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Biomedical subjects

R Flores

Publications and source records attributed to R Flores.

At least 37 records · Page 2Linked to original sources
PubMed2001

Multilocation trial of ceftiofur for treatment of postpartum cows with fever.

OBJECTIVE: To evaluate the effect of ceftiofur for treatment of postpartum cows with fever. DESIGN: Multilocation randomized complete block design trial. ANIMALS: 330 cows. PROCEDURE: Cows with rectal temperature > or = 39.5 C (103.1 F) during the first 10 postpartum days were randomly assigned to a treatment (ceftiofur; 1 mg/kg [0.45 mg/lb] of body weight daily for 3 days) or untreated control group. Cure (no additional or alternative antimicrobial treatment used, rectal temperature < 39.5 C, and no other concurrent clinical signs of disease when evaluated at 9 or 10 days after enrollment), milk production, and rectal temperature were evaluated. RESULTS: Ceftiofur-treated cows were significantly more likely to be cured than control cows (56.0 vs 28.9%, respectively), with an odds ratio of 3.14 when vaginal discharge (a factor with moderate interaction with treatment) was present at enrollment. Among cows that had an abnormal calving (a significant interaction factor), treated cows had first milking yield 2.27 kg (5 lb) greater than control cows. Treated cows had a significantly greater reduction in rectal temperature (1.19 C [2.14 Fl), compared with control cows (1.04 C [1.87 F]). CONCLUSIONS AND CLINICAL RELEVANCE: Parenteral administration of ceftiofur significantly improved cure rate, milk yield, and rectal temperature in postpartum cows with fever and vaginal discharge or dystocia. These findings provide information to determine appropriate treatment for postpartum cows, which for years has been debated in the dairy industry.

Animals↗
PubMed2001

An extra nucleotide in the consensus catalytic core of a viroid hammerhead ribozyme: implications for the design of more efficient ribozymes.

Hammerhead ribozymes catalyze self-cleavage of oligomeric RNAs generated in replication of certain viroid and viroid-like RNAs. Previous studies have defined a catalytic core conserved in most natural hammerheads, but it is still unknown why some present deviations from the consensus. We have addressed this issue in chrysanthemum chlorotic mottle viroid (CChMVd), whose (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. Effects of insertions at this position on hammerhead kinetics have not hitherto been examined. A10 caused a moderate decrease of the trans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, whereas A10-->C and A10-->G substitutions had major detrimental effects, likely because they favor catalytically inactive foldings. By contrast, A10-->U substitution induced a 3-4-fold increase of the rate constant, providing an explanation for the extra U10 present in two natural hammerheads. Because A10 also occupies a singular and indispensable position in the global CChMVd conformation, as revealed by bioassays, these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. Incorporation of the extra U10 into a model hammerhead also caused a similar increase in the rate constant, providing data for a deeper understanding of the hammerhead structural requirements and for designing more efficient ribozymes.

Base Sequence↗
PubMed2001

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Computational Biology↗
PubMed2001

Phylogenetic analysis of viroid and viroid-like satellite RNAs from plants: a reassessment.

The proposed monophyletic origin of a group of subviral plant pathogens (viroids and viroid-like satellite RNAs), as well as the phylogenetic relationships and the resulting taxonomy of these entities, has been recently questioned. The criticism comes from the (apparent) lack of sequence similarity among these RNAs necessary to reliably infer a phylogeny. Here we show that, despite their low overall sequence similarity, a sequence alignment manually adjusted to take into account all the local similarities and the insertions/deletions and duplications/rearrangements described in the literature for viroids and viroid-like satellite RNA, along with the use of an appropriate estimator of genetic distances, constitutes a data set suitable for a phylogenetic reconstruction. When the likelihood-mapping method was applied to this data set, the tree-likeness obtained was higher than that corresponding to a sequence alignment that does not take into consideration the local similarities. In addition, bootstrap analysis also supports the major groups previously proposed and the reconstruction is consistent with the biological properties of this RNAs.

Evolution, Molecular↗
PubMed2001

Polymorphism of the 5' terminal region of Citrus tristeza virus (CTV) RNA: incidence of three sequence types in isolates of different origin and pathogenicity.

Sequences of the 5' terminal region of the genomic RNA from eight isolates of Citrus tristeza virus (CTV) were previously classified into three types (I, II and III), with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. Sequencing of an additional 58 cDNA clones from 15 virus isolates showed that all sequences could be unequivocally assigned to one of the three types previously established. The relative frequency of each sequence type was assessed in 57 CTV isolates of different geographic origin and pathogenic characteristics by RT-PCR with sets of type-specific primers using CTV dsRNA as template. None of the isolates yielded amplification of the type I or II sequences alone, but in 19 of them type III sequences were the only amplification product detected. Within isolates containing more than one sequence type, eight had type II and III sequences, 11 had type I and III sequences, and 19 had sequences of the three types. Isolates containing only type III sequences caused only mild to moderate symptoms in Mexican lime, an indicator species for most CTV isolates, whereas isolates causing stem pitting in sweet orange an/or grapefruit, generally contained sequences type II. None of the sequence types could be traced to a precise geographic area, as all types were detected in isolates from at least nine of the 12 countries from which samples were taken.

5' Untranslated Regions↗
PubMed2001

A naked plant-specific RNA ten-fold smaller than the smallest known viral RNA: the viroid.

Viroids are subviral plant pathogens at the frontier of life. They are solely composed by a single-stranded circular RNA of 246-401 nt with a compact secondary structure. Viroids replicate autonomously when inoculated into their host plants and incite, in most of them, economically important diseases. In contrast to viruses, viroids do not code for any protein and depend on host enzymes for their replication, which in some viroids occurs in the nucleus and in others in the chloroplast, through a rolling-circle mechanism with three catalytic steps. Quite remarkably, however, one of the steps, cleavage of the oligomeric head-to-tail replicative intermediates to unit-length strands, is mediated in certain viroids by hammerhead ribozymes that can be formed by their strands of both polarities. Viroids induce disease by direct interaction with host factors, the nature of which is presently unknown. Some properties of viroids, particularly the presence of ribozymes, suggest that they might have appeared very early in evolution and could represent 'living fossils' of the precellular RNA world that presumably preceded our current world based on DNA and proteins.

Biological Evolution↗
PubMed2001

Morphological and quantitative study of the Leydig cells of pigs fed with anabolic doses of clenbuterol.

The effects of clenbuterol administered at anabolic doses on the testicular interstitium were studied in 30 pigs allocated to three experimental groups. The diet of two groups was supplemented with clenbuterol (Clb) (1 ppm), but whereas in the Clb+ group the treatment was given until slaughter (treatment period: 3 months), in the Clb- group the clenbuterol was withdrawn 2 weeks before slaughter (treatment period: 2-5 months); in the control group, the pigs were fed without clenbuterol. For histological procedures, a fractional sampling scheme was applied and routine techniques for light and transmission electron microscopy were used. The results of subjective morphology and morphometrics showed slight differences between the treated and the control groups. Conversely, the stereological results identified a prominent hyperplasia of the Leydig cells and ultrastructural analysis of these cells revealed a conspicuous increase in the organelles related to testosterone production, suggesting a functional activation of the interstitial cells in response to the clenbuterol treatment.

Adrenergic beta-Agonists↗
PubMed2000

Simultaneous multiorgan presence of human herpesvirus 8 and restricted lymphotropism of Epstein-Barr virus DNA sequences in a human immunodeficiency virus-negative immunodeficient infant.

Because a profound dysregulation of the immune system occurs in primary immunodeficiencies, viral infections are not uncommon. Human herpesvirus (HHV)-8 DNA was detected by polymerase chain reaction (PCR) analysis, Southern blotting, and in situ hybridization (ISH) in peripheral blood mononuclear cells and lymphoid organs (bone marrow, spleen, and lymph nodes) and endothelial and epithelial cells and macrophages from several organs (skin, lung, esophagus, intestine, choroid plexus [but not in brain or cerebellum], heart, striated muscle, liver, and kidney) of a human immunodeficiency virus-negative infant with DiGeorge anomaly who died of disseminated infection. Epstein-Barr virus DNA sequences were detected in the spleen and lymph nodes (by PCR and ISH) and in bone marrow (only by ISH) but not in blood or nonlymphoid organs. This report is believed to be the first of multiorgan dissemination of HHV-8 in a primary immunodeficiency.

Blotting, Southern↗
PubMed2000

Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.

Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons.

Base Sequence↗
PubMed2000

The 23-kDa protein coded by the 3'-terminal gene of citrus tristeza virus is an RNA-binding protein.

The 23-kDa protein (p23), encoded by the 3'-proximal gene of the RNA of Citrus tristeza virus (CTV), was overexpressed in Escherichia coli fused to the maltose-binding protein and purified by affinity chromatography. Gel retardation and UV crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded RNA in a non-sequence-specific manner. Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable at high salt concentrations, suggesting that interactions other than those between the negatively charged RNA and the basic region of p23 are involved. Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-stranded and double-stranded DNA. By use of a series of artificially generated mutants, the RNA-binding domain of p23 was mapped between positions 50-86, a region containing several basic amino acids and a putative zinc-finger domain. Additional p23-derivatives lacking the conserved residues presumably involved in coordinating the zinc ion showed RNA-binding activity, but with an apparent dissociation constant higher than the wild-type protein. These conserved residues might confer binding specificity or increase binding stability in vivo. Within the Closteroviridae family, p23 is the only protein characterized so far showing RNA-binding activity.

Chromosome Mapping↗
PubMed2000

A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid.

Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases. On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids. The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) was a 25% inhibition observed. These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded.

Amanitins↗
PubMed2000

Geographic targeting of nutrition programs can substantially affect the severity of stunting in Honduras.

The effect of nutrition intervention programs in developing countries is likely to vary with the degree to which the program can be successfully targeted at the most vulnerable. In Honduras, the existence of a recent census of the height of first-grade children makes it possible to assess a priori the effect of different targeting strategies, holding constant other features of a hypothetical program. We simulate a nutrition intervention with 20% national coverage and uniform gains of 0.5 Z-scores for all beneficiaries, with a number of different approaches to targeting. The VIIth National Census of First-Graders' Heights provides the baseline scenario and permits identification of priority departments, municipalities, schools and individuals, for a total of six alternative targeting mechanisms. Effect is assessed on the basis of changes in the prevalence of stunting (less than -2 Z-scores) and in two different measures of the severity of stunting adapted from the economics literature (the malnutrition gap and the quadratic malnutrition gap). We find that the simulated program has the potential to substantially improve the severity, but not the prevalence of stunting in Honduras. Household targeting with an imperfect indicator of vulnerability could reduce the malnutrition gap by >20% and the quadratic malnutrition gap by >30%, but would be very expensive to implement. "Broad stroke" geographic targeting could reduce the same measures by 15 and 20%, respectively, and would be much less expensive to implement. We conclude that geographic targeting has the potential to substantially enhance the effect of nutrition programs on the severity of stunting in Honduras.

Body Height↗
PubMed2000

Sequences of Citrus tristeza virus separated in time and space are essentially identical.

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

Base Sequence↗
PubMed2000

The DNA of a plant retroviroid-like element is fused to different sites in the genome of a plant pararetrovirus and shows multiple forms with sequence deletions.

Carnation small viroid-like RNA (CarSV RNA) and its homologous DNA are the two forms of a unique plant retroviroid-like system. CarSV RNA is a 275-nucleotide noninfectious viroid-like RNA, present in certain carnation plants, which can adopt hammerhead structures in both polarity strands and self-cleave accordingly. CarSV DNA is organized as a series of head-to-tail multimers forming part of extrachromosomal elements in which CarSV DNA sequences are fused to sequences of carnation etched ring virus (CERV), a plant pararetrovirus. Analysis of more than 30 CarSV-CERV DNA junctions showed that distinct regions of the viral genome seem able to interact with CarSV DNA. All these junctions were short nucleotide stretches common to both CarSV and CERV DNAs. This suggests a polymerase-driven mechanism for their origin involving an enzyme with low processivity, most likely the viral reverse transcriptase. This view was further supported by the observation that most of CarSV sequences forming part of the junctions correspond either to strong secondary structure motifs in the conformation proposed for CarSV RNA or to its self-cleavage sites, which may have facilitated polymerase jumping. Accompanying the most-abundant CarSV RNA, a series of CarSV RNAs with sequence deletions were previously characterized. Here we have identified some of their corresponding DNA forms, together with other CarSV DNA forms with deletions not found in any CarSV RNA species identified so far. Some of these CarSV DNA forms have also been detected fused to CERV sequences. The existence of these shortened CarSV DNA versions may provide a continuous input of their corresponding transcripts and explain the persistence of CarSV RNAs with defective hammerhead structures for which an RNA-RNA model of amplification seems unlikely.

Base Sequence↗
PubMed1999

Reactive oxygen intermediates during programmed cell death induced in the thymus of the Ts(1716)65Dn mouse, a murine model for human Down's syndrome.

Down's syndrome (DS) is one of the most frequent genetic disorders in humans. It has been suggested that overexpression of copper-zinc superoxide dismutase (SOD-1) in DS may be involved in some of the abnormalities observed, mainly neurodegenerative and immunopathological processes. One of the consequences is early thymic involution. Recently, Ts(1716)65Dn mice (Ts65Dn mice), made segmentally trisomic for a chromosome 16 segment, fulfill the criteria for a DS model. To study the possible role of SOD-1 overexpression in thymocyte biology, we analyzed the role of reactive oxygen intermediates during in vivo and in vitro programmed cell death (PCD) induced in the thymus of Ts65Dn mice. Our main findings can be summarized as follows. Ts65Dn thymuses exhibit greater PCD activity than controls, as ascertained by a combination of morphological, histochemical, and ultrastructural procedures. Ts65Dn thymocytes were highly susceptible to PCD induced by both LPS (in vivo) and dexamethasone, a synthetic glucocorticoid agonist (both in vivo and in vitro). Thymus abnormalities were probably caused by SOD-1 hyperexpression in Ts65Dn cells, in that reactive oxygen intermediate generation (specifically H2O2 production) is enhanced in thymocytes and clearly correlates with apoptosis. Similarly, oxidative injury correlated with the formation of lipid peroxidation by-products and antioxidants which partly inhibit PCD in thymocytes.

Animals↗
PubMed1999

Mapping the molecular determinant of pathogenicity in a hammerhead viroid: a tetraloop within the in vivo branched RNA conformation.

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398-399 nt that can adopt hammerhead structures in both polarity strands. We have identified by Northern-blot hybridization a nonsymptomatic strain (CChMVd-NS) that protects against challenge inoculation with the symptomatic strain (CChMVd-S). Analysis of CChMVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the mutations observed in CChMVd-NS molecular variants were previously identified in CChMVd-S RNA, but others were never found in this RNA. When bioassayed in chrysanthemum, cDNA clones containing the CChMVd-NS specific mutations were infectious but nonsymptomatic. Site-directed mutagenesis showed that one of the CChMVd-NS-specific mutations, a UUUC --> GAAA substitution, was sufficient to change the symptomatic phenotype into the nonsymptomatic one without altering the final accumulation level of the viroid RNA. The pathogenicity determinant-to our knowledge, a determinant of this class has not been described previously in hammerhead viroids-is located in a tetraloop of the computer-predicted branched conformation for CChMVd RNA. Analysis of the sequence heterogeneity found in CChMVd-S and -NS variants strongly supports the existence of such a conformation in vivo, showing that the rod-like or quasi-rod-like secondary structure is not a universal paradigm for viroids.

Asteraceae↗