Analysis of ochratoxin B alone and in the presence of ochratoxin A, using carboxypeptidase A

A method is described for ochratoxin B analysis, which is adapted to the earlier described method of ochratoxin A analysis, using carboxypeptidase A (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976). The fluorescence spectra of ochratoxins A and B coincide too much to allow direct discrimination of the two compounds. A method using the differences in kinetic parameters of the enzymatic hydrolysis of the two compounds is suggested for the analysis of mixtures of the ochratoxins.     

Separation of Met-tRNAf and Met-tRNAm by chromatography on Sepharose 4B columns

Prolonged apnea after succinyldicholin is a pharmacogenetic phenomenon. Estimation of genetically determined cholinesterase variants of patients with a prolonged apnea after succinyldicholin or in patients with cholinesterase deficiency respectively are described. The importance of atypical cholinesterase phenotypes in anaesthesia and the possibility of a therapy of prolonged apnea are mentioned. Biochemical data of atypical cholinesterase variants as cause of the prolonged degradation of succinyldicholin as well as formal genetic aspects of cholinesterase polymorphism are discussed.     

Self-joining of Si3N4 using metal interlayers

This work focuses on various aspects of diffusion bonding of Ti-foil and Nb-foil interlayers during the self-joining of Si₃N₄. Joints were diffusion joined by hot-uniaxial pressing at temperatures ranging from 1200 °C to 1600 °C using different holding times. The microstructural characterization of the resulting interfaces was carried out by scanning electron microscopy, electron-probe microanalysis (EPMA), and X-ray diffraction. The results showed that Si₃N₄ could not be bonded to Ti at temperatures lower than 1400 °C; however joining was successful at higher temperatures. On the other hand, Si₃N₄ was solid-state bonded to Nb at temperatures ranging from 1200 °C to 1600 °C. Joining occurred by the formation of a reactive interface on the metal side of the joint. Ti₅Si₃, TiSi, and TiN were detected at the Si₃N₄/Ti interface, and Nb₅Si₃ and NbSi₂ at the Si₃N₄/Nb interface, resulting from high-temperature reaction between Ti or Nb and Si₃N₄. Four-point bending testing gave a maximum joint strength of 147 MPa for Si₃N₄/Ti/Si₃N₄ samples hot pressed at 1500 °C and 120 minutes. However, strong joints were obtained above 1450 °C (>100 MPa). These results indicated that there is a strong relationship between the thickness of the interface and the mechanical strength of the preceding joints. This article is based on a presentation made in the symposium entitled “Processing and Properties of Structural Materials,” which occurred during the Fall TMS meeting in Chicago, Illinois, November 9–12, 2003, under the auspices of the Structural Materials Committee.     

Chemical modification of carboxypeptidase A crystals. Nitration of tyrosine-248

The physiological adjustments to aerobic work (5.6 km/h, up a 9% grade) and to exhausting treadmill work of former champion middle-distance runners were determined in 1971, at ages 47-68 yr, 25-43 yr after their competitive careers in track. In the resting state the former athletes as a group are very much like nonathletes of the same ages. Efficiency in the aerobic walk was the same in both groups and did not change with age in either, but the former athletes on the average performed the walk with less strain as indicated by lower blood lactates, "ventilatory equivalents," and heart rates than nonathletes at corresponding ages. Mean VO2max of the runners declined from 71.4 ml/min-kg-1 in youth to 41.8 at a mean age of 56.6 yr, as compared with mean values of 50.6 and 36.5 ml/min-kg-1 in nonathletes at corresponding ages. VO2max had declined below the average of nonathletic men in only two of the former runners. Mean maximal heart rate declined with age from 186 to 180 in the runners, and from 199 to 186 in nonathletes at corresponding ages. Ventilatory responses of men in both groups were closely related to the increases of blood lactate in both aerobic and maximal work.     

四氧化三锰生产废水处理和循环利用

采用沉淀法除锰和离子交换法去除钙、镁离子,两步法处理四氧化三锰生产废水后,可返回系统循环使用。考察了碱和氧化剂加入量及反应时间对除锰效果的影响。结果表明:处理后的废水中锰离子含量低于0.05 mg/L,锰的去除率达到99.99%。使用阳离子交换树脂能完全去除废水中的钙、镁离子。经过半年工业生产试验表明:废水处理成本低,工艺方法切实可行,产品全部合格。     

Pancreatic secretion of zinc and carboxypeptidase A and B in growing pigs

Responses to supplemental dietary L-carnitine of broilers fed on diets with different levels of metabolizable energy (ME) were investigated using growth performance and some carcass measurements. Three isonitrogenous diets containing 13.5, 12.8 or 12.2 MJ ME/kg were formulated, with or without supplemental L-carnitine (50 mg/kg) and fed ad libitum from 18 to 53 d of age. Supplemental L-carnitine increased body-weight gain (BWG) and improved feed conversion (FC) during the first 2 weeks of study. FC was also improved during the fourth week of the experiment. Weights of breast yield and thigh meat yield were significantly increased, whereas quantity and percentage of abdominal fat were reduced by supplemental L-carnitine. A significant interaction between supplemental dietary L-carnitine and dietary energy level was noted for BWG and FC during the second week of study.     

A t(3;8) chromosomal translocation associated with hepatitis B virus intergration involves the carboxypeptidase N locus

Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.     

Thermal stabilization of carboxypeptidase A as a function of pH and ionic milieu

Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).     

粗Na2WO4溶液的净化分离研究进展

对粗钨酸钠溶液中的几种常见而又典型的杂质，如砷、磷、硅、钼等的净化分离研究成果及几种常用的分离净化方法作了较详细的回顾，对本课题未来的研究方向进行了讨论。     

Crystal structure of carboxypeptidase A complexed with an inactivator in two crystal forms

Two different crystal forms of carboxypeptidase A (CPA) complexed with an inactivator were obtained by the method of hanging drop vapor diffusion. The inactivator, 2-benzyl-3-iodo-propanoic acid (BIPA), binds covalently to an active site residue Glu270 of CPA. The complexes were crystallized in the space group P2(1) (CPA-I) and P2(1)2(1)2(1) (CPA-II), respectively. The structures of both crystal forms were determined by molecular replacement using the native CPA crystal structure as the search model. The final crystallographic residuals are 0.163 for CPA-I and 0.152 for CPA-II. Except for the modification of Glu270, the inactivator exhibits normal binding mode compared with other ligand complexes of CPA. In the final electron density difference maps (2Fo-Fc, Fo-Fc), the density of the iodo ion could not be found in both crystal forms while the conserved water molecule remains coordinated to Zn2+ as in the native CPA. Comparisons of the complexes of CPA-BIPA with the native CPA and the CPA-D-Phe complex are presented. The mechanism of the inactivation of CPA and its implication for catalytic mechanism were discussed.     

Antiprogestin-mediated inactivation of cytochrome P450 3A4

Interleukin-12 (IL-12) is a heterodimeric cytokine that is central to the development of T helper 1-dependent cellular immunity. Although this cytokine has potential therapeutic application as an antineoplastic agent, the systemic infusion of IL-12 has led to toxic fatalities; hence, restriction of expression of IL-12 to the microenvironment of target tumor cells has obvious appeal. In this study, we examined whether tumor cells that were liposome-transfected with IL-12 could enhance the induction of cytolytic lymphocyte immunity to the native tumor. The plasmid expression vector that we used has several useful features including replication to high copy number as an episome and a polycistronic message enabling the production of both the p35 and p40 subunits of IL-12 without alternative splicing; up to 3 ng/mL/10(6)/48 hours of IL-12 was produced following transfection. Tumor cells transfected with IL-12 were superior to untransfected cells in the induction of lymphocyte-mediated cytolysis. IL-12 transfectants induced a heterogeneous population of natural killer, lymphokine activated killer, and cytolytic T lymphocytes, the latter of which exhibited tumor-specific activity. Our studies suggest that liposome-mediated transfection of tumor cells with an episomal, high copy number plasmid vector expressing both IL-12 subunits is a promising approach to cancer vaccination, a strategy that could be implemented ex vivo in treating malignancies such as metastatic ovarian cancer.     

Role of CYP3A4 in human hepatic diltiazem N-demethylation: inhibition of CYP3A4 activity by oxidized diltiazem metabolites

The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabolism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ metabolites in serum occurs during prolonged therapy and leads to decreased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhibition. This study assessed the role of human CYPs in microsomal DTZ oxidation and the capacity of DTZ metabolites to inhibit specific CYP activities. DTZ N-demethylation varied 10-fold in microsomal fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was correlated with testosterone 6beta-hydroxylation (r = 0.82) and, to a lesser extent, tolbutamide hydroxylation (r = 0.59) but not with activities mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsomes catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also active (approximately 20% and 10% of the activity supported by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased microsomal DTZ oxidation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produced no inhibition. Some inhibition was produced by alpha-naphthoflavone, a chemical that inhibits CYP1As and also interacts with CYP3A4. In further experiments, the capacities of DTZ and three metabolites to modulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vitro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selectively inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibitory. The IC50 value of DTZ against CYP3A4-mediated testosterone 6beta-hydroxylation (substrate concentration, 50 microM) was 120 microM. The N-desmethyl (IC50 = 11 microM) and N,N-didesmethyl (IC50 = 0.6 microM) metabolites were 11 and 200 times, respectively, more potent. From kinetic studies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive inhibitors of CYP3A4 (Ki = approximately 2 and 0.1 microM, respectively). CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent biotransformation in NADPH-supplemented hepatic microsomes in vitro, supporting the contention that inhibitory metabolites may be generated in situ. These findings suggest that N-demethylated metabolites of DTZ may contribute to CYP3A4 inhibition in vivo, especially under conditions in which N-desmethyl DTZ accumulates, such as during prolonged DTZ therapy.     

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.     

四氧化三锰干燥系统控制

干燥系统的工况条件是保证四氧化三锰质量稳定的前提,通过引入自动控制系统,控制尾气出口温度、炉膛氧化性气氛、炉膛正压、干燥料浆浓度的变化,避免这些因素变化造成干燥工况的波动,从而保证干燥过程的高效和四氧化三锰质量的稳定.