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Biomedical subjects

G Grimaldi

Publications and source records attributed to G Grimaldi.

At least 55 records · Page 3Linked to original sources

Members of the zinc finger protein gene family sharing a conserved N-terminal module.

We report the isolation of human members of a sub-family of structurally related finger protein genes. These potentially encode polypeptides containing finger motifs of the Krüppel type at the C-terminus, and a conserved amino acid module at the N-terminus; because of its invariant location the latter is referred to as finger preceding box (FPB). The FPB, detected also in previously described finger proteins from human, mouse and Xenopus, extends over approximately 65 amino acids and appears to be composed of two contiguous modules: FPB-A (residues 1-42) and FPB-B (residues 43-65). The latter is absent in some of the members analyzed. Elements A and B and the zinc finger domain are encoded by separate exons in the ZNF2 gene, a human member of this sub-family. The positioning of introns within this gene is remarkable. One intron flanks and a second interrupts the first codon of the FPB-A and FPB-B modules, respectively. A third intron occurs a few nucleotides downstream of FPB-B marking its separation from the remainder of the coding sequences. This organization, together with the absence of FPB-B in some cDNAs, supports the hypothesis that mRNAs encoding polypeptides that include one, both or none of the FPB-A and FPB-B modules may be assembled through alternative splicing pathways. Northern analyses showed that members of this sub-family are expressed as multiple transcripts in several cell lines. The sequences of distinct cDNAs homologous to the ZNF2 gene indicate that alternative splicing events adjoin either coding or non coding exons to the FPB sequences.

Amino Acid Sequence↗

Isolation and expression analysis of a human zinc finger gene (ZNF41) located on the short arm of the X chromosome.

We have isolated a novel human zinc finger gene, ZNF41, from a human X-chromosome-specific library. Nucleotide sequence analysis reveals that ZNF41 potentially encodes a polypeptide featuring an array of 18 contiguous zinc fingers of the C2H2 type. Multiple polyadenylated transcripts homologous to ZNF41 are present at different levels in several distinct cell types. Southern analyses of somatic cell hybrids containing either intact or rearranged X chromosomes confirm the genomic origin of the isolated gene and establish that it is localized between Xcen and Xp22.1.

Amino Acid Sequence↗

Characterization and classification of leishmanial parasites from humans, wild mammals, and sand flies in the Amazon region of Brazil.

Ninety-four leishmanial isolates from the Brazilian Amazon Region (Amapá, Amazonas, Pará, and Rondônia) were identified and classified using specific monoclonal antibodies and an indirect radioimmunoassay (serodeme analysis); eighty-two were also characterized by enzyme electrophoresis (zymodeme analysis), the results of which were subjected to a numerical phenetic analysis. Six isolates from humans (3), Didelphis marsupialis (1), Lutzomyia olmeca nociva (1), and Lu, reducta (1) showed reactivity patterns and isoenzyme profiles similar to those obtained with the Leishmania amazonensis reference strains, and were identified as this species. Eighty-six stocks were classified as members of the L. braziliensis complex; of these, 61 were L. guyanensis or variants, which presented three serodeme subtypes, but whose isoenzyme profiles were all similar to the reference strain. A total of 15 isolates were distinguished as L. braziliensis or variants and were classified into five serodeme subtypes. The isolate from Psychodopugus davisi appeared, from the numerical analysis, to be a distinct parasite species. Ten isolates showed reactivity patterns and isoenzyme profiles similar to those obtained with the L. naiffi reference strain. A parasite isolated from Ps. claustrei appeared to be different from all reference strains by both techniques, and was classified as probably being a new species. The importance of these results with respect to the taxonomic status of the New World Leishmania, and their implications for both clinical and epidemiologic data are discussed.

Animals↗

Characterization of Leishmania colombiensis sp. n (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Adult↗

Lutzomyia ovallesi (Diptera: Psychodidae) as a vector of cutaneous leishmaniasis in Venezuela.

In the search for vectors of cutaneous leishmaniasis in the Barro Negro forest, Duaca, Lara State, Venezuela, 4,864 wild-caught Lutzomyia females were dissected and examined for promastigotes. Natural infection was found in 25 (0.5%) Lu. ovallesi. By biological parameters and enzyme electrophoresis all isolates from Lu. ovallesi were indistinguishable from those obtained from humans and dogs in the same Region. The isoenzyme profile of these isolates appear to indicate that strains were a hybrid between Leishmania braziliensis and L. guyanensis.

Animals↗

Population heterogeneity among clones of New World Leishmania species.

Cell cloning techniques and schizodeme analysis were used to detect mixtures of subpopulations in Leishmania parasites, isolated from humans and a reservoir host. Clones were obtained by plating promastigotes, at limited dilution, on solid medium. The resultant colonies were analysed by the restriction profile of kinetoplast DNA minicircles (schizodeme analysis). The efficiency of plating was around 40-60%, and subpopulations of different schizodemes, in stocks of Leishmania isolated from 2 cases of human cutaneous leishmaniasis were detected. The presence of different schizodemes in one isolate from a sylvatic animal suggested the possibility of a mixed natural infection.

Animals↗

Spacer promoters are orientation-dependent activators of pre-rRNA transcription in Drosophila melanogaster.

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

Animals↗

Identification, using isoenzyme electrophoresis and monoclonal antibodies, of Leishmania isolated from humans and wild animals of Ecuador.

Six strains of Leishmania isolated from wild mammals and humans on the Pacific Coast of Ecuador were identified by isoenzyme electrophoresis and by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. Single isolates from Sciurus vulgaris, Potos flavus, and Tamandua tetradactyla were identified as Leishmania amazonensis. Three other strains, isolated from cutaneous lesions of humans, were identified as Leishmania panamensis.

Animals↗

A review of the geographic distribution and epidemiology of leishmaniasis in the New World.

A review of the epidemiologic aspects of the New World leishmaniases, including their known geographic distribution, etiologic agents, zoonotic reservoirs, and insect vectors, based on biological and molecular characterization of Leishmania isolates is presented. Data summarized in this paper on parasite taxonomy and geographic distribution come from our studies of greater than 1,000 New World Leishmania isolates identified by species-specific monoclonal antibodies using an indirect radioimmune binding assay and from scientific literature.

Animals↗

Multiple repeated units in Drosophila melanogaster ribosomal DNA spacer stimulate rRNA precursor transcription.

Drosophila melanogaster ribosomal DNA (rDNA) transcriptional units are separated by nontranscribed spacer (NTS) segments consisting of tandemly arranged repeats 95, 330, and 240 base pairs long. NTS sequences stimulate transcription from the rRNA precursor (pre-rRNA) promoter. Primer extension analysis of RNA from cells cotransfected with plasmids carrying NTS sequences of various lengths shows that the activity of the pre-rRNA promoter is directly correlated with the number of 240-base-pair repeats; NTS sequences upstream of these units also stimulate pre-rRNA transcription. The NTS effect might depend upon transcription from duplicated promoters present within the 240- and 330-base-pair repeats. The strength of the pre-RNA promoter correlates in each construct with the level of spacer transcription. The action of spacer sequences, although able to take place over a large distance, is not independent of orientation: stimulation of pre-rRNA transcription is abolished in plasmids carrying inverted NTS segments. Removal of a putative transcription termination site located upstream of the pre-rRNA promoter has no effect on pre-rRNA initiation nor does it substantially alter spacer enhancement.

Animals↗

Epidemiological studies on American leishmaniasis in Ceará State, Brazil. Molecular characterization of the Leishmania isolates.

Two methods of molecular characterization, using monoclonal antibodies and enzyme electrophoresis, were employed in the identification of 36 stocks of Leishmania isolated from human and canine cases of American visceral (AVL) and cutaneous (ACL) leishmaniases in the northern part of Ceará State, Brazil. Molecular homogeneous strains of Leishmania donovani (chagasi) isolated from both human and canine hosts were detected in 14 municipalities. Two more parasite species, L. braziliensis braziliensis and L. mexicana amazonensis, were also detected in the state. The implication of these results with respect to both the clinical and epidemiological data are discussed.

Animals↗

Canine American cutaneous leishmaniasis: a clinical and immunological study in dogs naturally infected with Leishmania braziliensis braziliensis in an endemic area of Rio de Janeiro, Brazil.

Clinical and immunological findings from 35 dogs infected with Leishmania braziliensis braziliensis are described. The majority of the dogs had ulcerated single lesions on the ears. Sera from all infected dogs showed detectable Leishmania-induced antibodies using an indirect fluorescent antibody test. Antimonial therapy resulted in prompt healing of the lesions in 80.9% of the animals followed by a significant reduction in the anti-Leishmania antibody titers. However, treatment follow-up showed recurrences at the site of the primary lesion in 42.8% of the cases. These data were correlated with a persistence of the parasite in clinically healed lesions as well as with a negative intradermal test (leishmanin-delayed type hypersensitivity) observed in all animals but one.

Animals↗

Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies.

Five hundred thirty stocks of Leishmania isolated from human and domestic and wild reservoir hosts, representing a wide geographic distribution of endemic foci of American cutaneous (ACL) and visceral leishmaniases (AVL) were characterized and identified at species and/or subspecies levels based on their reactivity to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. This study confirms and extends our preliminary results on the high specificity of some of these monoclonals for the L. braziliensis, L. mexicana, and L. donovani complexes. This study also demonstrates the relative stability of these molecular markers and the general usefulness of the method for parasite identification. Two hundred ninety-two of 420 isolates of ACL were classified as members of the L. braziliensis complex. Two hundred twenty-seven were L. b. braziliensis; these showed the widest geographical distribution (Brazil: Amazonas, Bahia, Ceara, Espirito Santo, Goias, Minas Gerais, Para, Paraiba, Rio de Janeiro, and Sao Paulo; Honduras: Santa Barbara and Yoko; Peru: Ancash, Piura, and Ucayali; and Venezuela: Cojedes, Distrito Federal, Lara, Portuguesa, Vale Hondo, Yaracuy, and Zulia). Forty-one stocks were identified as L. b. guyanensis (from North Brazil: Amazonas, Amapa, Para, and Rondonia). Twenty-one stocks were identified as L. b. panamensis (from Costa Rica: Alajuela, Guanacasten, Limon, Puntarenas, and San Jose; and Honduras: El Paraiso, and Olancho). Out of 128 isolates classified as members of the L. mexicana complex, 74 were differentiated as L. m. amazonensis (from Bolivia; Brazil: Amazonas, Bahia, Ceara, Goias, Maranhao, Mato Grosso do Norte, and Para; Peru: Pasco Forest and Van Humboldt; and Venezuela: Carabobo, Guarico, and Merida). Forty-four stocks were identified as L. m. venezuelensis (from Venezuela: Lara). Six stocks were L. m. mexicana (from Belize; and Mexico: Campeche [corrected] and Quintana Roo, Yucatan). One hundred ten isolates from AVL were identified as L. donovani chagasi (from Brazil: Bahia, Ceara, Maranhao, Minas Gerais, Mato Grosso do Sul, Piaui, Rio de Janeiro, and Sergipe; and Honduras: Valle). The implications of these results with respect to both the clinical and epidemiological data (including the detection of seven unusual characterized stocks) are discussed.

Antibodies, Monoclonal↗

Transient expression of Drosophila melanogaster rDNA promoter into cultured Drosophila cells.

Recombinant plasmids that carry the bacterial CAT gene under the transcriptional control of the D. melanogaster rDNA promoter have been introduced by transfection into cultured Schneider II Drosophila cells and their template activity followed at the RNA and protein level. While no CAT enzyme activity is measurable 48 hrs after transfection, high levels of hybrid rRNA-CAT transcripts that originate at the authentic rRNA start site are detected by S1 mapping analysis. The interval -180/+34 of a rDNA transcriptional unit is sufficient to ensure faithful polymerase I transcription. However, the presence of a complete NTS (non transcribed spacer) region greatly enhances the transcriptional activity of exogenously added rDNA templates. Competition experiments between constructs carrying different amounts of NTS sequences indicate that spacer segments confer a transcriptional advantage efficiently attracting necessary transcription factors and/or polymerase I molecules.

Acetyltransferases↗

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.

Animals↗