Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.     

Fluorogenic derivatization reagents suitable for isolation and identification of cysteine-containing proteins utilizing high-performance liquid chromatography-tandem mass spectrometry

The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-4-sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl), are proposed for use in proteomics studies. Following derivatization of protein mixtures with these reagents, a series of standard processes of isolation, digestion, and identification of the proteins were performed utilizing high-performance liquid chromatography-fluorescence detection and tandem mass spectrometry with the probability-based protein identification algorithm. Both DAABD and TAABD derivatives were detected fluorometrically at the femtomole level and showed more than 100-fold improvement in sensitivity compared to the underivatized original compounds with an electrospray ionization ion trap mass spectrometer analysis. The modification of the MASCOT database search system memorized with the fragment information of a DAABD-attached Cys residue allowed the identification of the proteolytic peptide fragments of the derivatized bovine serum albumin (BSA) with an estimated 38% sequence coverage of BSA. Utilizing DAABD-Cl as a derivatization reagent, identification of several proteins was also possible in a soluble extract of Caenorhabditis elegans (10 microg of protein). Consequently, for identification of proteins in the complex matrixes of proteins, DAABD-Cl could be a more appropriate reagent than ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate as reported previously.     

Enhancing effect of nonionic surfactant on the inactivation of lipopolysaccharide by steam-heat treatment II

n-alkylpolyoxyethylene surfactants (CnH2n+1O(CH2CH2O)mH; CnEm) showed a strong enhancing effect on the inactivation of lipopolysaccharide (LPS) by heat treatment over a wide range of temperatures. The effect of CnE8 (n = 10-16) was observed above the critical micelle concentration (CMC) and above the cloud point, and was influenced by the length of the alkyl chains. The efficacy of the surfactants was in the order C10E8 < C12E8, C16E8 < C14E8. However, the hydrophilic moiety seemed to have no influence. An 80-95% solution of n-butanol showed a similar effect, indicating that LPS was more effectively inactivated in the oily phase of the surfactants than in water. The effect of surfactant on the hydrodynamic diameter of LPS was the same before and after steam-heat treatment for 20 minutes at 121 degrees C. Each surfactant disaggregated LPS without alteration of the activity of LPS before the heat treatment. We consider that the surfactants interact with LPS in the region of lipid A in a manner that favors loss of the activities of LPS during heating.     

Lindeman's melting law and solidus curve under pressure of Al–Si and Al–Ge solid solutions

Using the pressure dependence of the mean-square displacement, the compression effect on the melting temperature of matrix Al is studied by Lindeman's melting law. The melting curve obtained increases as a function of the pressure and is in good agreement with the observed tendency for Al. The contribution of the transverse-like acoustic modes to the mean-square displacement is important for Al and becomes more predominant under pressure. Then, considering the contributions from the band and local mode frequencies, the temperature- and pressure-dependent mean-square displacement is quantitatively calculated for Al–Si and Al–Ge solid solutions. The concentration dependence of the Debye temperature at high and low temperatures is estimated, and the pressure effect on the solidus curves is presented by applying Lindeman's melting law to the Al–Si and Al–Ge alloy systems. © 1998 Chapman & Hall     

Clinical assessment of complement activation and leukocyte kinetics during cardiopulmonary bypass: the effect of cepharanthine

Complement activation and leukosequestration to the lung was studied during cardiopulmonary bypass in eleven patients undergoing cardiac operation without blood transfusion. Six patients (group I) received methylprednisolone before and after the bypass, and the other five (group II) received cepharanthine (biscoclaurin alkaloid) before the bypass associated with methylprednisolone therapy. Leukosequestration to the lung was observed at the time of reperfusion of the lung only in group I, although complement activation estimated by the increase of C 4 a and C 3 a was quite similar in both group. Cepharanthine prevented the sequestration of the leukocyte to the lung may be by its membrane stabilizing effect.     

Incidence and influence of GB virus C and hepatitis C virus infection in patients undergoing bone marrow transplantation

Markers of GB virus C (GBV-C) and hepatitis C virus (HCV) were sought in 80 patients before and after they underwent BMT in a metropolitan hospital in Tokyo between 1990 and 1996. RNA of GBV-C was detected in 14 (18%) patients before BMT. Of the 55 patients who had been transfused, 14 (25%) possessed GBV-C RNA at a frequency significantly higher than in the 25 untransfused patients who were all negative (P < 0.01). HCV RNA was detected in three of the 55 (5%) transfused patients, but in none of the 25 untransfused patients. Sera at 3 months after BMT were available for 57 patients. GBV-C RNA persisted in all 10 patients who were infected before BMT, while it was detected in five of the remaining 47 (11%) patients who were not. However, persistent and/or ongoing GBV-C infection had no appreciable influence on patient morbidity or mortality. Two of the 57 patients were positive for HCV RNA before BMT and this persisted after BMT in both. HCV RNA became positive in eight of the remaining 55 (15%) patients who were negative before BMT. Of the 14 patients who received transfusions screened by the first-generation test at BMT, seven (50%) became positive for HCV RNA, a rate significantly higher than the one of 41 (2%) patients who received transfusions screened by the second-generation test (P < 0.001). These results indicate that BMT patients are at increased risk of GBV-C infection transmitted by transfusions received before and at the time of BMT, and that the risk of HCV infection has decreased after the implementation of the second-generation anti-HCV test.     

Relationship between metabolic hormones and ovulation of dominant follicle during the first follicular wave post-partum in high-producing dairy cows

Recent studies suggest that IGF-I is a crucial regulatory factor in follicular growth during early post-partum period. The aim of the present study was to determine in detail the changing profiles of metabolic and reproductive hormones in relation to ovulation of the dominant follicle (DF) of the first follicular wave post-partum in high-producing dairy cows. Plasma concentrations of related hormones in 22 multiparous Holstein cows were measured from 4 weeks pre-partum to 3 weeks post-partum, and the development of DF was observed with colour Doppler ultrasound. Thirteen cows showed ovulation by 15.2 days post-partum. Anovulatory cows showed higher GH and lower IGF-I levels than those in ovulatory cows during the peri-partum period. Each DF developed similarly, and a clear blood flow in the follicle wall was observed despite ovulation or anovulation. In addition, detailed endocrine profiles were analyzed in 9 out of the 22 cows. Five cows showed an increase in plasma oestradiol-17beta (E2) with follicular growth followed by E2 peak, LH surge and ovulation. In these cows, plasma IGF-I concentrations remained high until 10 days post-partum followed by a gradual decrease. Subsequently, the insulin level increased together with the E2 peak towards ovulation. These profiles were not observed in anovulatory cows. In conclusion, our data strongly support the concept that IGF-I and insulin represent 'metabolic signals' of the resumption of ovarian function post-partum in high-producing dairy cows. Moreover, we provide the first visual evidence that both ovulatory and anovulatory DFs of the first follicular wave post-partum are similarly supplied with active blood flow.