Genetic and biochemical analysis of the cysteinyl residues of isopenicillin N synthase from Streptomyces clavuligerus

Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalyses the oxidative cyclization of the acyclic tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of the cysteine residues found in this enzyme were mutated individually into serine residues, either by the polymerase chain reaction or by single-strand site-directed mutagenesis. Functional analysis of these single mutants showed that the C104S mutant lost more than 96% of its activity, while the remaining C37S, C142S, and C251S mutants each lost 30-50% of their activity. Treatment with the thiol-group-specific reagent N-ethylmaleimide confirmed the importance of the cysteine 104 residue. Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S), prepared by recombining fragments of the IPNS-encoding pcbC gene from each of the three single mutants, showed that it had lost more than 90% of its activity. Conformational analysis by circular dichroism spectroscopy indicated that the IPNS triple mutant was structurally different from the wild type, suggesting that the loss of activity may be due to conformational changes rather than active site modifications.     

制革生态学(续七)

The dyes used in finishing are mainly metalcomplex dyes dissolved in an organic solvent, such as our Eukesolar 150 liquid dyes. Dyes of this type have high water resistance. Dyes employed for dyeing leather in the drum such as our Luganil and Lurazol liquid dyes can also be used, buttheir water resistance is inferior to that of the Eukesolar 150 liquid dyes.     

Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freund's). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.     

Maternal respiratory arrests, severe hypotension, and fetal distress after administration of intrathecal, sufentanil, and bupivacaine after intravenous fentanyl

The transjugular intrahepatic portosystemic shunt procedure is an effective intervention for patients with bleeding esophageal varices. Potential complications are numerous but rarely serious. A case of hemopericardium, cardiac tamponade, and death caused by perforation of the right atrium and aorta by a stent after embolization from the liver during stent placement is reported.     

Generation and Detection of Pairing Bosons--A New Method to Determine the Mechanism of Superconductivity in the Oxides

We propose a new method to determine the coupling boson in the cuprate superconductors based on the generation and detection of the appropriate boson analogous to the experiments performed on the generation and detection of phonons by conventional BCS superconductors. The appropriate choice of substrate and junction detector will allow the unambiguous determination of the boson that is formed by the recombination of energy gap edge quasiparticles produced by excitations into the normal state of the cuprates.     

The estrogen receptor from a tamoxifen stimulated MCF-7 tumor variant contains a point mutation in the ligand binding domain

The nonsteroidal antiestrogen tamoxifen (TAM) is the most commonly used endocrine treatment for all stages of breast cancer in both pre- and postmenopausal women. However, the development of resistance to the drug is common, as most patients treated with TAM eventually experience a recurrence of tumor growth. One of the potential mechanisms of treatment failure is the acquisition by the tumor of the ability to respond to TAM as a stimulatory rather than inhibitory ligand. We (Gottardis and Jordan, Cancer Res 48:5183-5187, 1988; Wolf et al., J Natl Cancer Inst 85:806-812, 1993) and others (Osborne et al., Eur J Cancer Clin Oncol 23: 1189-1196, 1987; Osborne et al., J Natl Cancer Inst 83: 1477-1482, 1991) have extensively described the reproducible development of TAM stimulated growth in a laboratory model system using MCF-7 human breast cancer cells grown as solid tumors in athymic mice. In this paper we report on the isolation of an estrogen receptor (ER) from a TAM stimulated tumor (MCF-7/MT2) which contains a point mutation that causes a tyrosine for aspartate substitution at amino acid 351 in the ligand binding domain. The mutant appears to the major form of ER expressed by this tumor. We also report that only wild type ER was detected in three other TAM stimulated MCF-7 tumor variants, suggesting that multiple mechanisms are possible for the development of TAM stimulated growth. The implications of these findings are discussed.     

Effect of resistance exercise training on cortical and cancellous bone in mature male rats

The thermal inactivation kinetics of Salmonella enteritidis PT4 between 49 and 60 degrees C were investigated. Using procedures designed to eliminate methodological artifacts, we found that the death kinetics deviated from the accepted model of first-order inactivation. When we used high-density stationary-phase populations and sensitive enumeration, the survivor curves at 60 degrees C were reproducibly biphasic. The decimal reduction time at 60 degrees C (D60 degrees C) of the tail subpopulation was more than four times that of the majority population. This difference decreased with decreasing temperature; i.e., the survivor curves became more linear, but the proportion of tail cells remained a constant proportion of the initial population, about 1 in 10(4) to 10(5). Z plots (log D versus temperature) for the two populations showed that the D values coincided at 51 degrees C, indicating that the survivor curves should be linear at this temperature, and this was confirmed experimentally. Investigations into the nature of the tails ruled out genotypic differences between the populations and protection due to leakage from early heat casualties. Heating of cells at 59 degrees C in the presence of 5 or 100 micrograms of chloramphenicol per ml resulted in reductions in the levels of tailing. These reductions were greatest at the higher chloramphenicol concentration. Our results indicate that de novo protein synthesis of heat shock proteins is responsible for the observed tailing. Chemostat-cultured cells heated at 60 degrees C also produced biphasic survivor curves in all but one instance. Cells with higher growth rates were more heat sensitive, but tailing was comparable with batch cultures. Starved cells (no dilution input) displayed linear inactivation kinetics, suggesting that during starvation a rapid heat shock response cannot be initiated.     

Ada-Based support for programming-in-the-Large

Specialized language features and an integrated set of tools can help developers of a large software system describe, analyze, organize, and manage its many modules.