic shock (40 survivors and 20 non-survivors). We analyzed HMGB-1 and IL-6 levels before and after PMX-DHP and defined organ failure as two or more SOFA points.There was a significant positive correlation between SOFA score and HMGB-1 level (P0.05). The HMGB-1 level before PMX-DHP significantly increased as the number of organ failures increased (P0.01: comparing 2 versus 5 organ failures). IL-6 levels decreased after PMX-DHP (P0.05 compared with before PMX-DHP), but HMGB-1 levels remained unchanged. HMGB-1 levels of survivors with organ failure in liver decreased after PMX-DHP, but those of non-survivors significantly increased 24h after PMX-DHP compared with before PMX-DHP (P0.01). In non-survivors with organ failure in liver, HMGB-1 levels were significantly higher than among survivors 24h after PMX-DHP (P0.01).Our results indicate that HMGB-1 is a useful prognostic biomarker in sepsis-induced organ failure in patients undergoing PMX-DHP.

Endotoxin-adsorbing fibers have been applied to treat septic shock patients. The limitations of endotoxin hemoadsorption therapy (PMX-DHP) and the optimal time to start PMX-DHP were examined in patients with septic multiple organ failure with hypercytokinemia (interleukin-6 = 1000 pg/mL). Subjects were separated into those who survived more than 28 days after the start of PMX-DHP therapy (S group) and those who did not (N-S group). Severity of symptoms and background factors, blood biochemical parameters, hemodynamic parameters, PaO(2)/FiO(2), pathogens, endotoxin, cytokines, and vascular endothelial cell function-related markers were examined before and after PMX-DHP. Number of days from onset of shock (or symptom development) to PMX-DHP initiation was longer in the N-S group than in the S group. These results suggest that PMX-DHP could save more lives in patients with septic multiple organ failure with IL-6 = 1000 pg/mL when applied early after the onset of shock.

Summary form only given. Since the introduction of magnetic wood, the authors have proposed various functions such as heating, attraction and electromagnetic wave absorption. This paper aims to clarify, through experimentation, the relationships between magnetic wood construction and their magnetic characteristics. The three types of magnetic wood (an impregnated type, a powder type and a coating type) have differences in their construction ratios of wood parts and magnetic particles. The authors show the experimental results for DC magnetic characteristics of magnetic wood measured by a vibrating sample magnetometer. They then compare the experimental results of the main parameters for DC magnetic characteristics and specific gravity of magnetic wood. The results show that the magnetic characteristics rely heavily on magnetic density and the dispersal of magnetic particles. Among the three types of magnetic wood, the coating type magnetic wood, which has a dense existence of magnetic particles, shows the best results in terms of DC magnetic characteristics.

Previous studies have demonstrated that rats treated with a proteinase inhibitor, leupeptin (Leu), show an accumulation of lipofuscin (Lf)-like dense bodies in the neurons. The present study examines the quantitative and ultrastructural changes in the Lf-like dense bodies of Purkinje (P) cells and Bergmann (B) glia over 20 days following a 4-day regimen of Leu administration by intracisternal infusion. Toluidine blue staining revealed considerable Lf-like granules in both types of cells 1 day after Leu infusion. However, these granules had almost disappeared by 20 days after infusion. Quantitative analysis revealed a decrease in the amount of accumulated Lf-like granules in P cells whereas the granules in B glia increased 10 days after Leu infusion. Electron microscopic examination revealed the presence of Lf-like dense bodies on the plasma membranes of both the P cells and B glia. The present results suggest that Leu-induced dense bodies in P cells are transferred to B glia, indicating a possible route for the removal of Lf.

We evaluated the regional distribution of 62Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM), a potential PET perfusion agent, in the rat brain and observed hyperfixation in transient global ischemia in rats.The distribution of 62Cu-PTSM was examined in comparison with that of 123I-labeled p-iodophenyl-N-isopropylmethanphetamine (123I-IMP) as a reference blood flow marker. Brain uptake of these two tracers was measured in Wistar rats subjected to 30-min four-vessel occlusion followed by recirculation for 10 min, 1 hr or 1, 3 or 5 days. Tracers were injected intravenously into rats 10 min before decapitation. The activities of Complex I and Complex I-III of mitochondria and the concentration of sulfhydryl (SH) groups were also measured.Copper-62-PTSM showed accelerated accumulation in the brain at 1 hr and 1 day after reperfusion when compared with that of 123I-IMP (p0.01), and this enhancement was considered to be due to hyperfixation. At these time points, SH concentration was significantly decreased (p0.01). On the other hand, the activity of Complex I was not influenced by ischemia/reperfusion, but that of Complex I-III was decreased to 65-70% of the control level (p0.01).Copper-62-PTSM showed hyperfixation most possibly as a result of increased NADH concentration, caused by disturbed electron transport in mitochondria.

An ideal hypoxia imaging agent should have high membrane permeability for easy access to intracellular mitochondria and low redox potential to confer stability in normal tissue, but it should be able to be reduced by mitochondria with abnormally high electron concentrations in hypoxic cells. In this context, nitroimidazole residues are not considered to be essential. In this study, Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), a 62Cu-bisthiosemicarbazone complex, with high membrane permeability and low redox potential, was evaluated as a possible hypoxia imaging agent, using electron spin resonance spectrometry and the Langendorff isolated perfused rat heart model as well as rat heart left anterior descending occlusion model.Nonradioactive Cu-ATSM was incubated with rat mitochondria, after which reduction of Cu(II) to Cu(I) was measured with electron spin resonance. As a model of hypoxic mitochondria, rotenone (Complex I inhibitor)-treated mitochondria were used.In this study, Cu-ATSM was reduced by hypoxic but not by normal mitochondria.Thus, retention of 62Cu-ATSM was studied serially in perfused rat hearts under conditions of normoxia (95% O2 + 5% CO2), hypoxia (95% N2 + 5% CO2) and reoxygenation (95% O2 + 5% CO2). In normoxia and reoxygenation, 62Cu-ATSM injected as a single bolus showed low retention (23.77% and 22.80%, respectively) 15 min after injection, but retention was increased markedly under hypoxic conditions (81.10%). Also, in the in vivo left anterior descending occluded rat heart model, 62Cu-ATSM retention was inversely correlated with accumulation of 201Tl, a relative myocardial blood flow marker.

There is substantial evidence to suggest that oxidative stress is associated with cerebral vasospasm following subarachnoid hemorrhage (SAH). Urinary 8-OHdG is the most common biomarker of DNA damage by oxidative stress. The aim of this study was to determine whether 8-OHdG is a good indicator of vasospasm occurrence following SAH.

Summary form only given. Since the introduction of magnetic wood, the authors have proposed various functions such as heating, attraction and electromagnetic wave absorption. The impregnated type magnetic wood, which is manufactured by impregnating magnetic fluid through conduit pipes, has the best woody characteristics among the three types of magnetic woods (impregnated type, powder type and coating type). In spite of the merits, the manufacturing methods up until now have been unsuccessful in producing an impregnated type magnetic wood with good magnetic characteristics, because it is difficult to impregnate a sufficient amount of magnetic fluid into the wood board. The purpose of this paper is to suggest a new impregnating method and report on the experimental results of DC and AC magnetic characteristics. The manufacturing method involves taking a thin layer of film from the wood and impregnating this section with a magnetic fluid. Next, these layers are laminated and adhered to create a board. Comparative measurements were made of the specific gravity of the laminated impregnated type and impregnated type magnetic woods. The experimental results for the proposed laminated impregnated type show that uniform magnetic characteristics are obtained and apparently good characteristics are shown from DC to 13.5 GHz.

The endotoxin activity assay (EAA) is a rapid whole-blood chemiluminescent test for endotoxin that has proven clinical utility in the detection and risk stratification of clinically ill patients with suspicion of sepsis.

The potentially envisaged actions of intravenous immunoglobulin (IVIg) on severe infectious disease include virus or toxin neutralizing action, opsonic effect, complement bacteriolytic activity, and enhancement of sensitivity to antibiotics. In the case of severe infectious disease, antibiotics are often supplemented with administration of IVIg. The aim of this study is that the changes in sepsis markers followed by IVIg administration are investigated with severe sepsis or severe septic shock patients.

The endotoxin adsorption method polymyxin B immobilised fiber with direct hemoperfusion (PMX-DHP) has been used for treatment of patients with severe sepsis and septic shock primarily caused by Gram-negative infections in Japan [1]. One hundred and twenty-six septic patients who had severe sepsis or septic shock due to intra-abdominal infections were treated with PMX-DHP.

The endotoxin adsorption method (PMX-DHP: Toray Industries, Inc., Tokyo, Japan) is used for treatment of patients with sepsis and septic shock primarily caused by Gram-negative infections in Japan. Procalcitonin (PCT) levels may be a good marker of infection and levels exceeding 10 ng/ml occur almost exclusively in severe sepsis. The aim of this study was to evaluate PMX-DHP for severe sepsis or septic shock patients according to PCT values. Patients were classified as a group in which PCT is higher than 10 ng/ml (H group) or a group in which PCT is lower than 10 ng/ml (L group).

The limitations of endotoxin hemoadsorption therapy (PMX-DHP) and the optimal time to start PMX-DHP were examined in patients with septic multiple organ failure with hypercytokinemia (IL-6 > 1000 pg/ml). This study included 66 patients with infectious systemic inflammatory response syndrome in whom IL-6 > 1000 pg/ml before PMX-DHP therapy. These subjects were separated into two groups, those who survived for more than 28 days after the start of PMX-DHP therapy (S group; 38 patients) and those who did not survive (N-S group; 28 patients). Severity of symptoms and background factors, hemodynamic parameters, PaO2/FiO2, endotoxin, cytokines (TNF-α, IL-6, IL-1ra), and vascular endothelial cell function-related markers (ICAM-1, ELAM-1, PAI-1) were examined before and after PMX-DHP. Statistical analyses were performed by chi-squared test for background factors, with Wilcoxon's signed rank test for comparison within a group, and Mann–Whitney's U test for comparison between groups. This study was approved by the IRB of Tokyo Medical University.

Changes in hemodynamics and a variety of mediators such as cytokines were compared with respect to the number of dysfunctional organs in MODS cases treated with blood purification.

Polymyxin B immobilized fiber (PMX-R; Toray industries, Inc) has been developed in Japan for direct removal of endotoxin (PMX-DHP). PMX-DHP is used for treatment of patients with sepsis and septic shock primarily caused by Gram-negative infections, and its effectiveness of hemodynamic changes has been evaluated. In 119 cases (62 ± 16 years old, 82 men and 37 women) treated with PMX-DHP, changes in pulmonary oxygenation before and after PMX-DHP were examined, using PaO2/FiO2 as an oxygenation indicator, separately according to the outcome (80 survivors and 39 who died). Improvement of PaO2/FiO2 was identified in 64% of all cases. In the survival group, there appeared to be a trend for PaO2/FiO2 improvement as blood pressure increased. In the non-survival group, however, no improvement of PaO2/FiO2 was obtained, regardless of an increase of blood pressure. There was no significant correlation between PaO2/FiO2 and the rate of change in endotoxin levels, TNF-α, and IL-6. Granulocyte elastase increased significantly after PMX-DHP treatment in both groups. In the survival group, PaO2/FiO2 appeared to increase as macrophage inflammatory protein (MIP-1) 1-α decreased, suggesting the possibility that changes in MIP-1-α influenced PaO2/FiO2. As the mechanism for the improvement of pulmonary oxygenation by PMX-DHP has been shown clearly, it remains to be examined further.

A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).

The previous studies have shown that (a) noncovalent interactions of the ferro-heme fragment of residues 1-38 and apoprotein (1-104) of horse cytochrome c simultaneously and specifically form two isomeric complexes, types I and II resembling the native protein (the redundant residues flexibly protruding from the ordered structure); (b) the type II form but not type I appears to bind to CO; and (c) residues 39-55 are more flexible for type II form than type I (Parr, G. R., and Taniuchi, H. (1981) J. Biol. Chem. 256, 125-132). In the present study, we investigated 1) kinetics and thermodynamics of interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104); 2) the properties of the CO binding population; 3) the rate of dissociation of complexes ferri- and ferro-(1-38)-H.(39-104) (mimicking type II form); and 4) thermal transition of the 695-nm absorption band and biological activity of complexes. The results indicate (a) interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104) occurs without going through dissociation (t1/2 less than or equal to 12 min at 10 degrees C) and is associated with delta H (= -7.2 +/- 3.7 kcal/mol at 10 degrees C) favoring type I form and delta S (= 23 +/- 13 e.u. at 10 degrees C) favoring type II; (b) the CO-binding population correlates with type II; and (c) change from the ferrous to the ferric state of heme appears to perturb the thermodynamic relationship between type I and II forms. Interpreting the results and available evidence, we suggest that "intramolecular" flip between ferro-type I and ferro-type II forms would establish the Boltzmann distribution of these two distinctly different energy states, type I form having more strengthened interatomic interactions and type II more pronounced internal motion.

To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation constant by more than 400-fold for the ferrous complex. In contrast, replacement of leucine 35 with isoleucine seems to increase it only by a small degree. Since both leucine 32 and leucine 35 are completely buried within the structure, hydrophobic interaction would not explain this striking difference. However, thermodynamic analyses and absorption spectra of the ferric complex have indicated that replacement with norvaline of leucine 32 increases both delta H and delta S (more positive) associated with formation of an intermediate three-fragment complex and decreases both delta H and delta S (more negative) associated with transformation from the intermediate to the ground state, resulting in weakening the methionine 80--S--heme-Fe bond formed in the latter step. Taking the results together with the fragment exchange studies on the ferrous complex and available evidence, we suggest that the interaction involving leucine 32 would be coupled not only with the methionine 80--S--heme-Fe bond but also with the energy state of other distant residues such as tryptophan 59, generating extra energy for modulating the binding of the complex, i.e. the force of folding. In contrast, leucine 35 would be less important even if it were involved in such coupling.

The kinetics of formation of noncovalently bound ferrous complexes derived from fragments of horse heart cytochrome c have been investigated. When the reactions are initiated by combining ferrous heme fragments with an appropriate apofragment, in the presence of 50 mM imidazole, second order rate processes are observed with rate constants essentially the same as those reported with ferric heme fragments (Parr, G. R., and Taniuchi, H. (1979) J. Biol. Chem. 254, 4836-4842). An additional, probably consecutive, kinetic process is also demonstrated. If imidazole is not present in the reaction buffer, the kinetic profiles are dramatically altered. While this is partially due to aggregation (dimerization) of the ferrous heme fragments, it can nevertheless be demonstrated that the complementation reactions with apofragments are much faster than those observed with the corresponding ferric heme fragments (in the absence of imidazole). These results reflect the effect of the oxidation state of the heme iron on the folding mechanism and, thus, the manifold nature of protein folding pathways. The rate of reduction of productive ferric complexes by sodium ascorbate was investigated and biphasic reactions were found in all cases. The data indicate an equilibrium between two forms of the ferric complexes. The results of an experiment in which the complementation of ferric (1-25)H and (23-104) was carried out in the presence of sodium ascorbate indicate that the intermediate complex (Parr, G. R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 8914-8918) is not reducible by ascorbate. Thus, the increase in oxidation-reduction potential occurring on formation of the productive complex from the unbound heme fragment occurs at a late stage of the overall reaction, possibly coinciding with ligation of methionine 80 to the heme iron.

A noncovalently bound, ordered complex consisting of fragments (1-25)H and (14-103) derived from tuna cytochrome c has been prepared and characterized. The equilibrium properties of this complex are indistinguishable from those of the corresponding complex, (1-25)H X (23-104), of horse cytochrome c. The tuna species possesses a second tryptophan residue (at position 33 of the amino acid sequence and replacing the histidine residue of the horse protein) whose fluorescence is also essentially fully quenched in the native form and in the complex. It is found that for the tuna complex, the fluorescence of both tryptophans is fully quenched during the second order phase of the reaction mechanism. This provides evidence for the compact nature of the intermediate complexes formed at this stage of the complementation process (Parr, G. R., and Taniuchi, H. (1982) J. Biol. Chem. 257, 10103-10111). The observed second order rate constant for the tuna fragments is reduced by a factor of two from that for the horse fragments. During the first order kinetic phase corresponding to the transition from the intermediate complexes to the native complex, qualitative as well as quantitative differences are also observed between the tuna and the horse species. The kinetics of complementation of the "hybrid" complexes is also reported. This exchangeability of both the heme fragment and the apofragment of the complex between these two species indicates close similarity of the folding mechanism. Nonetheless, the results also serve to highlight the subtle effects exerted on the folding process by a relatively small number of changes (17%) in the amino acid sequence.

The reactions of ferric heme-containing fragments with apofragments to form ordered complexes resembling native horse heart cytochrome c have been studied under conditions which resolve the overall process into consecutive second order and first order kinetic steps. In the initial, second order step the two fragments combine to form an intermediate complex which exhibits tryptophan 59 fluorescence quenching similar to native cytochrome c, but which has not yet achieved the native ligation state of the heme iron. The existence of first order processes following the second order step is demonstrated by absorbance changes in the Soret region. the entire absorbance change at 695 nm, relating to ligation of the sulfur atom of methionine 80 to the heme iron, is also associated with these first order processes. Thus, ligation of methionine is a late event in this self ordering of the polypeptide chains. Since the conformational energy is assumed to distinctly decrease in this late process of folding (Parr, G.R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 2616-2623), it would follow that small spatial rearrangements of the polypeptide chains in the late stage of folding (as manifested by the ligation of methionine) are associated with a specific decrease in energy.

Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.

The rate of unfolding of Nuclease-T at pH 8,20 degrees was determined as a function of concentration of the ligands deoxythymidine 3',5'-diphosphate (pdTp) and Ca2+ on the basis of the rate of exchange between free fragment, Nuclease-T(50-149) and labeled fragment, Nuclease-T-(50-149) incorporated in the structure of nuclease-T (Taniuchi, H. (1973) J. Biol. Chem. 248, 5164-5174). The rate constant of unfolding of unliganded Nuclease-T' was 4.6 times 10-4s-1. Those of Nuclease-T' bound with pdTp, with Ca2+, and with both pdtp and Ca2+ were 9.0 times 10-5, 1.6 times 10-4, and 2.2 times 10-5s-1, respectively. The association constants of pdTp and Ca2+ with Nuclease-T' were found to be 1.0 times 10-4 and 2.0 times 10-2 m-1, respectively. Those of pdTp with Nuclease-T' plus Ca2+ and of Ca2+ with Nuclease-T' plus pdTp were 4 times 10-5 and 1.4 times 10-4M-1, respectively. The calculation of free energy change on the basis of the association constants shows that the magnitude of negative free energy change involved in the binding of either of the two ligands increases by approximately 2 kcal when the other ligand is already bound. There is a correlation between the free energy change and the specifically coupled with the cooperative interacions operating throught the three-dimensional structure resulting in strengthening of the interactions throughtout the structure, including those with the ligands, without a large change in conformation.

The kinetics of complementation of ferric heme fragments and apofragments of horse heart cytochrome c have been investigated using stopped flow techniques. When changes in fluorescence are monitored, a single phase exhibiting second order kinetics is observed for all cases of productive complexes. The rate constants are independent of the presence of overlapping amino acid sequences in the apofragments but are strongly dependent on the amino acid sequences of the heme fragments. Thus for heme fragment (l-38)H, a rate constant of 2.1 X 10’ M-' s-l is obtained with apofragments (l-104), (23-104), and (56-104). For (l-53)H with (l-104) or (54-104), it is 1.5 X 10’ M-’ s-l. The complementation of (l-25)H with (l-104) or (23-104) results in a rate constant of 3.2 x 10’ M-’ s-l, which is greater by an order of magnitude than those for the other heme fragments. In the presence of 10 mM imidazole, the rate constants dramatically increase to 3.1 x 106, and 2.4 x lo6 M-’ s-l for heme fragments (l-38)H and (l-53)H, respectively, but only by approximately 2-fold for (l25)H. This result is attributed to a change in the axial coordination group of the heme iron atom in fragments (l-38)H and (l-53)H from histidine 33 (or 26) to an imidazole molecule, which in turn results in a major perturbation of the interactions between the heme moiety and its host peptide. When the complementation of the productive complexes is monitored by changes in the Soret absorbance, the data cannot be fit to either first order or second order kinetic equations. However, the results for a nonproductive complex ((l-25)H plus apofragment (39-104)) are identical in both absorbance and fluorescence modes. The data can be fit to an equilibrium process with a rate constant of formation of 1.3 x lo6 M-’ s-l and that of dissociation of 0.95 s-l. Thus, the formation of productive complexes appears to involve a second phase of the reaction which does not occur in the case of the nonproductive complex and which causes no additional fluorescence change but does result in further absorbance changes.

Two forms of yeast cytochrome c synthetases with different specificities were resolved, one (synthetase I), solubilized from mitochondria or the cell debris with Triton X-100, recognizing not horse apocytochrome c but yeast apo-iso-1-cytochrome c as a substrate and the other (synthetase II) still bound with the particulate fraction from mitochondria after treatment with Triton, recognizing both horse and yeast apocytochromes c. The activity with labeled yeast apo-iso-1-cytochrome c as a substrate of cytochrome c synthetase I can be quantitatively inhibited by nonlabeled Candida krusei apocytochrome c and partially by nonlabeled tuna apocytochrome c but not by nonlabeled horse apocytochrome c indicating a specific amino acid sequence being recognized. However, an enzyme similarly solubilized from beef heart mitochondria recognized both horse apocytochrome c and yeast apo-iso-1-cytochrome c for attachment of heme. In view of the fact that the yeast synthetase II and the beef synthetase can both utilize either horse apocytochrome c or yeast apo-iso-1-cytochrome c as substrates, we suggest that these enzymes may also be involved in biosynthesis of cytochrome c1, that is, the ability to attach heme to apocytochrome c and apocytochrome c1 may have been conserved in eucaryotic cells, and that both synthetases may therefore be homologous.

Seven monoclonal antibodies, prepared to yeast holo- or apo-iso-1-cytochrome c by the method of Kohler and Milstein (Goding, J. W. (1983) Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando, FL) were characterized by cross-reaction with a panel of evolutionarily related cytochromes c, apocytochromes c, fragments and homologous and hybrid fragment complexes, inhibition, competitive inhibition, and complementation and fluorescence titration. The results have permitted us to assign the specifically recognized amino acids as follows. IgG1 monoclonals: 4-74-6, Leu-63 and/or Asn-67 and/or Asn-68; 4-128-6, Glu-93; 4-145-10, Thr-74; 2-96-12, Asp-65; 2-34-19, Lys-59; and 10-28-86, trimethyl-lysine 77. IgM monoclonal 39-14, Pro-30 and His-31. With mAb 4-128-6 substitution of glutamic acid 93 with alanine, as it occurs in Candida cytochrome c, has resulted in a decrease in affinity by a factor of 10(4). A calculation appears to show that this value is too large to be accounted for solely by the sum of energy losses due to disruption of charge neutralization and changes of hydrophobic interaction including van der Waals interaction. This and similar results with mAb 10-28-86 have led us to the idea that some new extra interatomic interaction sensitive to differences in configuration of atomic groups may be present and perturbed in the substitution. Furthermore, the assumption of the presence of such interaction can explain the striking similarity between the antigen-antibody interaction (e.g. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002) and a model system of horse cytochrome c three-fragment complex (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711) with respect to the high specificity of interacting residues in the interface. Thus, by analogy to the hypothesis developed in the model system (Fisher, A., and Taniuchi, H. (1988) FASEB J. 2, A1338), we hypothesize that a closed interaction loop would be formed on the basis of contacting groups including glutamic acid 93 across or within the interface between the antigen and mAb 4-128-6 and mediate delocalized interaction to generate extra binding energy.

To investigate the amino acid sequence of apocytochrome c recognized by yeast mitochondrial cytochrome c synthetase, a labeled apofragment containing residues 1 to 25 of horse cytochrome c, N alpha-[3H]acetyl-(1-25), and an analog containing glycine in place of cysteines 14 and 17, N alpha-[3H]acetyl-[14-Gly, 17-Gly] (1-25), have been synthesized using the Merrifield's improved solid phase method (Mitchell, A. R., Ericksen , B. W., Ryabtsev , M. N., Hodges , R. S., and Merrifield, R. B. (1976) J. Am. Chem. Soc. 98, 7357-7362) and then purified to homogeneity. Upon incubation with yeast mitochondria in the presence of hemin, a radioactive species, produced from N alpha-[3H]acetyl-(1-25) and not from N alpha-acetyl-[14-Gly, 17-Gly] (1-25), formed a complex with native apofragment (23-104). This semisynthetic complex was indistinguishable from the native complex in resistance to trypsin upon reduction with ascorbate and by ion-exchange chromatography. The radioactive species, dissociated from the complex was identical with native heme fragment N alpha-acetyl-(1-25)H by reverse-phase high pressure liquid chromatography. Treatment of this radioactive heme fragment with silver sulfate and then with dithiothreitol generated the original apofragment . Thus, if it is assumed that the mitochondrial enzyme catalyzing this covalent attachment of heme to synthetic apo-N alpha-[3H]acetyl-(1-25) is a cytochrome c synthetase, the results may be interpreted as indicating that the amino acid sequence of residues 1 to 25 of horse cytochrome c would contain the principal recognition site of the enzyme.

Two forms of yeast cytochrome c synthetases with different specificities were resolved, one (synthetase I), solubilized from mitochondria or the cell debris with Triton X-100, recognizing not horse apocytochrome c but yeast apo-iso-1-cytochrome c as a substrate and the other (synthetase II) still bound with the particulate fraction from mitochondria after treatment with Triton, recognizing both horse and yeast apocytochromes c. The activity with labeled yeast apo-iso-1-cytochrome c as a substrate of cytochrome c synthetase I can be quantitatively inhibited by nonlabeled Candida krusei apocytochrome c and partially by nonlabeled tuna apocytochrome c but not by nonlabeled horse apocytochrome c indicating a specific amino acid sequence being recognized. However, an enzyme similarly solubilized from beef heart mitochondria recognized both horse apocytochrome c and yeast apo-iso-1-cytochrome c for attachment of heme. In view of the fact that the yeast synthetase II and the beef synthetase can both utilize either horse apocytochrome c or yeast apo-iso-1-cytochrome c as substrates, we suggest that these enzymes may also be involved in biosynthesis of cytochrome c1, that is, the ability to attach heme to apocytochrome c and apocytochrome c1 may have been conserved in eucaryotic cells, and that both synthetases may therefore be homologous.

The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X (39-104) of horse cytochrome c, have shown that invariant leucine 32 and partially invariant leucine 35, both buried in the interior, exhibit a striking difference in perturbation of binding fragment (28-38) by substitution with isoleucine. Then the idea has been proposed that the energy states of leucine 32, the Met-80-S-heme-Fe bond and other distant residues such as tryptophan 59 would be coupled to generate extra force while leucine 35 would be less important for such coupling if it were involved. In the present studies we synthesized three (28-38) analogs substituting invariant proline 30 with glycine or invariant glycine 34 with alanine or serine. Thermodynamic and kinetic studies and UV CD and biological activity measurements were carried out on binding of the analogs to complex (1-25)H X (39-104). The results with the ferric form show that perturbations of delta G, delta H, and delta S associated with formation of the intermediate complex and with the ensuing process by the Gly34----Ala or Ser substitution result in weakening the Met-80-S-heme-Fe bond formed in the latter process; in contrast, perturbation by the Pro30----Gly substitution is small. However, the biological activity is more perturbed by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in the Gly34----Ala or Ser substitution the complex appears to be more readily activated for both formation and disruption of the Met-80-S-heme-Fe bond at 20 degrees C and below than without substitution. In all cases reduction of the heme strengthens the binding of fragment (28-38). However, striking are the increases in perturbation (less negative) of both delta H and delta S for binding of fragment (28-38) to form the ground state on reduction of the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies), and Leu32----norvaline (the previous studies) substitutions. It is known that fluctuation of the atomic positions of most residues of tuna ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation of the heme and that these three residues are among those showing the least fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26, Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT 400 WORDS)

Nuclease-(1-126), although containing 89% of the amino acid sequence which folds to the ordered structure of nuclease A, is disordered and highly flexible (Taniuchi, H., and Anfinsen, C. B. (1969) J. Biol. Chem. 243, 4778-4786). On the other hand, Sachs et al. (Sachs, D. H., Schechter, A. N., Eastlake, A., and Anfinsen, C. B. (1974) Nature 251, 242-244) have demonstrated intrinsic enzymatic activity for nuclease-(1-126). To attempt to learn whether or not the active population of nuclease-(1-126) has the native conformation, we have examined nuclease-(1-126) with respect to enzymatic kinetics with and without the competitive inhibitor deoxythymidine 3',5'-diphosphate (pdTp), effect of temperature on enzymatic activity, binding of pdTp in the presence of Ca2+ and intrinsic viscosity, Stokes radius, CD, and response to trypsin action in the presence and absence of pdTp and Ca2+. The results indicate that the conformation of nuclease-(1-126) bound with pdTp in the presence of Ca2+ is partially constrained but still highly flexible below 30 degrees C, outside the range of thermal transition exhibited by the ordered elements of nuclease-(1-126). Thus, formation or stabilization of active site of nuclease-(1-126) by binding with ligands is not associated with cooperative folding of the entire polypeptide chain. Considering that nuclease-(1-126) does not bind to nuclease-(127-149) but does to nuclease-(111-149), the results are consistent with the idea that the specific cooperative interactions, providing extra stabilizing energy required for maintaining the polypeptide chain in the ordered state of nuclease A, may be disrupted for nuclease-(1-126) primarily due to cleavage of the peptide bond between residues 126 and 127. Then, it may be thought that binding with ligands does not compensate for this disruption.