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Biomedical subjects

R Franco

Publications and source records attributed to R Franco.

At least 199 records · Page 11Linked to original sources
PubMed1986

A computer program for enzyme kinetics that combines model discrimination, parameter refinement and sequential experimental design.

A method of model discrimination and parameter estimation in enzyme kinetics is proposed. The experimental design and analysis of the model are carried out simultaneously and the stopping rule for experimentation is deduced by the experimenter when the probabilities a posteriori indicate that one model is clearly superior to the rest. A FORTRAN77 program specifically developed for joint designs is given. The method is very powerful, as indicated by its usefulness in the discrimination between models. For example, it has been successfully applied to three cases of enzyme kinetics (a single-substrate Michaelian reaction with product inhibition, a single-substrate complex reaction and a two-substrate reaction). By using this method the most probable model and the estimates of the parameters can be obtained in one experimental session. The FORTRAN77 program is deposited as Supplementary Publication SUP 50134 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5.

Computers↗
PubMed1986

Enzyme kinetic studies from progress curves.

A method is described for fitting the velocities obtained from progress curves to a steady-state rate equation. It is based on the method of Markus & Plesser [(1981) in Kinetic Data Analysis: Design and Analysis of Enzyme and Kinetic Data (Edrenyi, ed.), pp. 317-339, Plenum Press, New York]. The obstacle of needing good initial estimates of kinetic parameters is removed by using the parameters provided graphically by a minor modification of the method of Yun & Suelter [(1977) Biochim, Biophys. Acta 480, 1-13]. This progress-curved-based method allows the same discrimination among rival models as do the initial-velocity-based methods, with a great saving of experimental time. The BASIC and FORTRAN 77 programs are deposited as Supplementary Publication SUP 50132 (17 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5-6.

Adenosine Deaminase↗
PubMed1986

Enzymes of the purine metabolism in rat brain microsomes.

Rat brain microsomes, when they are suspended in moderate ionic strength medium, released enzyme activities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), adenosine deaminase (ADA, E.C.3.5.4.4), guanine deaminase (GAH, E.C.3.5.4.3), and purine nucleoside phosphorylase (PNP, E.C.2.1.2.4). The activities released decreased when the saline concentration of the medium was increased and the opposite occurred when 50 mM, pH 7.4 sodium phosphate medium was used. Rat brain microsomes that had been extracted previously by moderate ionic strength solutions still had activities of all the enzymes tested, and released these activities upon sonication or deoxycholate (DOC) treatment. The proportion of the activity released was similar for all the enzymes. DOC treatment released higher enzymic activities and a smaller amount of protein than sonication did. The proportion of activities released was similar to that found in the 105,000 g supernatant. The suspension of microsomes still retained activities of the above-mentioned enzymes after consecutive extractions with increasing concentrations of detergent solutions (DOC and Triton X-100). The amount of enzymic activities released from the microsomes by sonication or DOC treatment did not depend on the protein composition of the homogenization medium. Thus, on increasing the enzyme concentration in the homogenization medium, the activities released did not increase in parallel. The set of results obtained showed that the microsomal fraction is as useful as the cytosolic one for studying purine catabolism in rat brain. Furthermore, the conditions in which purine enzymes are attached to the microsomal fraction are probably closer to "in vivo" conditions than those in which these enzymes are found in the soluble fraction.

Animals↗
PubMed1986

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum.

The activity of guanine deaminase (GAH, E.C.3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C.3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C.2.1.2.4), 5'-nucleotidase (5'N, E.C.3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5'-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.

5'-Nucleotidase↗
PubMed1986

Kinetics of the 5'-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain.

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5'-nucleotidase (5'N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.

5'-Nucleotidase↗
PubMed1986

An improved method for the preparation of rat brain microsomes.

The rat brains homogenized with different media (sucrose, ethylene glycol, dimethyl sulfoxide and urea) yielded different amounts of microsomal fractions. The dielectric constant, density and viscosity of the homogenization media did not correlate with the amount of microsomes separated by differential centrifugation. The homogenization media containing dimethyl sulfoxide were the most efficient for the isolation of rat brain microsomes. The increase in the yield was up to 4-fold when 50% (v/v) dimethyl sulfoxide was employed. Microsomes isolated in this manner were analogous to those obtained from isotonic sucrose solution, as was demonstrated by their chemical and enzymatic (5'-nucleotidase, adenosine deaminase, guanine deaminase, purine-nucleoside phosphorylase, lactate, malate and glutamate dehydrogenases, amine oxidase fumarate hydratase, acid and alkaline phosphatase, acetylcholinesterase, NADPH-cytochrome c reductase, catalase and thiamine-diphosphatase) characterization.

Animals↗
PubMed1985

Computer-based learning of cooperativity and allostery.

The aim of this article is to facilitate the understanding of enzyme cooperativity and allostery by undergraduate and postgraduate students with the aid of a graphic microcomputer. For this purpose the molecular models of Monod-Wyman-Changeux (MWC) and of Koshland-Nemethy-Filmer (KNF) are tested by showing how the different plots, direct, reciprocal, Scatchard and Hill, vary as do the parameters considered in these models. The programs used (one for each model) progress from easy aspects to complicated ones without the intervention of the user (student). Nevertheless, ultimately, with the MWC model, after the introduction of heterotropic effectors, the users can select the parameters in order to further their knowledge. This can be useful also for testing the kinetic behavior of multisubunit enzymes which present cooperativity and which have been extensively described in the literature.

Allosteric Regulation↗
PubMed1984

Computer simulation of purine metabolism.

A computer model of purine metabolism, including catabolism, salvage pathways and interconversion among nucleotides, is given. Steady-state rate equations corresponding to metabolic enzymes are written based on information from the literature about their kinetic behaviour. Numerical integration of this set of equations is performed employing selected parameters taken from the literature. After stabilization of purine compound concentrations is reached, simulation of enzyme deficit and enzyme overproduction is carried out. The latter is calculated by varying specified maximum velocities in the numerical integration. A pattern of intermediate metabolite concentrations is found. These results form a basis for the comparison of normal patterns or patterns reflecting the effects of inborn errors of metabolism. The aim of this paper is to demonstrate the usefulness of this computer simulation method in complex metabolism pathways.

5'-Nucleotidase↗
PubMed1984

Storage of chemotherapy drugs for use in the human tumor stem cell assay.

Tumor chemosensitivity assays require the frequent in vitro use of antineoplastic drugs. Economy and convenience are greatly enhanced if these drugs are stored in aliquots for use as needed. This study investigated the stability of eight common antineoplastic agents at -60 degrees C. Activity was measured as inhibition of colony formation in a commonly used soft agar culture system using an experimentally induced murine sarcoma. Our results indicate no loss of activity with these drugs under the specified storage conditions.

Animals↗
PubMed1983

Polypeptide hormone regulation of gene expression. Thyrotropin-releasing hormone rapidly stimulates both transcription of the prolactin gene and the phosphorylation of a specific nuclear protein.

The tripeptide hypothalamic releasing factor, thyrotropin-releasing hormone (TRH), rapidly increases prolactin gene transcription 7- to 12-fold in the GH4 rat pituitary cell line. The maximal rates of transcription are achieved within minutes and begin to attenuate within 1 h following addition of TRH. This transcriptional response appears to account for the observed effects of TRH on the accumulation of prolactin mRNA and the stimulation of prolactin biosynthesis. The rapid transcriptional effects of TRH include a large number of polymerase II-catalyzed transcription units. Addition of TRH to GH4 cell cultures also rapidly induces the phosphorylation of a 23,000-dalton chromatin-associated basic protein, distinct from known high mobility group or histone proteins and referred to as basic regulated phosphoprotein. The time course of TRH-stimulated prolactin transcription and basic regulated phosphoprotein phosphorylation demonstrates that the signal generated by TRH binding to its plasma membrane receptor rapidly reaches the cell nucleus.

Animals↗
PubMed1983

Inhibition of gastric acid secretion in the gastric brooding frog, Rheobatrachus silus.

The female gastric brooding frog Rheobatrachus silus broods its young in its stomach. A substance that inhibits gastric acid secretion in a toad stomach preparation in vitro appears to be secreted by the developing young. This substance has been identified as prostaglandin E2. Rheobatrachus silus may thus have developed a mechanism whereby prostaglandin secreted by the larvae inhibits acid secretion in the stomach of the female until the larvae have completed development and emerged as juvenile frogs by way of the female's mouth.

Animals↗