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Biomedical subjects

M A Alonso

Publications and source records attributed to M A Alonso.

At least 55 records · Page 3Linked to original sources
PubMed1997

Structural and biochemical similarities reveal a family of proteins related to the MAL proteolipid, a component of detergent-insoluble membrane microdomains.

The MAL gene encodes a proteolipid protein displaying a cell type-specific pattern of expression, including T lymphocytes, myelin-forming cells, and epithelial renal MDCK cells, which has been recently identified as a component of detergent-insoluble membranes known to be enriched in glycolipids and cholesterol. Sequence alignment revealed a high degree of conservation of the MAL protein across species and evidenced the existence of a significant level of overall identity between MAL and two other proteins, BENE and the plasmolipin proteolipid. Moreover, using subcellular fractionation of transiently transfected COS-7 cells, both MAL and BENE were identified in detergent-resistant membranes. These results suggest the existence of a novel family of MAL-related proteins (including MAL, BENE, and plasmolipin) with primary structure homologies and with the distinctive features of having unusual biochemical properties such as lipid-like behaviour and/or the ability to reside in glycolipid-enriched membrane microdomains.

Amino Acid Sequence↗
PubMed1997

The MAL proteolipid is a component of the detergent-insoluble membrane subdomains of human T-lymphocytes.

The human mal gene, identified during a search for cDNAs selectively expressed during T-cell development, encodes a highly hydrophobic protein belonging to a group of proteins, termed proteolipids, characterized by their unusual property of being soluble in organic solvents used to extract cell lipids. To study the localization of the MAL protein we have prepared stable transfectants expressing the MAL protein tagged with a c-myc epitope (MAL/c-myc) using human epithelial A-498 cells. Immunofluorescence analysis suggested that MAL/c-myc is localized mainly to cholesterol-enriched structures with a post-Golgi location and, at low levels, in early endosomes. Moreover, extraction of A-498 cell membranes with Triton X-100 (TX100) and fractionation by centrifugation to equilibrium in sucrose gradients demonstrated the presence of MAL/c-myc in the detergent-insoluble buoyant fraction, known to be enriched in glycolipids and cholesterol. To compare the behaviour of MAL in T-cells with that in epithelial A-498 cells, we prepared stably transfected cells expressing MAL/c-myc using human Jurkat T-cells. When TX100 extracts from Jurkat cells were subjected to centrifugation to equilibrium in sucrose gradients we found MAL exclusively in the floating fractions, together with molecules characteristic of the T-cell insoluble complexes, such as the tyrosine kinase p56lck, the glycosylphosphatidylinositol-anchored protein CD59 and the ganglioside GM1. These results, taken together, indicate that the MAL proteolipid is a component of the detergent-resistant membrane microdomains present in T-lymphocytes, and suggest that MAL might play a role in modulating the function of these microdomains during T-cell differentiation.

Animals↗
PubMed1997

[Community-acquired pneumonia (CAP) with hospital treatment. The value of the clinical picture and complementary exams in predicting its etiology].

We studied 162 patients with community-acquired pneumonia admitted for hospital treatment, in order to determine the utility of clinical and ancillary examinations for predicting etiology and guiding the most appropriate empirical treatment. Acute first appearance of symptoms, purulent expectoration, chest sounds indicating lung condensation, pleuritic chest pain and leukocytosis over 12,500/ml were statistically significant in differentiating typical pneumonias from those with atypical behavior patterns. The last two features were the most relevant according to multivariate analysis. We conclude that careful taking of case histories and basic blood testing continue to be relevant and must not be considered anachronistic for the differential diagnosis of community-acquired pneumonias.

Adolescent↗
PubMed1997

A tandem array of Sp-1 sites and a reverse initiator element are both required for synergistic transcriptional activation of the T-cell-specific MAL gene.

We have characterized the three cis elements responsible for promoter strength present in the 5'-flanking proximal region of MAL, a human T-cell-specific gene encoding a proteolipid protein present in detergent-insoluble complexes of high molecular weight. The first element consisted of an initiator sequence that, curiously, was present in reverse orientation compared to that of the standard initiator elements. The other two elements were contained in a region of 126 bp upstream of the mRNA initiation site, and consisted of a tandem array of one GC box and one GA box. The GC box corresponds to a consensus site for the nuclear factor Sp1, whereas the GA box deviates from this consensus, although it was able to compete for the binding of Sp1 in vitro and to respond to trans-activation by Sp1 in vivo. This simple promoter lacks an apparent TATA box and lost more than 99% of its activity when a fragment of 60 bp containing the GC and GA boxes was deleted. A synergistic effect on transcriptional activation was observed in the presence, but not in the absence, of the initiator element when both GC and GA boxes were present.

Base Sequence↗
PubMed1997

Nitric oxide production induced by herpes simplex virus type 1 does not alter the course of the infection in human monocytic cells.

Undifferentiated U937 cells were not susceptible to herpes simplex virus type 1 (HSV-1) infection, but after differentiation with phorbol 12-myristate 13-acetate an increase in the permissivity to the virus was observed accompanied by the production of significant levels of viral particles. High levels of nitric oxide (NO) were produced in differentiated U937 cells infected with HSV-1. This production was comparable to that observed after addition of the NO donor glycerine trinitrate. The levels of NO drastically decreased when the cells were incubated with L-monomethyl arginine (L-NMA), an inhibitor of NO synthase. Although similar levels of NO were sufficient to decrease susceptibility of U937 cells to other viruses, neither incubation with NO donors nor addition of L-NMA altered the permissiveness to HSV-1 infection. Thus, these results suggest that NO does not interfere with the replication of HSV-1 in U937 cells.

Cell Differentiation↗
PubMed1997

The role of verb tense and verb aspect in the foregrounding of information during reading.

In three experiments, we explored the role of verb tense and verb aspect in subjects' construction of mental models while reading. In the first and second experiments, subjects read paragraphs in which verb tense was manipulated to assign current and past attributes (job descriptions) to a main character. The results showed that on-line accessibility was faster for current attributes. The results of the third experiment showed that verb aspect manipulations regarding character actions affect the speed at which the characters are available for recognition.

Humans↗
PubMed1996

Use of messenger RNA differential display to identify interleukin-11-responsive genes in human umbilical cord blood mononuclear cells: IL-11 upregulates the expression of the hMAL gene.

Human umbilical cord blood (HUCB) mononuclear cells represent a source of hematopoietic stem and progenitor cells, including cells responsive to interleukin-11 (IL-11). To investigate the molecular mechanisms associated with IL-11 action, we have used HUCB mononuclear cells as a model system to identify genes that are transcriptional targets of IL-11. Using the technique of messenger RNA differential display, we have identified 17 candidate cDNA differentially expressed in mononuclear cells incubated without and with IL-11. Fifteen of these cDNA were recovered, and 11 were sequenced. DNA sequence analysis has identified one of these cDNA as being the human MAL gene, originally identified as a marker for intermediate stages of T cell differentiation. Northern analysis using a MAL-specific probe confirms the upregulation of MAL by IL-11 in HUCB cells.

Cells, Cultured↗
PubMed1996

Differential susceptibility of aeromonads and coliforms to cefsulodin.

Volumn 62, no. 6, p. 1885, Abstract, line 3: "For 102 of 129 Aeromonas isolates" should read "For 102 of 130 Aeromonas isolates." Page 1887, column 1, lines 16 and 17: "and for 13 of 21 coliform isolates" should read "and for 13 of 94 coliform isolates." [This corrects the article on p. 1885 in vol. 62.].

Journal Article↗
PubMed1996

Differential susceptibility of aeromonads and coliforms to cefsulodin.

Cefsulodin was evaluated as a potential selective agent for aeromonads. Resistance of Aeromonas and coliform isolates was determined by using a standard disk diffusion technique. A total of 119 Aeromonas and 78 coliform strains were isolated. For 102 of 130 [corrected] Aeromonas isolates (environmental and reference strains), the MIC of cefsulodin was < 8 micrograms/ml. Results of MIC tests by the agar dilution method showed that a concentration of cefsulodin of 10 micrograms/ml or less inhibited the growth of 96% of isolates. In comparison, for 81 of 94 coliform isolates (environmental and reference strains), the MIC of cefsulodin was > 32 micrograms/ml. Because cefsulodin suppresses growth of Aeromonas and other oxidase-positive organisms, total coliform (TC) and Escherichia coli counts on Chromocult Coliform agar (CC agar) without cefsulodin and on CC agar with 10 mg of cefsulodin per liter (CC-CFS) were compared. Variance analysis of data from 14 sewage-polluted irrigation water specimens did not demonstrate any statistically significant difference in the enumeration of E. coli with CC and CC-CFS media. On average, the CC agar recovered 2.46 times as many TCs as CC-CFS. However, Aeromonas colonies made up an average of 58.6% of the TC counts on CC agar. Because no Aeromonas spp. were recovered on CC-CFS, background interference was eliminated and the counts that were obtained reflected more accurately the number of TCs. Results of this study suggest that cefsulodin may be a useful selective agent against Aeromonas spp. which should be included in coliform chromogenic media when high levels of accompanying flora are expected.

Aeromonas↗
PubMed1994

Alternative splicing of human T-cell-specific MAL mRNA and its correlation with the exon/intron organization of the gene.

Sequence analysis of the T-cell-specific MAL gene revealed four exons, each encoding a hydrophobic, presumably membrane-associated, segment and its adjacent hydrophilic sequence. Amplification by the polymerase chain reaction of cDNA from different T-cell samples indicated the existence of four different forms of MAL mRNA, termed MAL-a, -b, -c, and -d, that arise from differential usage of exons II and/or III. As the three introns were located between complete codons, the reading frame was maintained in all the transcripts. A model resembling the structures postulated for different proteolipid proteins is proposed for the protein encoded by each alternative mRNA species.

Alternative Splicing↗
PubMed1994

Genomic structure and subcellular localization of MAL, a human T-cell-specific proteolipid protein.

Genomic DNA clones containing the T-cell-specific human MAL gene were isolated. Restriction and sequence analysis revealed four exons and three introns. Each hydrophobic segment of MAL together with its adjacent hydrophilic sequence correlates closely with one exon of the gene. RNase protection analysis revealed that the previously described MAL mRNA, which contains the sequences present in the four exons, is the mRNA species predominant in T-cells. A remarkable similarity was found between the hydrophobicity pattern of MAL and those of the peripheral membrane protein 22 (PMP-22) and the 16-kDa proteolipid of vacuolar H(+)-ATPase. Direct evidence supporting that MAL is a proteolipid was obtained by extracting bacterial lysates expressing recombinant MAL protein with lipophilic solvents used to extract lipids. The use of two different antibodies raised against distinct peptides from the MAL molecule has allowed the localization of MAL in the endoplasmic reticulum of T-cells. This subcellular localization is in agreement with the presence of a RWKSS motif in the COOH-terminal tail of MAL, next to its last putative transmembrane domain, that fits with one of the consensus sequences (RXKXX) for residency in the endoplasmic reticulum for transmembrane proteins. A possible function for MAL protein in T-cells is discussed based on its subcellular localization and the unique lipid-like properties of the proteolipid proteins.

Amino Acid Sequence↗
PubMed1993

Intracellular retention and degradation of human mutant variant of a alpha 1-antitrypsin in stably transfected Chinese hamster ovary cell lines.

Normal (PiM) and mutant (PiZ) variants of human alpha 1-antitrypsin (alpha 1-AT) cDNA, cloned into the pTnd eucaryotic expression vector, were used to derive recombinant Chinese hamster ovary cell lines permanently expressing the corresponding proteins. Secretion, accumulation and glycosylation of PiM and PiZ alpha 1-AT proteins were studied in the presence of various transport-impairing drugs. Pulse-chase, followed by immunoprecipitation as well as immunofluorescence experiments showed that the PiZ alpha 1-AT undergoes continuous degradation that was prevented by Brefeldin A but not by incubation of cells at 16 degrees C. Moreover, monensin partially impaired the glycosylation of both PiM and PiZ alpha 1-AT but not their secretion nor the degradation of PiZ alpha 1-AT. Those results suggest that PiZ alpha 1-AT degradation occurs in the cis-Golgi network, a compartment located between the endoplasmic reticulum and the Golgi stack. The process did not apparently involve lysosomes since it was insensitive to chloroquine. In addition, inhibition of PiM and PiZ alpha 1-AT glycosylation and secretion by tunicamycin did not result in the accumulation of the protein, but instead in its rapid lag-free degradation. Treatment of cells with the A23187 ionophore, for a short (60 min) but not a long (24 h) period, improved the secretion of PiZ alpha 1-AT in a similar way as it affects retention of naturally endoplasmic-reticulum-resident proteins, suggesting that the small proportion of PiZ alpha 1-AT which is not degraded or secreted, but accumulates in the endoplasmic reticulum, is back transported as a partially glycosylated species from the post endoplasmic reticulum compartment in which degradation takes place.

Animals↗
PubMed1993

Feeding status-related effects of 17 beta-estradiol on liver 3-hydroxy-3-methylglutaryl CoA reductase.

Total and expressed 3-hydroxy-3-methylglutaryl CoA reductase was determined in hepatic microsomes prepared from fasted and fed control rats and 17 beta-estradiol-treated rats. Plasma, liver and microsome cholesterol levels were also measured. Fasting resulted in significantly lower total and expressed specific activity of HMG-CoA reductase such that the expressed/total activity ratio doubled regardless of the endocrine status of the animals. Rats treated for either 3 or 21 days with 50 micrograms 17 beta-estradiol and starved for 24 hours exhibited greater reductase activity than untreated animals, the percentage of enzyme in effective form being maintained. When rats were fed on a commercial diet or a 4% cholestyramine-diet for five days, thus presenting higher basal HMG-CoA reductase levels, the stimulatory effect of 17 beta-estradiol was not statistically significant. In contrast, when total reductase was reduced by dietary cholesterol, the estrogen effect was accentuated. Long-term estradiol treatment was associated with increases in liver weight, decreases in free and total plasma cholesterol, and microsome cholesteryl ester accumulation; while short-term estradiol treatment increased plasma esterified and total cholesterol as well as the microsomal content of cholesteryl esters. These findings suggest (i) that the two 17 beta-estradiol treatments employed in this study are associated with higher HMG-CoA reductase levels and this is particularly apparent when cholesterol synthesis occurs at a low rate and (ii) that plasma cholesterol levels are not strictly correlated with hepatic HMG-CoA reductase activity.

Animals↗
PubMed1993

High level expression in Escherichia coli cells and purification of poliovirus protein 2Apro.

The poliovirus protease 2Apro has been produced to high levels in Escherichia coli using the inducible system that utilizes T7 RNA polymerase. The protease coding sequences that contained an additional AUG to start translation were cloned in pET vectors. Synthesis of 2Apro was induced by IPTG or IPTG plus rifampicin, the levels of the protein made being higher when IPTG alone was used. The expression of the protein is not toxic for E. coli cells and can be readily visualized by Coomassie blue staining of total bacterial protein extracts separated in polyacrylamide gels. Centrifugation of the broken bacterial cells sediments more than 95% of the 2Apro synthesized at a 95% purity level after sarkosyl treatment. Antibodies raised against 2Apro in E. coli recognize a 16K protein in poliovirus-infected cells. In addition, 2Apro shows activity in trans as measured by the cleavage of p220 in HeLa cell extracts and by cleavage of a poliovirus protein substrate that contains the junction between the P1 and P2 polypeptides.

Base Sequence↗
PubMed1992

CD4 gene transcription is transiently repressed during differentiation of myeloid cells to macrophage-like cells.

The CD4 glycoprotein, which serves as receptor for human immunodeficiency virus (HIV), is expressed in several types of cells of hematopoietic origin, including T lymphocytes and monocytes. Triggering differentiation of peripheral blood monocytes, monocytic U-937 or promyelocytic HL-60 precursor cells to macrophage-like cells by phorbol ester treatment transiently induced both a rapid reduction in surface CD4, demonstrated by flow-cytometry analysis, and a gradual loss of CD4 mRNA, revealed by Northern-blot analysis. Experiments in HL-60 cells to determine the cause of the observed decay in CD4 mRNA levels suggested that the half-life of CD4 transcripts did not diminish but increased after phorbol ester stimulation. Direct measurement of CD4 gene transcription by run-on analysis indicated that the rate of synthesis of new CD4 mRNA molecules was reduced approximately 10-fold after phorbol ester stimulation, whereas the rate of synthesis of c-fos mRNA resulted in a 2.5-fold increase. These data suggest that phorbol ester treatment specifically reduces CD4 mRNA levels by repressing CD4 gene transcription. These findings may be relevant to understand the regulation of CD4 gene expression during differentiation.

Blotting, Northern↗
PubMed1992

Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1.

The induction of the AP-1 transcription factor has been ascribed to the early events leading to T cell differentiation and activation. We have studied the regulation of AP-1 activity in human peripheral blood T lymphocytes stimulated through the activation inducer molecule (AIM)/CD69 activation pathway. Phorbol esters are required to induce AIM/CD69 cell-surface expression as well as for triggering the proliferation of T cells in conjunction with anti-AIM mAb. Mobility shift assays showed that addition of anti-AIM mAb to PMA-treated T lymphocytes markedly enhanced the binding activity of AP-1 to its cognate sequence, the phorbol ester response element. In contrast, anti-AIM mAb did not induce any change in the binding activity of NF-kappa B, a transcription factor whose activity is also regulated by protein kinase C. The increase in AP-1-binding activity was accompanied by the marked stimulation of the transcription of c-fos but not that of c-jun. Blockade of the DNA-binding complexes with an anti-Fos mAb demonstrated a direct participation of c-Fos in the AP-1 complexes induced by anti-AIM mAb. Most of the AP-1 activity could be eliminated when the anti-AIM mAb was added to the culture medium in the presence of cycloheximide, suggesting that de novo protein synthesis is crucial for the induction of AP-1-binding activity. These data provide the evidence that activation of human peripheral blood T cells through the AIM activation pathway regulate the activity of AP-1. Therefore, this pathway appears as a crucial step in the initiation of early T cell activation events.

Antibodies, Monoclonal↗
PubMed1992

Tumor necrosis factor-alpha production induced in T lymphocytes through the AIM/CD69 activation pathway.

Human activation inducer molecule (AIM/CD69), a dimeric glycoprotein of 33 and 27 kDa, is the earliest inducible cell surface antigen expressed during lymphocyte activation, which has been also involved in lymphocyte proliferation. Although AIM is absent from peripheral blood resting lymphocytes, it is expressed by in vivo activated lymphocytes infiltrating sites of chronic inflammation in several pathologies, as well as by lymphocytes after in vitro activation with different stimuli. We have investigated the possibility that tumor necrosis factor-alpha (TNF-alpha) gene expression and protein secretion could be induced in peripheral blood T cells through the AIM/CD69 molecule. Anti-AIM monoclonal antibodies (mAb) were able to induce TNF-alpha secretion in T cells when protein kinase C (PKC) was simultaneously activated by treatment with phorbol esters. TNF-alpha secretion was detected at 24 h and peaked at day 3 upon T lymphocyte activation with anti-AIM mAb. Immunoprecipitation studies with an anti-TNF-alpha mAb from surface iodinated T cells activated through AIM, demonstrated that TNF-alpha first appeared as a cell surface molecular form of 26 kDa, which is subsequently released to the extracellular medium as the 17-kDa molecular form of TNF-alpha. AIM stimulation dramatically increased TNF-alpha mRNA levels, and this mRNA induction and subsequent TNF-alpha secretion were virtually abrogated by the immunosuppressive drug cyclosporin A. Taken together these results indicate that AIM constitutes a novel molecular pathway in T lymphocytes for induction of TNF-alpha, and suggest a relevant pathologic role for AIM+ lymphocytes located at sites of tissue injury in the pathogenesis of different chronic inflammatory diseases.

Animals↗