Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain.

A mutant of papain, where an inter-domain hydrogen bond between the side chain hydroxyl group of a serine residue at position 176 and the side chain carbonyl oxygen of a glutamine residue at position 19 has been removed by site-directed mutagenesis, has been produced and characterized kinetically. The mutation of Ser176 to an alanine has only a small effect on the kinetic parameters, the kcat/Km for hydrolysis of CBZ-Phe-Arg-MCA by the Ser176Ala enzyme being of 8.1 x 10(4) /M/s compared with 1.

A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain.

The controversy concerning the various suggested roles for the side chain of Asp158 in the active site of papain has been clarified by using site-directed mutagenesis. Both wild-type papain and an Asp158 Asn variant were produced in a baculovirus-insect cell expression system, purified to homogeneity from the culture, and characterized kinetically. With CBZ-Phe-Arg-MCA as substrate, the kcat/KM and kcat values obtained for the Asp158Asn papain are 20,000 M-1.

Kinetic and thermodynamic investigation of the (dien)Pd(II)-polycytidylic acid system.

The reaction between (dien)PdCl+ and polycytidylic acid was studied using spectroscopic and stopped-flow methods. In neutral solution, the palladium complex binds at the N3 site of the cytosine base and causes a noncooperative disruption of the ordered helical structure of poly(C). Interaction at the phosphate group of the polynucleotide was also demonstrated by using the dye acridine orange as an indicator.

Mémoire du CMFC, Section du Québec à la Commission Rochon.

The mandate of the Rochon Commission, a board of inquiry into Quebec's health and social services, is to analyse and rationalize the use made of those services in the Province of Quebec. The Quebec Chapter of CFPC presented a brief to the Commission, describing the place of the College in promoting high standards of quality in family practice. The brief submitted by the Chapter emphasizes, also, on the necessity of a specific two-year residency training program that all family physicians would be obliged to complete before they become eligible to obtain a licence to practice.

Strategies in aminoglycoside use and impact upon resistance.

Assessment of amikacin resistance over a 10-year period at our institution revealed that the number of resistant strains remained stable. Qualitatively, amikacin-resistant Enterobacteriaceae and Pseudomonas were fairly stable. There was a slight increase in amikacin-resistant Acinetobacter and staphylococci.

Interaction of (dien)Pd(II) with cytidine and cytidine 5'-monophosphate. Influence of the phosphate group on the kinetics and mechanism.

The kinetics and the equilibrium of (dien)PdCl+ interaction with cytidine (C) and cytidine 5'-monophosphate (CMP) were studied by spectrophotometry and by stopped-flow methods. In both cases, the mechanism implies a (dien)Pd(H2O)2+ intermediate with a significant contribution of the solvent path at low chloride concentrations. With CMP, the rate is affected due to the addition of a mechanistic path via an intermediate formed between (dien)Pd(II) and the phosphate group of CMP.

Monocistronic and polycistronic bacteriophage T4 gene 23 messages.

We studied transcription of T4 late genes by in vitro translation of size-fractionated late RNA and by hybridization of T4 late RNA to plasmids containing identified T4 late genes. We identified mRNA species that coded for the late proteins gp10, gp18, gp21, gp22, gp23, and gp24. Functional mRNA's that coded for the early proteins gp32 and IPIII were also detected after fractionation of late RNA.

Sizes of bacteriophage T4 early mRNA's separated by preparative polyacrylamide gel electrophoresis and identified by in vitro translation and by hybridization to recombinant T4 plasmids.

We determined the sized of specific T4 prereplicative nRNA's by preparative polyacrylamide gel electrophoresis, and we used the following two techniques to identify specific gene transcripts; cell-free protein synthesis accompanied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to distinguish T4 polypeptides and hybridization to recombinant plasmids containing T4 DNA of known genetic composition. In our first analysis, the use of nonsense and in-phase deletion mutants allowed unambiguous identification of the functional transcripts that encoded genes 32, rIIB, and rIIA. In addition, we identified the functional transcript that encoded genes 43, 45, 30, 39, and 52, the beta-glucosyl transferase gene, and the deletion 293 region.

Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids.

The 13C chemical shifts for all the carbon atoms is spironolactone have been assigned. Assignments for nine additional steroids which include the C-7 beta isomer of spironolactone, its C-7 thiol hydrolysis product, the 7 alpha-thioacetate derivative of testosterone and its thiol hydrolysis product are also reported.

SC 25152: A potent mineralocorticoid antagonist with reduced affinity for the 5 alpha-dihydrotestosterone receptor of human and rat prostate.

It has previously been shown that spironolactone possesses antiandrogenic activity in the rat and interacts with rat prostate 5 alpha-dihydrotestosterone cytoplasmic receptors to block the nuclear uptake of this hormone. Current evidence suggests that this androgen receptor interaction may be an important mechanism through which spironolactone causes endocrine side effects in rat and man. We have analyzed the interactions of several spirolactone analogs with the androgen receptor of human and rat prostate and the mineralocorticoid receptor of human and rat kidney.

The effect of the administration of spironolactone on the concentration of plasma testosterone, estradiol and cortisol in male dogs (1).

The effect of the administration of spironolactone, deacetylspironolactone, aldadiene or soldactone on the concentration of plasma testosterone, estradiol, and cortisol was examined in male dogs. Decreases of 60 to 75% in plasma testosterone and estrodiol occur only at high doses (100 mg/kg) of spironolactone or deacetylspironolactone but not at low doses of spironolactone (5 to 10 mg/kg); they occur concomitantly with similar decreases of androgen formation by the testis. No decreases were detected with aldadiene or soldactone.

The intratesticular localization of cytochrome P-450 and cytochrome P-450-dependent enzymes in the rat testis.

Following separation of the seminiferous tubules from the interstitial cells in the rat testis, the amount of cytochrome P-450 and the activities of the cytochrome P-450-dependent enzymes, the 17alpha-hydroxylase and the C17-C20 lyase, were measured in the microsomes of the separated fractions. The amount of cytochrome P-450-dependent enzymes recovered in the microsomal fraction of the interstitial cells ranged from 71 to 86% of the whole testis. However, in some experiments lower recoveries of the activities of the enzymes were attributed to the breakdown of cytochrome P-450 to cytochrome P-420.

Effect of spironolactone on sex hormones in man.

Administration spironolactone at a dosage of 400 mg/day to healthy male volunteers for 5 days resulted in a significant rise in plasma progesterone and 17alpha-hydroxyprogesterone which persisted throughout the study. A transient increase in plasma FSH and LH concentration was observed after the second but not the third or fifth days of drug administration. There was no change in plasma concentration of testosterone, 17beta-estradiol, or prolactin.