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J M Requena

Publications and source records attributed to J M Requena.

67 records · Page 4Linked to original sources
PubMed1992

Characterization of a highly repeated interspersed DNA sequence of Trypanosoma cruzi: its potential use in diagnosis and strain classification.

In the present paper we report the characterization of a repetitive DNA sequence from the genome of the Maracay strain of Trypanosoma cruzi. The sequence is 1025 nucleotides long and represent about 7% of the total nuclear DNA of the parasite with a copy number of 1-2 x 10(4) copies per genome. It is also present in a high copy number in several strains of T. cruzi but absent from other trypanosomatids and from the human genome. The DNA sequence is interspersed and present on many chromosomes. We show that the different copies of this repeat present extensive endonuclease restriction polymorphisms. Using 2 conserved oligonucleotide primers derived from the repetitive sequence, together with the polymerase chain reaction technique, the presence of less than 1/30 of a single parasite can be readily detected by ethidium bromide staining.

Animals↗
PubMed1991

A potential Z-DNA-forming sequence is located between two transcription units alternatively expressed during development of Drosophila hydei.

Recent studies have demonstrated that Z-DNA exists in vivo in Escherichia coli as well as in Drosophila and mammalian cells. In the present paper, we show the existence in vivo of Z-DNA epitopes in the developmentally regulated subregion 4-75C of polytene chromosomes in Drosophila hydei. The Z-DNA epitopes were detected in subdivision C2 only during late third instar when the transcriptional activity of the locus was high. Accumulation of nonhistone chromosomal proteins in that locus was also detected during late third instar only at the time of the Z-DNA formation. Northern blot data and nucleotide sequence analysis indicated that the Z-DNA-forming sequence is located between two transcription units whose expression is regulated during the third instar. Our results suggest that in subdivision 4-75C2 a B- to Z-DNA flux occurs at a specific time during late third instar and that this flux may play a negative as well as a positive role in gene expression.

Animals↗
PubMed1991

DNA binding properties and antileukemic (L1210) activity of a Pt-pentamidine complex.

The DNA thermal stabilizing effect and the antileukemic properties of a Pt-pentamidine complex have been studied. The results indicate that the pentamidine ligands in Pt-pentamidine probably have an interaction with the DNA stronger than that of pentamidine alone because they are bound to the nucleic acid through the cis-PtCl2 residues. However, the cis-PtCl2 residues do not seem to significantly destabilize the helix. Two types of evidences are consistent with this hypothesis: (1) a decrease in the dielectric constant of the medium does not remove the pentamidine ligands from the Pt-pentamidine: DNA complex, and (2) the renaturation of the DNA in Pt-pentamidine:DNA complex is DNA concentration independent. 1H- and 13C-NMR spectroscopic data together with the elemental analysis indicate that the complex is a stoichiometric oligomer of formula [(cis-PtCl2)3(pentamidine)3] [PtCl4]2. This drug exhibits significant antineoplastic activity in BDF1 mice bearing i.p. L1210 leukemia. At a concentration of 50 mg/kg, about 15% of the LD50 for the 1,5 and 9 days schedule, the antitumor activity (T/C = 337%) is considerably superior to that of cis-DDP (T/C = 215%) or carboplatin (T/C = 220%) at doses representing 75% and 50%, respectively, of the LD50 for the same treatment schedule. Moreover, it was found that the nephro-hepatotoxicity of the complex is low.

Animals↗
PubMed1990

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA.

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.

Amino Acid Sequence↗
PubMed1990

The heat shock protein hsp70 binds in vivo to subregions 2-48BC and 3-58D of the polytene chromosomes of Drosophila hydei.

Multiple interactions of members of the hsp70 family with cellular components have already been described. We present, however, the first evidence that upon heat shock treatment hsp70 molecules interact with specific chromosomal subdivisions of the polytene chromosomes of Drosophila hydei. After a heat shock treatment of 20 min the protein binds to subdivision 3-58D1 and to the heat shock inducible subdivisions 2-48B3-6 and 2-48C1-2. Hsp70 molecules were also observed in subdivision 3-58D1 during recovery at 25 degrees C but not in subdivisions 2-48B3-6 and 2-48C1-2. Our data suggest that this interaction is stress specific. DNase and RNase experiments suggest, moreover, that the hsp70 molecules bind to RNA from ribonucleoproteins (RNPs) in subdivisions 2-48B3-6 and 2-48C1-2 and to DNA in subdivision 3-58D1. The DNA sequences in subdivision 3-58D1 seem to have the potential to adopt the Z-DNA conformation.

Animals↗
PubMed1989

Molecular cloning of a Drosophila potential Z-DNA forming sequence hybridizing in situ to a developmentally regulated subdivision of the polytene chromosomes.

We describe the selection of a group of plasmids with potential to form Z-DNA, from libraries of Drosophila hydei nuclear DNA using anti Z-DNA monoclonal (22) or polyclonal (10c) antibodies. The supercoiled closed circular forms of most of the selected recombinant plasmids from the 10c Z-DNA library show affinity to the polyclonal 10c antibody as indicated by DNA binding assays. One of these plasmids, pF17, was selected for further study. The insert in this plasmid adopts the Z conformation at bacterial supercoiled density. Analysis of deletion plasmids indicates that a Z-epitope is located within a short fragment of the insert in which 3 GC repetitions are found. The Drosophila DNA insert in pF17 hybridizes in situ with locus 4-75C1-2 of the polytene chromosomes, a locus whose transcriptional activity is developmentally regulated during the third instar.

Animals↗
PubMed1988

A head-to-tail tandem organization of hsp70 genes in Trypanosoma cruzi.

We describe the isolation and characterization of the T. cruzi hsp 70 DNA coding region which was found to be formed by multigene copies organized in a tandem array in a head-to-tail manner. The restriction pattern of one of the repetition units within the largest clone obtained from the genomic library, clone Tc70.6, shows that the hsp70 coding region should be formed by at least seven identical copies of 2.5 kb. We have found, however, the presence of restriction polymorphisms (Pvu II) within these repeats. Subsequent analysis of the time course of nuclear DNA digestion has revealed that the copy number per haploid genome could be as greater as 10. The analysis of the DNA and amino acid sequence of a fragment (70%) of one of the repetition units has shown the existence of a high homology with all the hsp70 genes of other organisms. The protein sequence homology of the fragment analyzed is as high as 88% when compared with that of the T. brucei hsp70. On the other hand, there are significant restriction site variations between both. The T. cruzi hsp70 contains at the C-terminal end a tetrapeptide repeat of the structure (GMPG)9.

Amino Acid Sequence↗
PubMedDate not supplied

Adjuvant guided polarization of the immune humoral response against a protective multicomponent antigenic protein (Q) from Leishmania infantum. A CpG + Q mix protects Balb/c mice from infection.

It has been shown that vaccination with three doses of the Leishmania infantum poly-protein Q containing five genetically fused antigenic determinants from the Lip2a, Lip2b, H2A and P0 proteins, mixed with BCG induces clearance of parasites in 9 out of 10 Leishmania infantum-infected Beagle dogs, in addition to clinical protection. In the present paper we analysed the immunogenic potential of the poly-protein Q and the specificity and polarization of the response against the antigenic determinants of Q when mixed with various adjuvants. The data showed that the Q protein had high intrinsic immunogenic potential and that it was able to induce a long-lasting IgG response. The IgM immunogenic potential of the poly-protein was mainly due to the LiP2a and LiP2b determinants, whereas the IgG immunogenic potential was mainly due to the LiP2a component. It was observed that the protein itself elicited a mixed IgG2a/IgG1 response and that the determinants of Q were endowed with different IgG2a/IgG1 potential. It was also observed that the adjuvants did not influence the intensity or specificity of the IgM response but that they modulated the intensity, the specificity and the polarization of the IgG response against the determinants of Q. CpG-ODN motifs or double-stranded DNA plasmids containing CpG motifs when mixed with Q induced a predominant IgG2a response mainly observed at early stages post-immunization. The data showed that a CpG + Q mix induced significant protection against L. infantum infection in Balb/c mice.

Adjuvants, Immunologic↗
PubMedDate not supplied

Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T. cruzi CL-Brener.

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.

Animals↗
PubMedDate not supplied

Genes and chromosomes of Leishmania infantum.

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes.

Animals↗
PubMedDate not supplied

[Conjunctival ophthalmomyiasis caused by Oestrus ovis].

BACKGROUND: Microbiologic diagnosis of conjunctival ophthalmomyiasis. MATERIAL AND METHODS: Material extracted from three patients' conjunctiva who complained of sudden presence of foreign body and were diagnosed as conjunctival ophthalmomyiasis has been studied. Patients were recruited over two months from the Emergency Department. Diagnosis was performed through observation using the light microscope. RESULTS: Oestrus ovis first-instance larvas were observed in all the samples studied. CONCLUSIONS: This observation of similar cases in a short period of time and the low frequency of diseased people who lived in a urban environment makes necessary to think in this agent as cause of ophthalmomyiasis in our area and therefore perform an adequate differential diagnosis.

Adolescent↗