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Biomedical subjects

A Roa

Publications and source records attributed to A Roa.

At least 19 recordsLinked to original sources
PubMed1997

Identification of the pac promoter from Kluyvera citrophila.

The nucleotide sequence of the 5'-terminal region of the pac gene encoding the penicillin G acylase from Kluyvera citrophila ATCC 21285 has been determined. The transcriptional start site has been identified by primer extension analysis in a different position to that previously found for the homologous pac gene of Escherichia coli W ATCC 11105. Two nucleotide changes in the -35 box appear to be responsible of the promoter displacement in K. citrophila. A putative upstream promoter element (A+T-rich enhancer sequence) and a binding site for the cAMP receptor protein (CRP) were located upstream of the -35 box. Transcriptional lacZ and cat fusions demonstrated that pac expression was subjected to catabolite repression mediated by cAMP and its receptor protein. Remarkably, phenylacetic acid which is a potent inducer of the penicillin G acylase from E. coli, was only able to cause a significant induction of the pac expression in CRP+ cells cultured in the presence of glucose, suggesting that this effect is CRP-dependent.

Base Sequence↗
PubMed1996

Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation.

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

Acylation↗
PubMed1995

New insights on the specificity of penicillin acylase.

In contrast with the general thought that penicillin G acylases (PGAs) were only able to hydrolyse amides or esters of higly hydrophobic acids, we have demonstrated that the PGA from Kluyvera citrophila catalysed the hydrolysis of 4-nitrophenyl esters of acetic, propionic, butyric and valeric acids. Values of kcat. and kcat./Km were greatest for the first compound and less than values for benzylpenicillin by factors of 30 and 7, respectively. 4-Nitrophenyl acetate was hydrolysed faster than 2-nitrophenyl acetate but slower than phenyl acetate. The pH dependence of the reaction was similar to that of benzylpenicillin. Several experiments showed that hydrolysis of 4-nitrophenyl acetate was not catalysed by contaminating esterase activity. The implications for the structural basis of substrate binding are discussed. These substrates open, for the first time, a way to investigate the kinetic parameters of PGA at the presteady-state and provides a new perspective about the role of PGA in nature.

Esters↗
PubMed1995

[Presumptive organ donations for transplants agreement of the Ethics Committee of the University of Chile Medical School].

The ethics committee of the Faculty of Medicine, University of Chile was consulted about the ethical aspects of presumptive organ donation for transplantation. After analyzing the problem, the committee concluded that every human being has the right to make use of his organs freely, voluntarily and according to his own discernment. The society has no right to make obligatory this donation, even after death. The foundations of this agreement were laid in a series of reasons. In fact, the corpse is not a juridical but a ethical asset and deserves respect for whom it was. It cannot be commercialized and is the only non-religious object susceptible of profanation. It is also object of popular affective and religious manifestations. Beliefs and affects must be respected. Organ donation is an act of charity and cannot be compulsory. The organ donation consent must be explicit, voluntary and solemn.

Chile↗
PubMed1994

Changing the substrate specificity of penicillin G acylase from Kluyvera citrophila through selective pressure.

Escherichia coli (muT, mutD, Leu-) cells transformed with plasmid pYKD59 harbouring the pac gene encoding penicillin acylase (PA) from Kluyvera citrophila ATCC 21285 were exposed to environmental conditions that made expression of this enzyme essential for growth. Under these conditions, spontaneous mutants were isolated that used adipyl-L-leucine as the sole source of L-leucine. DNA sequencing of the mutant pac genes identified a transversion mutation of thymine to guanine at position 1163. This mutation was located in the beta-subunit of the enzyme and resulted in conversion of Phe-360 to valine. The assignment of this mutation to the shift in substrate specificity was further confirmed by site-directed mutagenesis. Secondary-structure prediction of the region surrounding Phe-360 suggests that this mutation should not produce any significant structural change. The purified mutant acylase was able to hydrolyse adipyl-, glutaryl-, valeryl-, caproyl-, heptanoyl- and phenoxyacetyl-L-leucine at pH 5 with greater efficiency than the wild-type enzyme. However, the mutant enzyme was not able to hydrolyse glutaryl-7-aminocephalosporanic acid and had lost 90% and 50% of activity on penicillin G and phenylacetyl-L-leucine respectively. Nevertheless, mutant PA retained its original activity on 6-nitro-3-phenylacetamidobenzoate and p-nitrophenylphenylacetate, suggesting that the binding specificity of PA by the acyl and amine moieties of the substrate are not independent phenomena. The small differences observed between the c.d. spectra of the mutant enzyme recorded at pH 5 and 8 suggest the existence of different conformational states at the two pH values, but these differences were indistinguishable from those observed in the native enzyme and cannot be correlated with the shift in substrate specificity. Our results demonstrate that it is possible to change the specificity of PA by laboratory evolution and use it to identify the amino acids involved in substrate recognition. However, the synchronous participation of the alpha- and beta-subunits in the complex induced-fit-like mechanism of acylases suggests that, to obtain new enzymes for industrial application, the selection pressure should be specifically designed for the compound of interest.

Amino Acid Sequence↗
PubMed1993

Characterization and distribution of cholinesterase activity in mouse uterine horns: changes in estrous cycle.

1. Both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are present in the mouse uterus, BChE being more abundant. 2. Their molecular forms were sequentially solubilized by different extraction media obtaining three ChE fractions whose specific activity was different, depending on the stage of the estrous cycle: hydrosoluble (estrous: 75.5 +/- 6.6 and diestrous: 47.9 +/- 8.7 mU/mg prot); detergent-soluble or amphiphilic (estrous 26.6 +/- 2.4 and diestrous 14.7 +/- 3.3 mU/mg prot.), and high ionic strength-soluble (estrous: 18.7 +/- 4.2 and diestrous 12.8 +/- 1.2 mU/mg prot.). 3. Histochemical procedures demonstrated a different distribution for both ChE activities. AChE was found in nerves next to smooth muscle cells of the circular layer and blood vessels, while BChE was concentrated in the longitudinal stratum surrounding the smooth muscle cells. Under the predominance of progesterone, BChE was also found in the endometrial glands. 4. Maximal contractions evoked by the addition of ACh to the isolated organ bath were concentration dependent and greater in estrous than in diestrous. Nevertheless the difference at the two stages of the estrous cycle disappeared when contractions were normalized to smooth muscle cross-sectional area. 5. BChE but not AChE inhibition augmented maximal contractions elicited by ACh in longitudinal but not in circular smooth muscle. 6. The effect of BChE inhibition on the contractile force developed was greater at lower concentrations of ACh and did not depend on the stage of the estrous cycle.

Acetylcholinesterase↗
PubMed1993

[Changes in ethical attitudes during medical studies].

The aim of this work was to explore the attitude changes towards ethico-medical issues in medical students as they went through medical school. A personal standardized questionnaire containing 14 closed and direct questions was applied to 27 freshmen, 38 interns and 14 post graduates. The most outstanding differences in ethical attitudes occurred between first year students and the older groups. The younger students adhered to ethical principles, valued justice towards patients and moral integrity in physicians. The older students and post graduates emphasized the professional role of physicians and preferred institutional ethical regulations over public control. They also considered more valuable therapeutic efficiency and paternal attitudes towards patients as well as social prestige and economical rewards. We conclude that there is a progressive erosion of humanistic principles along the medical studies, that are substituted by a model of professional competence, social recognition and internal ethical control.

Attitude of Health Personnel↗
PubMed1992

Nicotine stimulates adrenergic terminals and inhibits contractions of mouse uterine horns.

1. Nicotine (1-100 microM) stimulated both basal and electrically evoked release of 3H-norepinephrine and also caused a transient inhibition of contractions in an in vitro preparation of mouse uterine horns. 2. The inhibitory effect of nicotine on electrically evoked contractions was potentiated by aminophylline (89 micrograms/ml), and overcome by both propranolol (1 microM) and by omitting magnesium from the physiological solution. Acetylcholine (10 microM), in the presence of atropine (10 microM) was able to reproduce the inhibitory effect of nicotine. 3. These pharmacological findings suggest that the inhibitory action of nicotine on electrically evoked contractions in mouse uterus could be indirect, i.e. mediated through the action of this compound on presynaptic nicotine receptors located on adrenergic terminals.

Acetylcholine↗
PubMed1992

Assembly and disassembly of the Golgi complex: two processes arranged in a cis-trans direction.

We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt-1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans-Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans-Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae.

Animals↗
PubMed1992

[The embryo, the human and the humanized].

Since the moment of fecundation the human embryo is endowed with the properties of unity and uniqueness and its existence is therefore inviolable. Disputing arguments against this thesis are analyzed. Recent views of some biologists negate the human character to the embryo since the essence of a human being would be its cultural nature and ability to communicate. However, the embryo contains all the genetic information that will allow him to develop the ability to communicate. Any attempt to separate the 3 moments of time, past present and future is a definitive violation of ethics. A basic foundation of ethics is that present and future are implicit in the past and vice-versa. Finally, the idea that the unwanted child is not a cultural being should be discarded.

Abortion, Induced↗
PubMed1989

[Specialization and dehumanization of medicine].

A landmark of our times is the preeminence of technology. The old idea of man achieving a better destiny through effort and self improvement has been displaced by the use of technology which is right here to provide everything, to instantly convert future in present. Contemporary Medicine is committed to technology, but this leads to a fragmentation of man in ever smaller segments. As a consequence a real knowledge of the sick individual becomes impossible, his self consciousness, his intimal life and vital projects become inaccessible to the doctor.

Humanism↗