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Biomedical subjects

R Casado

Publications and source records attributed to R Casado.

10 recordsLinked to original sources
PubMed2006

CT and MR imaging findings in methanol intoxication.

We present the CT and MR imaging findings in acute methanol intoxication in a 35-year-old man who was admitted to the emergency department with weakness, blurred vision, mild bilateral areactive mydriasis, and a progressive decrease in the level of consciousness. CT and MR imaging showed bilateral putaminal hemorrhagic necrosis and subcortical white matter lesions with peripheral contrast enhancement. There was only partial improvement in patient's Glasgow Coma Scale score during follow-up.

Acidosis↗
PubMed1999

Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress.

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17. 5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37 degrees C to 50 degrees C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4 degrees C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress.

Amino Acid Sequence↗
PubMed1998

Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds.

Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.

Amino Acid Sequence↗
PubMed1997

Purification and in vitro chaperone activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds.

A 20-kD protein has been purified from cotyledons of recalcitrant (desiccation-sensitive) chestnut (Castanea sativa) seeds, where it accumulates at levels comparable to those of major seed storage proteins. This protein, termed Cs smHSP 1, forms homododecameric complexes under nondenaturing conditions and appears to be homologous to cytosolic class I small heat-shock proteins (smHSPs) from plant sources. In vitro evidence has been obtained that the isolated protein can function as a molecular chaperone; it increases, at stoichiometric levels, the renaturation yields of chemically denatured citrate synthase and also prevents the irreversible thermal inactivation of this enzyme. Although a role in desiccation tolerance has been hypothesized for seed smHSPs, this does not seem to be the case for Cs smHSP 1. We have investigated the presence of immunologically related proteins in orthodox and recalcitrant seeds of 13 woody species. Our results indicate that the presence of Cs smHSP 1-like proteins, even at high levels, is not enough to confer desiccation tolerance, and that the amount of these proteins does not furnish a reliable criterion to identify desiccation-sensitive seeds. Additional proteins or mechanisms appear necessary to keep the viability of orthodox seeds upon shedding.

Amino Acid Sequence↗
PubMed1996

Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons.

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.

Amino Acid Sequence↗
PubMed1994

2S Arginine-rich proteins from Pinus pinaster seeds.

Low molecular weight globulins, which are abundant proteins in the Pinus pinaster Ait. megagametophyte, were purified and characterized. They showed a dimeric structure formed of one large and one small subunit linked by disulfide bridges. They were characterized by a high Arg and Glx content and by a relatively high Cys content. A comparison of their characteristics with those of angiosperm 2S proteins suggests that there is homology between them.

Journal Article↗
PubMed1992

Basic Endochitinases Are Major Proteins in Castanea sativa Cotyledons.

Basic endochitinases are abundant proteins in Castanea sativa Mill. cotyledons. Three basic chitinases were purified with molecular masses of 25, 26, and 32 kD (Ch1, Ch2, and Ch3) and with isoelectric points between 8 and 9.5. Antibodies raised against Ch1 cross-reacted with Ch2 and Ch3. However, Ch3 showed differences when compared with the other two enzymes, especially in its higher cysteine content. The size, amino acid composition, and N-terminal sequence of Ch1 indicate that it is a class II endochitinase and, therefore, has no cysteine-rich hevein domain. Ch1 inhibits the growth of the fungus Trichoderma viride. The biological role of these endochitinases is discussed.

Journal Article↗
PubMed1984

[Immunologic responses following surgical resection of gastrointestinal carcinomas].

The amount of IgG, IgM, IgA, C3, C4 and C5 in serum, and the response to delayed hypersensitivity tests with PPD, varidase, candidin and mumps antigens were estimated in patients submitted to radical surgery for gastric and colorectal cancer (group I), before and at intervals after the operation was performed, and compared with cancer patients undergoing palliative abdominal surgery (group II), and with patients submitted to major abdominal surgery for diseases other than cancer (group III). Group I showed a decrease of all the immunoglobulins (Igs) and complement fractions (F) during the first week after operation, and a significant increase after one month. Similar changes were observed in group III, the augmentation of Igs and F after one month being somewhat smaller. Group II showed a larger number of positiveness than either group I or II in both the preoperative and early postoperative periods (p = 0.001). After 2 and 3 months, the number of positive cases was larger in group I than in group II (p = 0.05). According to these results, the immune response is recovered one month after the excision of gastric and colorectal malignant tumors, being this the best moment to receive a complementary cytoreductive therapy.

Complement C3↗
PubMedDate not supplied

Electrophoretic analysis of seed storage proteins from gymnosperms.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pore gradient gel electrophoresis (PGGE) followed by SDS-PAGE and Western blot analysis were used to characterize the seed storage proteins from seven gymnosperm species from the families Pinaceae (Abies alba, Cedrus atlantica and Picea abies), Cupressaceae (Biota orientalis, Chamaecyparis lawsoniana and Cupressus arizonica), and Taxaceae (Taxus baccata). SDS-PAGE and PGGE X SDS-PAGE indicate the presence of proteins with characteristics similar to the 7S globulins in all the species studied. Antibodies to a 7S globulin subunit from Pinus pinaster cross-reacted with homologous polypeptides from Pinaceae species, but not with corresponding subunits from species belonging to other families. Also detected in each of the studied species, with the exception of A. alba and T. baccata, were those of C. atlantica may be extracted by saline buffer, while the remainder require a dissociating agent. Antibodies raised against the small subunit from P. pinaster 11S protein recognized only the corresponding polypeptides from Pinaceae species. Overall, these results help clarify our knowledge of gymnosperm seed storage proteins.

Animals↗