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Biomedical subjects

M Freire

Publications and source records attributed to M Freire.

At least 55 records · Page 3Linked to original sources
PubMed1989

Purification and characterization of a cofactor that controls the oxidative phase of the pentose phosphate cycle in liver and other tissues of rat.

We have recently reported the presence, in rat liver, of a cofactor characterized as a protein of Mr 10(5), which cooperates with GSSG to prevent the inhibition of glucose-6-phosphate dehydrogenase by NADPH. The inhibition that this coenzyme also exerts on 6-phosphogluconate dehydrogenase is similarly prevented by a cofactor-GSSG system. The activity of the cofactor increases in the livers of rats fed on carbohydrate-rich diets. Purification of the components in rat liver homogenate by ion-exchange chromatography and preparative polyacrylamide gel electrophoresis showed that the deinhibitory effect on both dehydrogenases is exerted by the same cofactor. The purified cofactor appeared as a unique protein of Mr 37.10(3) in SDS-polyacrylamide gel electrophoresis. Rat kidney and adipose tissue were the only nonhepatic tissues showing a cofactor-GSSG deinhibitory effect on both dehydrogenases of the oxidative phase of the pentose phosphate cycle. The deinhibitory activity, also corresponding with a cellular component of Mr 10(5), was only diet-inducible in adipose tissue. The neutralization of the kidney and adipose tissue deinhibitory activity by rat liver cofactor antibodies suggested that there was a structural relationship between the cofactors prepared from these tissues.

Adipose Tissue↗
PubMed1989

Synthesis and apparent secretion of prothymosin alpha by different subpopulations of calf and rat thymocytes.

Prothymosin alpha (ProT alpha), a polypeptide containing the sequence of thymosin alpha 1 (T alpha 1) at its NH2-terminus, has been isolated from calf thymocytes in a concentration in the order of that found in the whole thymic gland. As deduced from the analysis of their tryptic peptides, calf ProT alpha differs from the rat polypeptide at least in an alanine residue replacing valine at position 92. Thymocytes cultured in a radioactive medium exhibit an important secretory activity, ProT alpha being one of the products synthesized and exported to the culture medium. Large and small thymocyte subpopulations from calf and rat differ in their capacity to synthesize ProT alpha. The polypeptide is produced in a major concentration by large thymocytes. However, all the calf and rat thymocyte subpopulations show a similar capacity to secrete ProT alpha, the amount of the newly synthesized polypeptide recovered from cell culture supernatants being 80-90% of that found in thymocyte extracts.

Animals↗
PubMed1988

Quantitative computer analysis of Golgi-impregnated neurons using a new 'expanded stick' model for coding; its application to the study of axons.

A computer program to obtain quantitative data from an expanded 'stick model' of the Golgi-impregnated neuron is described. The computer program allows complete three-dimensional morphometric analysis of neurons, including the size of varicosities and spines and their distribution on neuronal branches. Furthermore, the perimeter and area of the perikaryon as well as the length, width and area of neuronal processes can be obtained. The use of this computer program is illustrated by coding two types of axons.

Animals↗
PubMed1987

An "expanded stick" model for coding Golgi-impregnated neuronal morphology.

A new model for coding Golgi-impregnated neuronal morphology, which can be implemented in any computer-assisted optical microscope, is described. This model, in addition to storing the three-dimensional coordinates of the selected neuronal points and their topological identifiers, codes for "width", "nature", and "shape". The "width" code digitizes the width of a neuronal process. The "nature" code can identify structures such as perikaryon, axon, apical dendrite, basal dendrite, etc. The "shape" code defines nodules and spines. Computer graphics routines are described for drawing nodules and spines as well as neuronal processes with "width".

Animals↗
PubMed1987

A simple procedure for large-scale purification of plasmid DNA.

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.

Chromatography, Ion Exchange↗
PubMed1986

Regulation of the pentose phosphate cycle. Cofactor that controls the inhibition of glucose-6-phosphate dehydrogenase by NADPH in rat liver.

A cofactor of Mr 10(4), characterized as a polypeptide, was found to co-operate with GSSG to prevent the inhibition of glucose-6-phosphate dehydrogenase by NADPH, in order to ensure the operation of the oxidative phase of the pentose phosphate pathway, in rat liver [Eggleston & Krebs (1974) Biochem. J. 138, 425-435; Rodriguez-Segade, Carrion & Freire (1979) Biochem. Biophys. Res. Commun. 89, 148-154]. This cofactor has now been partially purified by ion-exchange chromatography and molecular gel filtration, and characterized as a protein of Mr 10(5). The lighter cofactor reported previously was apparently the result of proteolytic activity generated during the tissue homogenization. The heavier cofactor was unstable, and its amount increased in livers of rats fed on carbohydrate-rich diet. Since the purified cofactor contained no glutathione reductase activity, the involvement of this enzyme in the deinhibitory mechanism of glucose-6-phosphate dehydrogenase by NADPH should be ruled out.

Animals↗
PubMed1986

The distribution of N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid alpha-naphthyl acetate esterase and alpha-naphthyl acetate esterase in subpopulations of thymocytes, bone marrow cells and other lymphoid organs in mice.

Thymocytes, bone marrow lymphocytes, as well as lymphocytes from spleen, lymphoid nodes and peripheral blood were obtained from BALB/c mice. Subpopulations of BALB/c bone marrow T-lymphocyte precursors and immature (small) and mature (large) thymocytes, as established by the percentage of terminal deoxynucleotidyl transferase (TdT) and peanut agglutinin (PNA) positive cells, were obtained by centrifugation on discontinuous density gradients. The activities of N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (BG), acid alpha-naphthyl acetate esterase (ANAE) and alpha-naphthyl acetate esterase (NAE) were determined by enzymatic assays of cell extracts of the diverse T-lymphocyte subpopulations, in order to follow their evolution with the maturation of the T-lymphocytes in the thymus. These activities were compared with that determined in lymphocytes from spleen, lymphoid nodes and peripheral blood. The glucidases BG and NAG and the esterases ANAE and NAE present a high decrease in their activities from bone marrow T-lymphocyte progenitors to immature thymocytes. BG, NAG and ANAE activities undergo an about 3-fold increase with the evolution of the thymocytes from small to large cells. Whereas the level of the NAE activity decreases (2-fold) with that evolution of the thymocytes. Lymphocytes from spleen and lymphoid nodes exhibit activities of the glucidases and, specially, the esterases marked by higher than those of thymocyte populations. Peripheral blood lymphocytes also present NAG, ANAE and NAE activities higher than in thymocytes, but their BG activity is lower.

Animals↗
PubMed1986

An inexpensive and interactive microcomputer system for codifying Golgi-impregnated neuronal morphology.

An interactive microcomputer system has been developed for the quantitative analysis of Golgi-impregnated neuronal morphology (GINM). The system uses commercially available hardware: a 48K RAM microcomputer (Apple II), a TV monitor (NEC), an optical microscope (Reichert Biovar) with a camera lucida and a specially developed wheel attached to the focus adjustment knob and to a digital planimeter (Tamaya), allowing one to obtain the value of the z-coordinate. The x- and y-coordinates are obtained using the movement of a point plotted by the computer on the TV monitor. This point is superimposed upon the GINM point to be codified employing the camera lucida. The x- and y-coordinates are acquired automatically while the variations of the z-coordinate are introduced by the computer keyboard. The three-dimensional coordinates of the selected GINM points and different codes are stored by the computer in a six-dimensional array. The software is written in BASIC. The results demonstrate that the digitization and quantitative analysis of GINM can be achieved with reasonably inexpensive equipment.

Animals↗
PubMed1985

Evidence for the synthesis of thymosin alpha 1 by calf thymocytes and the production of this peptide by natural processing.

Thymus and thymocytes from calf were extracted under isotonic conditions in the presence of protease inhibitors or under severe denaturing conditions (after quick freezing and thawing in boiling 0.1 M NaCl). The extracts, as well as the medium in which the thymocytes were obtained from thymus fragments (thymocyte supernatants), were size-fractionated by ultrafiltration. As in whole thymus isotonic extracts, thymosin alpha 1 [A. L. Goldstein, T. L. K. Low, M. McAdoo, J. McClure, G. B. Thurman, J. Rossio, C-Y. Lai, D. Chang, S-S. Wang, C. Harvey, A. H. Ramel, and J. Meienhofer (1977) Proc. Natl. Acad. Sci. USA 74, 725-729] was contained in isotonic extracts from thymocytes and also in thymocyte supernatants, as determined by isoelectric focusing and reverse-phase HPLC analysis. The extraction under denaturing conditions mainly yielded products with molecular masses over 50,000, showing very similar isoelectric focusing patterns in both thymocytes and whole thymus extracts. As deduced by isoelectric focusing analysis of diverse size-fractionated products, a strong association capacity seems to be responsible for an apparently high molecular mass of the components of these extracts. According to the pI, two of these components were prothymosin alpha [A. A. Haritos, G. J. Goodall, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 1008-1011] and thymosin alpha 1. Prothymosin alpha was not detected in any isotonic extracts or thymocyte supernatants. These data suggest that calf thymocytes are capable of producing thymosin alpha 1, which would arise by natural processing of its precursor.

Animals↗
PubMed1985

Characteristics of the translation of thymosin alpha 1 precursor mRNA by cell-free wheat germ system. Evidence for the acetylation of thymosin alpha 1 precursor.

At the optimal concentrations of Mg2+ and K+ to translate total thymus PolyA+-RNA, the purified thymosin alpha 1 precursor mRNA saturate the protein synthetic activity at lower concentration than the total thymus mRNAs. The polyamine spermidine increases the translation rate of the messenger, which is modulated by magnesium, rather than improve the yield in full-length chains of thymosin alpha 1 precursor. As other eukariotic mRNAs, this messenger presents the "cap" modification at the 5'-end terminal position. The incorporation of [3H]acetate into the translation product of the messenger, shows an evidence for the acetylation of the thymosin alpha 1 precursor during its biosynthesis in vitro.

Acetylation↗
PubMed1984

The distribution of adenosine deaminase, purine nucleotide phosphorylase and 5'-nucleotidase in subpopulations of thymocytes, bone marrow cells and other lymphoid organs in mice.

Lymphocyte populations of BALB/c mice were obtained from bone marrow, thymus, spleen, peripheral blood and lymphoid nodes. Subpopulations of thymocytes and bone marrow T-lymphocyte precursors were separated by density gradient centrifugation. The activity of adenosine deaminase (ADA) undergoes a marked increase during the evolution of bone marrow T-cell precursors to immature thymocytes, and a decrease with thymocytes maturation. The peripheral blood lymphocytes (PBL) present the lower activity of the enzyme, and lymphocytes from spleen (SL) and lymphoid nodes (LNL) show activity in the order of that in mature thymocytes. The activity of purine nucleotide phosphorylase (PNP) in the different lymphocytes populations experiments a very little variation with the T-lymphocyte differentiation. With the evolution of T-lymphocyte precursors to immature thymocytes the 5'-nucleotidase (5'-NT) activity experiment a 2-fold decrease. The thymocytes maturation is correlated with an increase in the activity of 5'-NT. The PBL present the maximal activity of the enzyme, whereas in spleen and LNL its levels of activity are in the range of that in mature thymocytes and bone marrow T-cell precursors respectively.

5'-Nucleotidase↗
PubMed1983

Synthesis of thymosin alpha 1 precursor cDNA and purification of active mRNA by affinity chromatography.

1. Thymosin alpha 1 precursor [3H]cDNA-cellulose synthesis was carried out by reverse transcription with RNA-dependent DNA polymerase from avian myeloblastosis virus using oligo(dT)-bound to cellulose as a primer. 2. Unlabelled thymosin alpha 1 precursor cDNA-cellulose was synthetized in a preparative scale to be used in affinity chromatography. 3. Poly(A)-mRNA from calf thymus was subjected to cDNA-cellulose affinity chromatography and an active messenger obtained in a yield of 3% respect to the total thymus poly(A)-mRNA chromatographied. 3. The analysis of the translation products of the purified mRNA have shown it as the thymosin alpha 1 precursor messenger.

Animals↗
PubMed1982

Co-therapy in a clinic for schizophrenia.

In the outpatient treatment of schizophrenia, the routine practice of providing each patient with two co-therapists results in several benefits: reduced dependency, increased expertise, objective monitoring, added support and morale for the therapists, more obvious and therefore more reversible countertransference, more obvious and therefore more treatable "splitting" and "projecting." Despite these benefits, problems arise in the two-therapist arrangement. They are discussed under the following headings: loyalty conflicts; limit setting; crisis management; family work. The authors report on techniques of co-therapy problem prevention and problem resolution. Though not always successful, co-therapy continues to be a useful model of service delivery to schizophrenic outpatients.

Ambulatory Care↗
PubMed1981

Purification of thymus mRNA coding for a 16,000-dalton polypeptide containing the thymosin alpha 1 sequence.

A mRNA fraction purified by preparative polyacrylamide disc gel electrophoresis from calf thymus polysomes codes for a polypeptide(s) having a mass of 16,000-17,000 daltons. This polypeptide contains amino acid sequences corresponding to residues 11-18 and 19-25 of thymosin alpha 1. The yield of the octapeptide indicates that the 16,000-dalton peptide is the major product formed in the cell-free synthesis system containing the purified mRNA.

Animals↗
PubMed1978

Regulation of the oxidative phase of the pentose phosphate cycle in mussels.

1. The mechanisms that control the oxidative phase of the pentose phosphate cycle in mussel hepatopancreas were investigated. 2. The effects of GSSG (oxidized glutathione) on the inhibition of glucose 6-phosphate dehydrogenase by NADPH [Eggleston & Krebs (1974) Biochem. J. 138, 425-435] extend to 6-phosphogluconate dehydrogenase. 3. The effect of GSSG on both enzymes increases as the [NADP+1]/[NADPH] ratio decreases; greater percentage deinhibition always was obtained for 6-phosphogluconate dehydrogenase. 4. Increasing concentration of GSSG increased the percentage deinhibition. This effect is more pronounced with 6-phosphogluconate dehydrogenase. 5. We confirmed the apparent imbalance between the activities of the two enzymes [sapag-Hagar, Lagunas & Sols (1973) Biochem. Biophys. Res. Commun, 50, 179-185] in the presence of 10mM-Mg2+. 6. The imbalance practically disappears when the substrate concentrations are less than saturating and Mg2+ approaches physiological concentrations. 7. The addition of GSSG at physiological concentrations allows the activities of both enzymes to be measured at high [NADPH]/[NADP+] ratios ratios and the co-operative action of GSSG and Mg2+ on the imbalance between the two enzymes to be verified. 8. The control of the activity of the two enzymes of the pentose cycle could be carried out by deinhibition of the two dehydrogenases and by the intracellular concentrations of substrates and inorganic ions.

Animals↗
PubMed1978

Translation of mRNA from calf thymus in the wheat germ system: evidence for a precursor of thymosin alpha1.

When translated in the wheat germ system, mRNA from fresh calf thymus stimulates incorporation of radioactive amino acids into an acid-insoluble product, and 10--20% of the total radioactivity incorporated is precipitated with antisera to active thymosin fractions. In sodium dodecyl sulfate disc gel electrophoresis, radioactivity was recovered mainly in two peptides, corresponding to 16,000 and 11,000 daltons; the latter probably represents incomplete chains. Tryptic digests of each of these peptides yielded fragments corresponding to the sequence of residues 15--19 of thymosin alpha1; these peptides were characterized by cochromatography with digests of synthetic thymosin alpha1 and by Edman degradation. Thus, the 16,000-dalton peptide synthesized in the cell-free system appears to be q precursor of thymosin alpha1 and possibly of other peptides in the fractions isolated from calf thymus. The results support the conclusion that this peptide is synthesized in the thymus gland.

Cell-Free System↗