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R Sousa

Publications and source records attributed to R Sousa.

70 records · Page 4Linked to original sources
PubMed1992

Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein.

The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.

Amino Acid Sequence↗
PubMed1991

The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex.

The products encoded by the E2 open reading frame of the papillomaviruses are DNA-binding transcription factors involved in the positive or negative regulation of multiple viral promoters. To further understand the mechanisms by which the same transcription factor may act differentially, the full-length BPV-1 E2 protein was expressed and purified from yeast and assayed in vitro for its capacity to modulate transcription. E2 stimulated transcription of the HSV thymidine kinase (TK) promoter when E2-binding sites were positioned in an enhancer configuration approximately 100 bp upstream of the promoter start site. In contrast, the same full-length E2 protein repressed transcription of the HPV-18 E6/E7 P105 promoter. This repression was mediated through binding to the E2 DNA-binding site immediately upstream of the P105 promoter TATA box and could be abrogated by preincubation of the HPV-18 P105 promoter template with the nuclear extract allowing the formation of the preinitiation complex. In vitro DNA-binding experiments with purified E2 and TFIID showed that binding of E2 to its DNA target placed at different positions with respect to the TATA box differentially affects binding of TFIID to its cognate site. In these respects, E2 is similar to the bacteriophage lambda repressor, which can act either as a repressor or an activator of transcription depending on the position of its binding sites relative to the promoter sequences.

Animals↗
PubMed1990

Single crystals of a chimeric T7/T3 RNA polymerase with T3 promoter specificity and a nonprocessive T7 RNAP mutant.

Two RNA polymerases homologous to bacteriophage T7 RNA polymerase, bacteriophage Sp6 and T3 RNA polymerases, were screened for crystallization under conditions identical or similar to those reported for the growth of large single crystals of T7 RNA polymerase (Sousa, R., Rose, J. P., Chung, Y. J., Lafer, E. M., and Wang, B.-C. (1989) Proteins 5, 266; Sousa, R., Lafer, E. M., and Wang, B.-C. (1990) J. Crystal Growth, in press; Sousa, R., and Lafer, E. M. (1990) Methods 1, in press). A number of mutant T7 RNAPs were also screened under these conditions as were three chimeric RNA polymerases consisting of T7 RNAP N-terminal and T3 RNAP C-terminal sequences. One chimeric polymerase and two mutant polymerases crystallized readily under T7 RNAP crystallization conditions. The chimeric polymerase crystallized in a space group different from T7 RNA polymerase: orthorombic with unit cell parameters a = 75 A, b = 98 A, c = 159 A; space group P2(1)2(1)2(1) and 4 molecules/unit cell. This chimeric enzyme exhibits T3 promoter specificity and will make it possible to investigate how structural differences between the T3 and T7 RNA polymerase promoter recognition domains determine their different promoter specificities. One of the mutant polymerases successfully crystallized was an enzyme which can carry out promoter recognition and abortive transcription but cannot carry out processive transcription. Its structure may provide information on the nature of the conformational changes undergone by T7 RNAP in the abortive-processive switch. Crystals of the second mutant T7 RNA polymerase were unsuitable for x-ray analysis.

Chimera↗
PubMed1990

Two new monoclonal antibodies provide immunohistochemical evidence for the unique biochemical similarity of the mouse globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata.

Similarities in cellular morphology, afferentation, efferentation, and neurotransmitter content between the internal and external parts of the pallidum and the substantia nigra pars reticulata have long been noted. Here we present evidence that the globus pallidus, entopedunucular nucleus and substantia nigra pars reticulata are more closely related to each other antigenically than to any other anatomical subdivision in the murine central nervous system. In a monoclonal antibody library composed of 20 distinctive lines selected from 300 hybridomas screened immunohistochemically on mouse brain sagittal sections we found two antibodies whose staining patterns distinguish the pallidum and substantia nigra pars reticulata from all other brain gray matter regions but stain these two divisions similarly. One monoclonal antibody, F1-134, stains all brain gray matter regions moderately but gives intense staining of the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata only. Another monoclonal antibody, F1-20, stains different brain gray matter regions to varying degrees but shows a complete and exclusive exclusion of staining from the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. These results support the tripartite pallidum hypothesis. This study also provides an example of how the monoclonal antibody library strategy can be applied to general problems of brain organization.

Animals↗
PubMed1989

Preparation and use of a universal primed Sepharose for the purification of DNA-binding proteins.

We have devised a novel method for the construction of a DNA affinity matrix and tested its use in the purification of a sequence-specific DNA-binding protein from the yeast Saccharomyces cerevisiae. The matrix was prepared in two steps: first, a palindromic oligonucleotide containing an XhoI cohesive end was covalently linked via its loop to a Sepharose matrix; second, directly to this 'universal' primed Sepharose was ligated a 37-bp oligonucleotide, with XhoI cohesive ends, containing the sequence of the upstream activation sequence 1 (UAS1) site of the yeast iso-1-cytochrome c (CYC1) gene. After fractionating a yeast crude extract through DEAE-cellulose, heparin ultrogel and Mono Q columns, a single pass through the affinity matrix allowed the purification to apparent homogeneity of the 120-kDa protein factor P, which is responsible for the binding to the UAS1 site.

Base Sequence↗
PubMed1989

A point mutation in the CYC1 UAS1 creates a new combination of regulatory elements that activate transcription synergistically.

Dissection of the upstream activation site 1 (UAS1) of the yeast CYC1 gene showed that the A and B regions respond individually to regulation by the HAP1 protein, and that a point mutation in the B region converts this region to a translation upstream factor (TUF)-regulated element. Combinatorial analyses revealed that the transacting factors involved with these wild-type and mutant UAS1 target sites combine to activate transcription in a synergistic manner. Furthermore, combinations of heterologous factors, made possible by the point mutation, create a new specificity of regulation that differs from regulation by any one factor individually.

Base Sequence↗
PubMed1989

Single crystals of bacteriophage T7 RNA polymerase.

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.

Crystallization↗
PubMed1989

Mapping and characterization of an X-linked processed gene related to MYCL1.

A DNA sequence with homology to the myc family of proto-oncogenes has been characterized and found to be a processed gene related to L-MYC (MYCL1). This processed gene (MYCL2) was isolated by cross-hybridization to an oligonucleotide probe synthesized from the C-MYC (MYC) sequence in a highly conserved region of the myc gene family. Sequence analysis of MYCL2 revealed an open reading frame of 1194 bp with no intervening sequences and strong homology to the recently published DNA sequence of MYCL1. Southern and Northern blot analyses of DNAs and RNAs from small cell lung carcinomas confirmed its MYCL1 homology. Mapping of MYCL2 by somatic cell hybrids places this sequence on the long arm of the X chromosome in bands q22----q28.

Animals↗
PubMed1986

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium.

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.

Animals↗
PubMed1985

Anti-Z-DNA antibody binding can stabilize Z-DNA in relaxed and linear plasmids under physiological conditions.

It is shown that anti-Z-DNA antibody binding can stabilize sequences of d(CG/GC)n and d(CA/GT)n in the Z-DNA conformation in a plasmid in the complete absence of supercoiling. This effect is quantitated by using antibody preparations of different affinities and varying concentrations. The d(CG/GC)n sequence can be stabilized under physiological conditions. This is the first demonstration that a region of Z-DNA can be stabilized by protein binding in a completely relaxed plasmid under physiological conditions. The antibody-Z-DNA complex in the relaxed plasmid is shown to be an equilibrium state and not a long-lived kinetic intermediate since specific binding of the antibody to linearized plasmids containing Z-forming sequences is observed.

Antibodies↗
PubMed1985

Isolation and characterization of Z-DNA binding proteins from wheat germ.

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.

Animals↗
PubMed1985

[Benign tumors of the esophagus. Esophageal leiomyoma].

Benign neoplasms of the esophagus are rare. The most frequent symptom (75% of the cases) is dysphagia, and a very rare one is dyspnea. When a benign tumor of the esophagus is suspected, a radiological examination is made and the diagnosis confirmed by fibro-esophagoscopy and biopsy. The authors make a summarized review of the benign tumors of the esophagus, and describe a case of esophageal leiomyoma.

Esophageal Neoplasms↗
PubMedDate not supplied

Intracystic infection of liver hydatidosis.

BACKGROUNDS/AIMS: The aim of this study was to review the clinical presentation and management of patients with intracystic bacterial infection in liver hydatidosis. METHODOLOGY: The records of 480 patients suffering from liver hydatid disease treated at our institution were reviewed. Only 42 patients fulfilled intracystic bacterial infection criteria. We assessed the incidence, clinical and laboratory findings, diagnostic procedures and surgical approach in these cases. RESULTS: The incidence of intracystic bacterial infection was steady throughout the study period. Clinical and laboratory data are non-specific. Diagnostic procedures for intracystic bacterial infection, including liver computed tomography scan, are of limited value. Although both, radical and non-radical surgical approaches, were used in these patients, mortality was associated with non-radical surgery. The morbidity rate was high regardless of the procedure used. CONCLUSIONS: Our current goal in the management of intracystic bacterial infection, if overall condition of the patient is good, is to carry out a total or subtotal cystectomy in order to avoid mortality and hydatid disease relapses.

Adult↗
PubMedDate not supplied

[Cleft sternum in an infant].

The authors report the clinical case of a female black infant, one month 22 days old, with sternal malformation (complete sternal cleft). She didn't show other associated malformations. Early surgical repair was done with success. The relevant nomenclature of sternal defects is discussed.

Female↗