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Biomedical subjects

E Alvarez

Publications and source records attributed to E Alvarez.

At least 199 records · Page 11Linked to original sources
PubMed1989

Cloning and characterization of the acyl-coenzyme A: 6-aminopenicillanic-acid-acyltransferase gene of Penicillium chrysogenum.

A gene, aat, encoding acyl-CoA: 6-aminopenicillanic acid acyltransferase (AAT), the last enzyme of the penicillin (Pn) biosynthetic pathway, has been cloned from the genome of Penicillium chrysogenum AS-P-78. The gene contains three introns in the 5'-region and encodes a protein of 357 amino acids with an Mr of 39,943. It complements mutants of P. chrysogenum deficient in AAT activity. The aat gene is expressed as a 1.15-kb transcript and the encoded protein appears to be processed post-translationally into two nonidentical polypeptides of 102 and 255 aa, with Mrs of 11,498 and 28,461, respectively. Three proteins of 40, 11, and 29 kDa (the last one corresponding to the previously purified AAT), were identified in extracts of P. chrysogenum. The aa sequence of the N-terminal end of the 11-kDa polypeptide matched the nucleotide (nt) sequence of the 5'-region of aat. The N-terminal end of the 29-kDa polypeptide corresponded to the sequence beginning at nt position 916 of the sequenced DNA fragment (nt 441 of aat gene). The aat gene of P. chrysogenum resembles the genes encoding Pn acylases of Escherichia coli, Proteus rettgeri and Pseudomonas sp., all of which encode two nonidentical subunits derived from a common precursor, encoded by a single open reading frame.

Acyl Coenzyme A↗
PubMed1989

Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor.

The human transferrin receptor is expressed as a disulfide-linked dimer at the cell surface. The sites of intermolecular disulfide bonds are Cys-89 and Cys-98. We have examined the functional significance of the covalent dimeric structure of the transferrin receptor by substitution of Cys-89 and Cys-98 with serine residues. Wild-type and mutated transferrin receptors were expressed in Chinese hamster ovary cells (clone TF-) that lack detectable endogenous transferrin receptors. The rates of receptor endocytosis and recycling were measured and the accumulation of iron by cells incubated with [59Fe]diferric transferrin was investigated. No significant differences between these rates were observed when cells expressing wild-type and mutated receptors were compared. The structure of the mutant receptor lacking intermolecular disulfide bonds was investigated. The presence of a population of mutant receptors with a non-covalent dimeric structure was indicated by cross-linking studies using diferric [125I]transferrin and the bifunctional reagent disuccinimidyl suberimidate. However, sucrose density gradient sedimentation analysis of Triton X-100 solubilized transferrin receptors demonstrated that the mutant receptor existed as a monomer in the absence of diferric transferrin and as an apparent dimer in the presence of this receptor ligand. We conclude that the covalent dimeric structure of the transferrin receptor is not required for the expression of the dimeric state and functional activity of the receptor.

Animals↗
PubMed1989

Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78.

A gene (ips) encoding the isopenicillin N synthase of Penicillium chrysogenum AS-P-78 was cloned in a 3.9 kb SalI fragment using a probe corresponding to the amino-terminal end of the enzyme. The SalI fragment was trimmed down to a 1.3 kb NcoI-BglII fragment that contained an open reading frame of 996 nucleotides encoding a polypeptide of 331 amino acids with an Mr of 38012 dalton. The predicted polypeptide encoded by the ips gene of strain AS-P-78 contains a tyrosine at position 195, whereas the gene of the high penicillin producing strain 23X-80-269-37-2 shows an isoleucine at the same position. The ips gene is expressed in Escherichia coli minicells using the lambda phage PL promoter. Some similar sequence motifs were found in the upstream region of the ips gene of P. chrysogenum when compared with the upstream sequences of the ips genes of Cephalosporium acremonium and Aspergillus nidulans. Primer extension studies indicated that the start of the mRNA coincides with a T in position -11 which is located in a conserved pyrimidine-rich sequence, near two CAAG boxes. Clones of P. chrysogenum Wis 54-1255 transformed with the ips gene showed a five-fold higher isopenicillin N synthase activity than the untransformed cultures.

Amino Acid Sequence↗
PubMed1989

Two genes involved in penicillin biosynthesis are linked in a 5.1 kb SalI fragment in the genome of Penicillium chrysogenum.

Two genes, pcbC and penDE (also named ips and aat, respectively) encoding the enzymes isopenicillin N synthase and acyl-CoA:6-amino penicillanic acid (6-APA) acyltransferase, which are involved in the penicillin biosynthetic pathway in Penicillium chrysogenum, were cloned. Both genes are clustered together in a 5.1 kb SalI DNA fragment and are separated by a nontranscribed intergenic region of 1.5 kb. These genes are transcribed from different promoters in two separate transcripts of about 1.15 kb each. The penDE gene complements mutants of P. chrysogenum deficient in acyltransferase and the pcbC gene increases the level of isopenicillin N synthase in strains containing low levels of this enzyme. The clustering of penicillin biosynthetic genes is of great interest in the light of previous claims of horizontal transfer of the pcbC gene from beta-lactam producing Streptomyces to filamentous fungi.

Acyltransferases↗
PubMed1989

Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum.

The isopenicillin N synthase (pcbC) and acyl-CoA:6-APA acyltransferase (penDE) genes of Penicillium chrysogenum were located in a 19.5-kb DNA fragment that had been previously cloned in phage vector EMBL3. This 19.5-kb DNA fragment was mapped with several endonucleases, and the pcbC and penDE genes were located by hybridization with probes corresponding to internal fragments of each gene. A low penicillin producing strain (P. chrysogenum Wis 54-1255) and two high producing strains (AS-P-78 and P2) showed hybridizing fragments of identical sizes in their chromosomes. Dot-blot hybridization of serial dilutions of the total DNA of the three strains showed that the intensity of all the hybridizing bands was much higher in strains AS-P-78 and P2 than in Wis 54-1255. Hybridization of total DNA digestions with probes corresponding to fragments which mapped upstream or downstream of the pcbC-penDE region revealed that a fragment of at least 35 kb DNA has been amplified 9 to 14 fold in the high penicillin producing strains. The amplified region did not include the previously cloned pyrG gene that encodes OMP-decarboxylase, an enzyme involved in pyrimidine biosynthesis.

Acyltransferases↗
PubMed1989

Increased plasma free serotonin but unchanged platelet serotonin in bipolar patients treated chronically with lithium.

The effect of lithium salts administered chronically to bipolar patients on peripheral measures of the serotoninergic system has been studied. Plasma free serotonin (5HT), whole blood 5HT, plasma 5-hydroxyindoleacetic acid (5HIAA) and plasma total tryptophan (TP) have been analyzed in 22 patients treated with lithium carbonate (mean daily dose: 1280 mg, mean serum concentration: 0.73 mmol/l) and compared to 14 healthy controls and 11 patients treated chronically with antipsychotic drugs. Lithium salts induced significant increases in plasma free 5HT (+159% with respect to control values) and in plasma 5HIAA (+39%) without affecting 5HT contained in platelets. Plasma TP was also unchanged by chronic lithium treatment. The ratio between 5HT stored in platelets and 5HT free in plasma, a variable reduced after uptake inhibitors like clomipramine, was decreased in lithium-treated patients (-50%). These results are compatible with an enhanced synthesis of 5HT in the periphery (mainly enterochromaffin cells) as well as with an inhibition of platelet 5HT uptake (or increased 5HT efflux from intracellular stores) induced by lithium. The lack of effect of several antipsychotic drugs upon these variables is consistent with their predominant effect on the dopaminergic system and reinforces the specificity of the effect observed with lithium salts. Taken together, these results support the usefulness of using this "in vivo" 5HT peripheral model for the study of the actions elicited by drugs acting on the presynaptic components of the 5HT system.

Adult↗
PubMed1989

Effect of pinealectomy on liver insulin and glucagon receptor concentrations in the rat.

The studies described here were undertaken to characterize the hepatic insulin and glucagon receptors of control (C), pinealectomized (Pn), and melatonin-treated pinealectomized (Pn + Mel) rats. Compared with C rats, an increase in plasma glucose and glucagon levels and a reduction in circulating concentrations of insulin in Pn animals were observed. Melatonin treatment of Pn rats reverses all three parameters toward the normal values. In liver membranes, insulin binding was lower in Pn than in C rats, and glucagon binding was greater in Pn than in C animals; in Pn + Mel rats both insulin and glucagon binding reverse toward the normal values that were observed in C rats. The modifications in hormone binding reflect changes in the number of receptors but not in the affinity constants. The time courses of hormone association and dissociation from liver membranes were similar in all three experimental groups. The degradation of both hormones by liver membranes was similar in all three groups. Insulin receptor degradation also was similar in the three groups, while glucagon receptor degradation was similar in the liver membranes of C and Pn rats but smaller in Pn + Mel animals. These findings suggest that the pineal gland may modulate the circulating levels and liver receptor concentrations of insulin and glucagon. In addition, our results indicate that insulin and glucagon did not induce a down-regulation of liver receptors in Pn rats.

Animals↗
PubMed1989

Presence of melatonin in the umbilical cord blood of full-term human newborns.

The present study was designed to determine whether melatonin circulates in the umbilical cord blood of healthy human newborns and whether the concentrations of this hormone follow a circadian rhythm at birth. Umbilical cord blood was collected from full-term human newborns at the time of delivery. Serum melatonin was extracted with diethylether and determined by radioimmunoassay by using radioiodinated melatonin and rabbit antimelatonin antiserum. Significant amounts of melatonin were detected in the sera obtained from umbilical cord blood. When serum melatonin concentrations of human newborns were represented over 6-h periods or hour by hour over the 24 h of 1 d, similar hormone concentrations were found consistently. These results indicate that melatonin is present in quite high levels in the umbilical cord blood, which may be, at least in part, of maternal origin. Also, our results suggest the absence of a melatonin circadian rhythm in human newborns, which may reflect an immaturity of the components involved in melatonin synthesis.

Circadian Rhythm↗
PubMed1989

Effect of pinealectomy and of diabetes on liver insulin and glucagon receptor concentrations in the rat.

The studies described in this paper were undertaken to characterize the circulating and hepatic insulin and glucagon receptor concentrations of control (C), diabetic (Db), and pinealectomized-diabetic (Pn + Db) rats. Compared with C rats, an increase in plasma glucose and glucagon levels and a reduction in circulating insulin concentrations in Db animals was observed; these differences were greater in Pn + Db rats. In liver membranes, insulin binding was lower in Db and in Pn + Db than in C rats, and glucagon binding was greater in Db and in Pn + Db than in C rats. The modifications in hormone binding did not reflect changes in the affinity constants. The time courses of hormone association and dissociation from liver membranes were similar in all three experimental groups. The degradation of both hormones and their receptors was similar in all three groups. These findings indicate that in either pinealectomized-diabetic or diabetic rats there were significant changes in the circulating and liver insulin and glucagon receptor concentrations and that the changes in the circulating levels of both pancreatic hormones were more pronounced in pinealectomized-diabetic animals. In addition, the absence in Db and in Pn + Db rats of the insulin and glucagon down-regulation of their own receptors could further modify metabolic activities in these animals.

Animals↗
PubMed1989

Biochemical and ultrastructural approaches to the onset of the pineal melatonin rhythm in the rat.

To determine the pineal gland content of melatonin during rat development and the onset of the circadian rhythm, a biochemical and ultrastructural experimental model was designed. Body and pineal gland weights and DNA and protein contents of the pineal glands increased rapidly during ontogenic development. No significant differences in the values of these parameters were observed when the data were obtained during the day or at night. Pineal melatonin content as expressed by pineal gland or by micrograms of DNA, was very small in 21-day-old fetuses and 5- and 10-day-old suckling rats and the values determined were similar for both day and night. However, in 20-day-old suckling and adult rats higher melatonin concentrations and significant differences between the values obtained during the day and at night were observed. The onset of the circadian melatonin rhythm coincided with the time at which the components involved in melatonin synthesis reached adult levels and pinealocytes adopted an adult morphological appearance as observed with the electron microscope. These results indicate that the appearance of a circadian melatonin rhythm in the rat pineal gland is delayed until the end of the suckling period, although the transfer of melatonin from the mother to the pups may be important for postnatal reproductive and somatic development.

Animals↗
PubMed1989

Expression of Lewis antigenic determinants in colorectal adenocarcinomas.

Expression of type 1 and type 2 chain Lewis antigens was studied in 32 rectal adenocarcinoma specimens; the results were correlated with the patients' Lewis phenotype and secretor status. In addition, the pattern of expression of these antigens was analyzed in adjacent and distant normal mucosa. We used an indirect immunofluorescence technique with p-phenylenediamine counterstaining (Oriol technique) and a panel of monoclonal antibodies directed against the different antigenic specificities. Normal distal colonic mucosa only expresses monofucosylated structures (Lea and X) arising from activity of the alpha 1-3,4-fucosyltransferase coded by the Le gene. Rectal adenocarcinomas also show Lea and X, but also reexpress blood group antigens ABH and exhibit difucosylated determinants (Leb and Y). The accumulation of mono- and difucosylated type 2 chain in neoplastic processes, independently of the Le and Se genes, could be due to the enzymes coded by reactivation of the H and X genes. Blood group antigens form a complex signal code, genetically regulated, which intervenes in differentiation, growth and cellular recognition processes, and which may undergo important modifications during malignant transformation. These alterations could be useful in the diagnosis and prognosis of some types of carcinoma.

Adenocarcinoma↗
PubMed1989

[Sepsis and neonatal meningitis].

The epidemiological, clinical and bacteriological data of 38 infants under 28 days of age with a diagnosis of neonatal meningitis (13 patients), early neonatal sepsis (17 patients) or late neonatal sepsis (8 patients) were retrospectively reviewed. 52.6% of patients were premature and/or low weight neonates, 76.3% had an unfavorable obstetrical history and 39.4% had one or more underlying diseases. The clinical features were predominantly neurological (81.5%), cardiac or respiratory (71%) and gastrointestinal (34.2); the most outstanding laboratory data were positive CRP, hyperbilirubinemia and abnormalities in leukocyte differential count. The major microorganisms involved were E. coli (11 patients), S. agalactiae (9), S. aureus (5), L. monocytogenes (3) and coagulase-negative Staphylococcus (3). The overall incidence of these disorders was 3.33 cases/1000 live neonates, and the mortality rate was 7.9%.

Female↗
PubMed1989

[Surface markers and humoral factors in spontaneous murine leukemia].

This paper presents a model of spontaneous BALB/c lymphoma (LB) useful for the study of the relationship between the tumor and the immune system. Characterization of this tumor was done by FACS and it was determined that the tumor cells are T lymphocytes which express both CD4 and CD8 membrane markers but not the viral protein gp 70. In addition, tumor cells express IL-2 receptors and class I, but not class II, H-2 antigens. Antibodies were not detected in sera or ascitic fluid of syngeneic mice bearing a tumor transplant while both sera and ascites contained a suppressor factor that inhibited in vitro the proliferation of tumor cells and blastogenesis of normal BALB/c splenocytes after Concanavalin A stimulation. The results indicate that this tumor may be used as an experimental model for adult T cell leukemia as well as for the isolation and characterization of cytokines which may be responsible for the immunological unresponsiveness found in certain cancer patients.

Animals↗
PubMed1988

Heritable high frequency modulation of antigen expression in neoplastic cells exposed to 5-aza-2'-deoxycytidine or hydroxyurea: analysis and implications.

A study was undertaken to determine the ability of the DNA hypomethylating drug 5-aza-2'-deoxycytidine (5-azadCyd) to induce antigen expression in a high proportion of treated tumor cells and to evaluate if this effect could be mimicked by the drug hydroxyurea (HU) which is a genomic DNA hypermethylating agent. Induction of heritable changes in gene expression by 5-azacytidine or the 2'-deoxy analogue (5-azadCyd), at least in some cases, may not be necessarily due to their hypomethylating properties, but to some other induced high frequency genetic change which occurs when DNA synthesis or repair is perturbed. A comparison of 5-azadCyd and HU effects on human and murine tumors was chosen for this study. The phenotypic properties examined with the above treatments were (a) induction of a Mr 110,000 antigen, detected with M111 antibody, in a variant subpopulation (SMeLus7) of human melanoma cells which fail to maximally express this antigen (M111). The parent cell line, MeWo, and other MeWo-derived variant cell cell lines do not demonstrate a similarly inducible phenotype; and (b) induction of class I major histocompatibility complex antigens in a population of mouse mammary adenocarcinoma cells which normally fail to express these antigens. The results showed that 5-azadCyd was effective in inducing antigen expression in both systems whereas HU was effective (and equally so) only in the human melanoma cell line system. In these treatments 5-azadCyd was demonstrated to transiently hypomethylate the same human melanoma cell line whereas HU hypermethylated genomic cytosines. The results suggest that some of the reported effects of 5-azacytidine or 5-azadCyd in inducing very high frequency heritable phenotypic alterations may not necessarily be related to the drugs' ability to cause DNA hypomethylation. The implications of these results are discussed in terms of the use of 5-azacytidine or 5-azadCyd and the effects of chemotherapy on tumor progression and metastasis.

Animals↗
PubMed1988

The effect of hypoxia on hepatic cytochromes and heme turnover in rats in vivo.

We evaluated the effect of hypoxia (7% v/v) on hepatic heme turnover in vivo and microsomal heme protein content in male Sprague-Dawley rats. Hepatic heme protein turnover, measured as 14CO-production during continuous infusion of 5-14C-aminolevulinic acid, a precursor of nonerythrogenic heme, was decreased 60% during hypoxia and returned to control levels promptly after reoxygenation. Hepatic cytochrome P-450 content was decreased in hypoxic and 24-h reoxygenated animals. We conclude that normobaric hypoxia decreases hepatic cytochrome P-450 which could contribute to decreased drug metabolism in hypoxia. This decrease is probably due to heme oxygenase-independent breakdown of hepatic heme.

Animals↗
PubMed1988

Urinary phenylacetic acid excretion in depressive patients.

Phenylacetic acid (PAA), a metabolite of phenylethylamine, has been found in the urine of depressed patients at lower levels than in control subjects. It has been suggested that the determination of PAA in urine could be used as a biological marker of the depressive condition. In this study the levels of PAA in urine were investigated by gas-liquid chromatography in 39 patients diagnosed as having major depression according to DSM-III criteria, and in 32 healthy subjects. The values found in the patients were markedly lower than in controls. No relationship was found between the decrease of PAA excretion and weight loss. Using a discriminant equation, a value of 69% was obtained for both sensitivity and specificity. The results suggest that the determination of urine PAA could be a biological parameter comparable to the dexamethasone suppression test.

Adult↗