Biomedical subjects

M Freire

Publications and source records attributed to M Freire.

At least 37 records · Page 2Linked to original sources

PubMed1994

Prothymosin alpha and factors from calf thymic cells decrease expression of Thy 1.2 antigen among small thymocytes from C57BL/6 mice.

In this work we studied the effect of Prothymosin alpha (ProT alpha) and other thymic factors on the expression of Thy 1.2 antigen (a T-cell marker) and the activities of adenosine deaminase (ADA, E.C. 3.5.4.4), N-acetyl-beta-D-glucosaminidase (NABG, E.C. 3.2.1.30), beta-glucuronidase (BG, E.C. 3.1.1.1) and serine-esterase (SE, E.C. 3.4.21)., the levels of which change during the T-cell differentiation process among small thymocytes obtained from C57BL/6 mice. Incubation of small thymocytes in the presence of ProT alpha, Thymus Extracts (TE) or supernatants prepared from thymic stromal cells (TSCS) or thymocytes (TS) reduced the proportion of cells killed by anti-Thy 1.2 monoclonal antibodies but did not affect the enzymatic activities studied. This is the first evidence that ProT alpha affects Thy 1.2 expression in vitro.

Acetylglucosaminidase↗

PubMed1994

Characterization of T cell responses to purified leishmania antigens in subjects infected with Leishmania chagasi.

T cell responses to lipophosphoglycan-associated protein (LPG-AP) and the rgp63 antigens were studied in subjects with either asymptomatic L. chagasi infection or cured visceral leishmaniasis. The [3H]-thymidine uptake of lymphocytes stimulated with LPG-AP and rgp63 (mean +/- SD) was 14275 +/- 5048 and 3523 +/- 1678 cpm, respectively, for subjects with asymptomatic L. chagasi infection and 20046 +/- 5102 and 5086 +/- 3500 cpm, respectively, for subjects cured of visceral leishmaniasis. The responses to LPG-AP in both asymptomatic and cured visceral leishmaniasis were higher (P < 0.01) than those observed with rpg63. LPG-AP induced IFN-gamma production in all subjects studied, while rgp63 did not induce lymphocyte proliferation or IFN-gamma production in the majority of the subjects tested. IFN-gamma levels in cultures stimulated with LPG-AP were 103 +/- 81 pg/ml in individuals with asymptomatic L. chagasi infection and 127 +/- 123 pg/ml in subjects cured of visceral leishmaniasis. IFN-gamma levels in cultures stimulated with LPG-AP from subjects with asymptomatic L. chagasi infection were comparable to those observed in subjects cured of visceral leishmaniasis (P > 0.05). These data indicate that LPG-AP is recognized and induces T cell proliferation and IFN-gamma production in subjects with protective immune response against Leishmania chagasi.

Adolescent↗

PubMed1993

Transcript levels of thymosin beta 4, an actin-sequestering peptide, in cell proliferation.

Thymosin beta 4 (beta 4) is an ubiquitous 5-kDa peptide that has been identified as an actin-sequestering peptide. In this work, Northern blot analysis was used to study the beta 4 mRNA levels during the cell cycle of rat thymocytes and hepatocytes as well as in human lymphocytes from patients with leukemia. beta 4 mRNA was found in all the stages of thymocyte and hepatocyte cell cycle, showing an increase in the S-phase which was maintained during the G2 and M phases. Incubation of splenic T-cells with concanavalin A, phorbol myristate acetate or the ionophore A23187 lead to a similar increase of beta 4 transcript during the S-phase. The increase in beta 4 mRNA observed in the G2/M boundary of the cell cycle, together with its ability to inhibit actin polymerization, suggests a possible role of beta 4 in the the morphological changes and actin redistribution occurring during the cytokinesis.

Actins↗

PubMed1993

Prothymosin alpha is phosphorylated in proliferating stimulated cells.

Prothymosin alpha is a widely distributed polypeptide whose function, though unknown, seems to be related to cell proliferation. In vitro, it is a substrate for casein kinase-2. In this work, extracts of mitogenically stimulated murine splenic lymphocytes labeled with [32P] orthophosphate were found to contain [32P]prothymosin alpha. Phosphorylation activity was highly dependent on mitogenic activation with concanavalin A plus interleukin-2. While cells remained viable, phosphorylation increased with stimulation time in the presence of [32P]orthophosphate. Structural analysis showed that prothymosin alpha was phosphorylated at Thr residues located among its first 14 amino acids, whereas its in vitro phosphorylation by casein kinase-2 affects both Ser and Thr residues in this fragment, apparently in similar proportions. Thus, casein kinase-2 seems not to be responsible for the phosphorylation of prothymosin alpha in vivo. Prothymosin alpha was also found to be phosphorylated in proliferating murine thymocytes and HeLa cells; the phosphorylation sites were the same as in splenic lymphocytes, but the rate of phosphorylation was about 5 times lower. In thymocytes and subconfluent HeLa cells, the [32P]prothymosin alpha concentrations of the cytosolic and nuclear fractions were similar; in splenic lymphocytes, [32P]prothymosin alpha was found mostly in cytosol.

Amino Acid Sequence↗

PubMed1993

Distribution of serine esterase activity in the lymphoid system of C57BL/6 mice, effect of aging on the enzymatic activity.

1. The activity of serine esterase (SE) was investigated in the lymphoid system of C57BL/6 mice. SE activity increased in the lymphoid tissues with their content of mature T-lymphocytes, except that high levels were also observed in various populations of bone marrow cells. 2. The maturation of T-lymphocytes in the thymus was accompanied by an increase in their SE activity. 3. Experiments on the influence of age on SE activity showed that while thymocytes were not affected, a three-fold increase in activity occurs in spleen lymphocytes between the ages of 26 and 78 wk.

Aging↗

PubMed1993

Studies on the control of visceral leishmaniasis: validation of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) for field diagnosis of canine visceral leishmaniasis.

The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA), the latest version of the enzyme-linked immunosorbent assay, uses antigen-coated beads. A 96-well plate can be run in 20 min without electricity or expensive equipment. We compared the FAST-ELISA, a standard ELISA, and an indirect immunofluorescent assay (IFA) for evaluation of canine leishmaniasis under field conditions using samples from 161 dogs from our longitudinal study in the endemic area of Jacobina, Bahia, Brazil. Organisms were isolated by culture (NN medium) or by inoculation of hamsters with samples from 59 of the dogs. When plasma were tested, we found a sensitivity of 88% and a specificity of 90% using the FAST-ELISA with a spectrophotometer. Using the same plasma samples, the IFA had a sensitivity of 75% and a specificity of 93%. The standard ELISA had a sensitivity of 90% and a specificity of 85%. When whole blood was tested with the FAST-ELISA, we found a sensitivity of 85%. There was no significant difference between visual and spectrophotometric results with plasma or whole blood. The FAST-ELISA system provides a sensitive, specific, and field-adaptable test for canine visceral leishmaniasis, which can be evaluated quickly without the use of a microscope or spectrophotometer.

Animals↗

PubMed1993

Percutaneous catheter drainage of tuberculous psoas abscesses.

Six patients with 7 tuberculous psoas or ilio-psoas abscesses were treated by CT-guided catheter drainage and chemotherapy. The abscesses (5 unilateral and 1 bilateral) were completely drained using a posterior or lateral approach. The abscess volume was 70 to 700 ml (mean 300 ml) and the duration of drainage 5 to 11 days (mean 7 days). Immediate local symptomatic improvement was achieved in all patients, and there were no procedural complications. CT follow-up at 3 to 9 months showed normalization in 5 patients, 2 of whom are still on medical therapy. One patient, who did not take the medication regularly, had a recurrent abscess requiring new catheter drainage after which the fluid collection disappeared. Percutaneous drainage represents an efficient and attractive alternative to surgical drainage as a supplement to medical therapy in the management of patients with large tuberculous psoas abscesses.

Adult↗

PubMed1992

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.

Amino Acid Sequence↗

PubMed1992

Thymosin alpha 1 is a native peptide in several tissues.

Failure to detect thymosin alpha 1 (T alpha 1) in tissue extracts prepared by procedures that prevent proteolytic activity has hitherto supported the suggestion that T alpha 1 is not a natural peptide, but the product of uncontrolled proteolysis of prothymosin alpha (ProT alpha), a polypeptide that includes T alpha 1 at its NH2 terminus. In this work, purification by isoelectric focusing of a product with the same isoelectric point as synthetic T alpha 1, and its further characterization, demonstrated that T alpha 1 is present as a native peptide in calf thymus and in several lymphoid and non-lymphoid rat tissues. T alpha 1 shows abnormal chromatographic behaviour which appears to be due to association with other components in tissue extracts. In all the tissues studied, T alpha 1 was present in higher concentration than ProT alpha (80-183 and 44-123 micrograms per gram of tissue, respectively). The ProT alpha/T alpha 1 ratio did not change when no measures were taken to prevent proteolysis during tissue homogenization.

Animals↗

PubMed1991

Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration.

Prothymosin alpha (ProT alpha) is a widely distributed acidic protein whose function has been related to cell proliferation. We have analyzed the expression of the rat ProT alpha gene in several proliferative systems: concanavalin A (ConA)/interleukin-2-stimulated thymocytes, ConA-stimulated splenic T-lymphocytes, and hepatocytes proliferating during liver regeneration. In these systems, ProT alpha mRNA was detected in all stages of the cell cycle, with maximal increments (2-4-fold) at the beginning of the S phase. By contrast, the mRNAs for proliferating cell nuclear antigen/cyclin and histone H3, two cell-cycle-regulated proteins, were hardly detected in resting cells but increased notably at the G1/S boundary and in the S phase, respectively. Treatment of T-cells with the calcium ionophore A23187 increased ProT alpha mRNA levels 2.5-fold, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, had no effect on ProT alpha gene expression. Incubation of ConA-stimulated T-cells with hydroxyurea, a DNA synthesis inhibitor, did not decrease the levels of ProT alpha mRNA, indicating that its expression is independent of DNA synthesis. These findings suggest that ProT alpha is required throughout all the stages of the cell cycle, resembling a constitutively expressed gene rather than one strictly involved in cell proliferation.

Animals↗

PubMed1991

New developments in an "expanded stick" model for coding, graphic representation and metric analysis of tracer-filled or Golgi-impregnated neurons, including spines and varicosities.

A new data model allowing the coding, graphic representation and metric analysis of dendritic and axonal processes including spines and varicosities, is here described. The model is implemented in an interactive light microscope-computer system and stores the three-dimensional coordinates of the selected neuronal points, their topological identifiers, and the width of the processes. In addition codes for "nature", and "shape" are stored in the data array. The "nature" code identifies structures such as perikaryon, axon, apical dendrite, basal dendrite, etc. The "shape" code defines varicosities and spines and allows their graphic representation. At present, the coding for metric analysis is made at a final magnification of x1875, with a resolution of 0.11 microns in the objective plane. The graphic representation of spines and varicosities is an ellipse, whose major axis is the length of spines and varicosities and the minor axis the width of these structures. From this "expanded stick" model a computer program calculates the length, area and form factor of the perikaryon; the mean length, width and area of each neuronal branch; the distribution of varicosity and spine number and their size (length and width) per length interval; the total number of processes, varicosities and spines; and the total length and area of the processes.

Animals↗

PubMed1991

[The bone manifestations in 94 cases of sarcoidosis].

94 patients diagnosed as having sarcoidosis were studied in Galicia in order to evaluate the frequency of bone manifestations, as well as to analyse the clinical and radiological features. Bone lesions were found in 10 patients (10.6%) with a median age of 47.4 years, the predominant sex being female in this group (70%). The lesions were of two types: a) osteolytic on metacarpal (4 cases), nasal bones (1 case), sacrum (1 case), femur (1 case); b) osteosclerosis on femur and fibula (1 case), acrosclerosis (3 cases), metacarpal (1 case). The first lesions were associated to chronic types of sarcoidosis with skin and lung involvements. The second lesions were associated to a sub-acute type of Löfgren. We highlight the behaviour of both types of bone lesions and comment on the physiopathology of the sclerotic lesions.

Age Factors↗

PubMed1990

HRP-filling of neurons and axonal arbors in fixed brain slabs.

Visually-guided introduction of HRP into perfusion-fixed hamster brain slabs was carried out by three procedures: pressure or extracellular iontophoretic injections, introduction of a small HRP-crystal on the closed tip of a micropipette, and by dropping a very small drop of HRP solution on the ventricular surface. The prefixation was made with a solution of 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer in time periods ranging from 4 to 26 days. The introduction of HRP-crystals into brain slabs did not produce HRP-filling of cells and processes, but only a granular background of HRP diffusion. Injections into adjacent brain slabs of an HRP-containing solution did produce, in every case, some degree of labelling of neuronal somata, dendrites, individual axons, and fibre bundles. Several cells showed dense HRP-filling of the soma and processes, including dendritic spines and axonal varicosities. Dense HRP-filled axonal terminal arbors were seen in the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) after iontophoretic injections in the optic tract. The good ultrastructural preservation achieved by perfusion of the fixative solution was not affected by the subsequent injection procedures, and the pre- and post-synaptic specializations of HRP-filled axons were recognizable. This approach may prove to be useful in correlative light and electron microscope studies of the synaptic relationships between HRP-filled neurons and axons.

Animals↗

PubMed1990

Study of Golgi-impregnated material using the confocal tandem scanning reflected light microscope.

The tandem scanning reflected-light microscope (TSM) is a real-time, direct-view confocal microscope. Only those points in the specimen situated in the focal plane contribute information to the image. A Tracor Northern TMS with piezo-electric control of the objective lens was used to generate 3-D images from Golgi-impregnated hamster cerebral cortex. Stereoscopic pairs of images were recorded as 35-mm colour film transparencies by photographing while automatically through-focusing along inclined axes. Transferring the image via a TV camera to the computer, stereo-pairs were obtained by oblique through-focusing and summing, displaying maximum intensity data in each line of sight. Pseudocolour topographic displays were generated by assigning the pixel value in a zeta map image as the focal depth at which the back-scattered light signal was maximal. The TSM was also modified so that a conventional transmitted-light image with a large depth of field could be obtained simultaneously as the very shallow depth of field confocal back-scattered-light image seen at any focus level. The conventional image is a silhouette of the impregnated neurons: the top surface of the cell is not visible and the relationships of processes that cross over cell bodies cannot be discerned. TSM gives a high-contrast image. The Golgi precipitate over the neuronal surface is resolved as globular or ovoid, coloured particles. The smaller particles also cover the dendritic spines. All the confocal range (extended focus) image display methods satisfactorily demonstrated the 3-D arrangement of cell bodies and processes in the chosen volume.

Animals↗

PubMed1990

Women as victims of torture.

This is a retrospective study on the effects of torture on Latin American refugee women in Toronto. Thirty-six cases of female torture victims are reviewed. The cases are divided in 2 groups, according to whether they experienced physical and psychological torture or only psychological assaults. Both groups are compared in terms of demographic characteristics, social and/or political involvement prior to the traumatic experiences, symptoms for which they sought psychiatric intervention and recovery rates. The symptoms presented by all women are consistent with those described in the literature for torture victims, regardless of their sex. The main findings are that women who experienced direct physical and psychological violence more frequently had persistence of symptoms than women who experienced only psychological violence.

Adolescent↗

PubMed1989

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes.

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.

Animals↗

PubMed1989

The expression of prothymosin alpha gene in T lymphocytes and leukemic lymphoid cells is tied to lymphocyte proliferation.

We isolated the cDNA for human prothymosin alpha (ProT alpha) from a human peripheral T-cell library using two synthetic oligonucleotides as probes. Hybridization studies with this cDNA showed that the ProT alpha mRNA is detectable in all the rat tissues studied but is most abundant in thymus and within this gland mainly synthesized by thymocytes. In the T-cell lineage, its expression is higher in proliferative immature thymocytes than in pre- and post-thymic T lymphocytes. A quite similar pattern was obtained with the proliferation-related protein proliferating cell nuclear antigen/cyclin. These data show that ProT alpha mRNA levels change with the maturation stage of T-cells. Moreover, the amount of ProT alpha transcript is increased in lymphocytes from human patients with leukemias. Our findings indicate a role for ProT alpha linked to lymphocyte proliferation.

Amino Acid Sequence↗

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↗