Studies of mercurated derivatives of acetylacetone and ethyl acetoacetate by solid state nuclear magnetic resonance and vibrational spectroscopies

Seven new mono- and/or dimercurated compounds involving acetylacetone (2,4-pentanedione) or ethyl acetoacetate (3-ethyl ketobutanoate) were synthesized in aqueous medium. In all cases, mercuration occurred at methylene carbon atoms. All compounds were carefully analyzed by solid state carbon-13 nuclear magnetic resonance. Assignments were confirmed by using selective sequences, which allowed a total editing of the spectra. It was shown that deshielding of ¹³C mercurated sites occurred when the rate of mercuration increased. It was also possible to measure direct ¹J(¹⁹⁹Hg¹³C) coupling constants. The main bands of the vibrational spectra (infrared and Raman) were assigned. It was proved that v(C = O) and δ(C(γ)-H) could be directly related to the mercuration rate of molecules.     

Thermodynamically specific gating kinetics of cardiac mammalian K (ATP) + channels in a physiological environment near 37°C

Elementary K⁺ currents through isolated ATP-sensitive K⁺ channels from neonatal rat cardiocytes were recorded to study their temperature dependence between 9°C and 39°C. Elementary current size and, thus, K⁺ permeation through the open pore varied monotonically with temperature with a Q₁₀ of 1.25 corresponding to a low activation energy of 3.9 kcal/mol. Open-state kinetics showed a complicated temperature dependence with Q₁₀ values of up to 2.94. Arrhenius anomalies of _open(1) and _open(2) indicate the occurrence of thermallyinduced perturbations with a dominating influence on channel portions that are involved in gating but are obviously ineffective in altering pore-forming segments. At 39°C, open-state exit reactions were associated with the highest activation energy (O₂ exit reaction: 12.1 kcal/ mol) and the largest amount of entropy. A transition from 19°C to 9°C elucidated a paradoxical kinetic response, shortening of both O-states, irrespective of the absence or presence of cAMP-dependent phosphorylation. Another member of the K⁺ channel family and also a constituent of neonatal rat cardiocyte membranes, 66 pS outwardly-rectifying channels, was found to react predictably since _open increased on cooling. Obviously, cardiac K _(ATP) ⁺ channels do not share this exceptional kinetic responsiveness to a temperature transition from 19°C to 9°C with other K⁺ channels and have a unique sensitivity to thermally-induced perturbations.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

On-line state recognition in a yeast fed-batch culture using error vectors

The physiological states with respect to cell growth and ethanol production in a yeast fed-batch culture expressed in linguistic form could be recognized on-line by fuzzy inferencing based on error vectors. The error vector was newly defined here in a macroscopic elemental balance equation. The physiological states for cell growth and ethanol production were characterized by error vectors using many experimental data from fed-batch cultures. Fuzzy membership functions were constructed from the frequency distributions of the error vectors and state recognition was performed by fuzzy inferencing. In particular, an unusual physiological state for a yeast cultivation, in which aerobic ethanol production was accompanied by very low cell growth, could be recognized accurately. According to the results of the state recognition, an energy parameter, the P/O ratio in the metabolic reaction model was adaptively estimated, and the cell growth was successfully evaluated with the estimated P/O. (c) 1995 John Wiley & Sons, Inc.     

Oxidative stress and heart failure

Various abnormalities have been implicated in the transition of hypertrophy to heart failure but the exact mechanism is still unknown. Thus heart failure subsequent to hypertrophy remains a major clinical problem. Recently, oxidative stress has been suggested to play a critical role in the pathogenesis of heart failure. Here we describe antioxidant changes as well as their significance during hypertrophy and heart failure stages. Heart hypertrophy in rats and guinea pigs, in response to pressure over-load, is associated with an increase in antioxidant reserve and a decrease in oxidative stress. Hypertrophied rat hearts show increased tolerance for different oxidative stress conditions such as those imposed by free radicals, hypoxia-reoxygenation and ischemia-reperfusion. On the other hand, heart failure under acute as well as chronic conditions is associated with reduced antioxidant reserve and increased oxidative stress. The latter may have a causal role as suggested by the protection seen with antioxidant treatment in acute as well as in chronic heart failure. It is becoming increasingly apparent that, anytime the available antioxidant reserve in the cell becomes inadequate, myocardial dysfunction is imminent.     

Prediction of titration properties of structures of a protein derived from molecular dynamics trajectories.

This paper explores the dependence of the molecular dynamics (MD) trajectory of a protein molecule on the titration state assigned to the molecule. Four 100-ps MD trajectories of bovine pancreatic trypsin inhibitor (BPTI) were generated, starting from two different structures, each of which was held in two different charge states. The two starting structures were the X-ray crystal structure and one of the solution structures determined by NMR, and the charge states differed only in the ionization state of N terminus. Although it is evident that the MD simulations were too short to sample fully the equilibrium distribution of structures in each case, standard Poisson-Boltzmann titration state analysis of the resulting configurations shows general agreement between the overall titration behavior of the protein and the charge state assumed during MD simulation: at pH 7, the total net charge of the protein resulting from the titration analysis is consistently lower for the protein with the N terminus assumed to be neutral than for the protein with the N terminus assumed to be charged. For most of the ionizable residues, the differences in the calculated pKaS among the four trajectories are statistically negligible and remain in good agreement with the data obtained by crystal structure titration and by experiment. The exceptions include the N terminus, which responds directly to the change of its imposed charge; the C terminus, which in the NMR structure interacts strongly with the former; and a few other residues (Arg 1, Glu 7, Tyr 35, and Arg 42) whose pKaS reflect the initial structure and the limited trajectory lengths. This study illustrates the importance of the careful assignment of protonation states at the start of MD simulations and points to the need for simulation methods that allow for the variation of the protonation state in the calculation of equilibrium properties.     

Two steps in the transition between the native and acid states of bovine α-lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

A few studies indirectly support the existence of an intermediate in the transition of Ca(2+)-saturated bovine alpha-lactalbumin (alpha-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of alpha-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to alpha-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, g(em)) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the alpha-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g(em) showed an additional conformational transition at pH 4-5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191-197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which alpha-LA exists in an intermediate state between the N- and A-state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the alpha-LA, native-like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the "critical activated state" theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547-561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins.     

Chemically crosslinked protein dimers: stability and denaturation effects.

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.     

鳜鱼的耗氧率及其池塘养殖

本文较详细地报道了鳜鱼的耗氧率和窒息点,并对鳜鱼和作其饲料的鱼同池饲养的可能性进行了探讨。结果表明,鳜鱼耗氧量和体重正相关（ｒ＝０．９９）,耗氧率与体重反相关（ｒ＝－０．９７）;在水温２０℃,鱼种耗氧率约为０．１４ｍｇ／ｇ．ｈ．,成鱼约为０．１２ｍｇ／ｇ．ｈ．;耗氧量和耗氧率均与水温正相关（ｒ＝０．９０,ｒ＝０．９４）,水温１３—３０℃时,体重２３０±１１．７ｇ的鳜鱼。耗氧量为１４．３１—４２．１３ｍｇ／尾。ｈ．,耗氧率为０．０５９—０．１７５ｍｇ／ｇ．ｈ．;鳜鱼耗氧率昼夜变化与家鱼相反,黄昏至凌晨是高峰期,为０．１２—０．１５ｍｇ／ｇ．ｈ．（Ｔ＝２０℃）,白天是低谷期,为０．０７—０．１０ｍｇ／ｇ．ｈ;;鳜鱼的窒息点与家鱼类较接近,变化范围为０．４５—０．７６ｍｇ／Ｌ;鳜鱼和作其饲料的鱼在同一池塘中饲养,既可持续不断地提供鳜鱼充足的适口饵料,又简单易行,成本低,效益高,有较大的价值。     

Magnetic field dependence of radical-pair decay kinetics and molecular triplet quantum yield in quinone-depleted reaction centers

Radical-pair decay kinetics and molecular triplet quantum yields at various magnetic fields are reported for quinone-depleted reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26. The radical-pair decay is observed by picosecond absorption spectroscopy to be a single exponential to within the experimental uncertainty at all fields. The decay time increases from 13 ns at zero field to 17 ns at 1 kG, and decreases to 9 ns at 50 kG. The orientation averaged quantum yield of formation of the molecular triplet of the primary electron donor, ³P, drops to 47% of its zero-field value at 1 kG and rises to 126% at 50 kG. Combined analysis of these data gives a singlet radical-pair decay rate constant of 5 · 10⁷s^?1, a lower limit for the triplet radical-pair decay rate constant of 1 · 10⁸s^?1 and a lower limit for the quantum yield of radical-pair decay by the triplet channel of 38% at zero field. The upper limit of the quantum yield of ³P formation at zero field is measured to be 32%. In order to explain this apparent discrepancy, decay of the radical pair by the triplet channel must lead to some rapid ground state formation as well as some ³P formation. It is proposed that the triplet radical pair decays to a triplet charge-transfer state which is strongly coupled to the ground state by spin-orbit interactions. Several possibilities for this charge-transfer state are discussed.