Integrative analysis of chromosome banding, telomere localization and molecular genetics in the highly variable Ctenomys of the Corrientes group (Rodentia; Ctenomyidae).

The genus Ctenomys comprises about 70 species with great chromosome diversity. The Corrientes group is one of the most chromosomally variable lineages in the genus, where the diploid number (2n) varies from 41 to 70. In this group, three nominal species and numerous polymorphic and polytypic populations have been described. In order to get insight into the chromosomal evolution of this species complex, we applied different banding and molecular cytogenetic techniques. The results were interpreted in an evolutionary context, based on mitochondrial cytochrome b analyses. Studied samples are representative of the broad chromosomal variability in the group, including specimens with 2n = 42 to 2n = 70. Heterochromatin was scarce but concentrated in a few chromosomes. Centromeric DAPI-negative heterochromatin was observed in some autosomal pairs, which differed among populations. Location and amount of DAPI-neutral heterochromatin within the Y chromosome varied among populations. The variable distribution of heterochromatin indicates its dynamic behavior. NORs were detected in one pair of autosomes, which also differed among some populations. Telomeric FISH signals were observed in all complements only at the chromosome ends. The Corrientes group belongs to a clade that also includes C. pearsoni, C. lami, C. minutus, C. ibicuiensis and C. torquatus. Almost all of these species are variable at the chromosomal level, suggesting that this is the ancestral condition of the clade. Within the Corrientes group, the observed low genetic divergence, in contrast with its high chromosomal variability, is indicative of decoupling between the rates of chromosomal and mitochondrial evolution.

Non-surgically related causes of erectile dysfunction after bilateral nerve-sparing radical prostatectomy.

BACKGROUND: Erectile dysfunction (ED) represents one of the most common long-term side effects in prostate cancer (PCa) patients treated with bilateral nerve-sparing radical prostatectomy (BNSRP). The aim of our study was to assess the influence of non-surgically related causes of ED in patients treated with BNSRP. METHODS: Overall, 716 patients treated with BNSRP were retrospectively identified. All patients had complete data on erectile function (EF) assessed by the Index of Erectile Function-EF domain (IIEF-EF) and depressive status assessed by the Center for Epidemiologic Studies-Depression (CES-D) questionnaire. EF recovery was defined as an IIEF-EF of ⩾22. Kaplan-Meier analyses assessed the impact of preoperative IIEF-EF, depression and adjuvant radiotherapy (aRT) on the time to EF recovery. Multivariable Cox regression models were used to test the impact of aRT on EF recovery after accounting for depression and baseline IIEF-EF. RESULTS: Median follow-up was 48 months. Patients with a preoperative IIEF-EF of ⩾22 had substantially higher EF recovery rates compared with those with a lower IIEF-EF (P<0.001). Patients with a CES-D of <16 had significantly higher EF recovery rates compared to those with depression (60.8 vs 49.2%; P=0.03). Patients receiving postoperative aRT had lower rates of EF compared with their counterparts left untreated after surgery (40.7 vs 59.8%; P<0.001). These results were confirmed in multivariable analyses, where preoperative IIEF-EF (P<0.001), depression (P=0.04) and aRT (P=0.03) were confirmed as significant predictors of EF recovery. CONCLUSIONS: Preoperative functional status and depression should be considered when counseling PCa patients regarding the long-term side effects of BNSRP. Moreover, the administration of aRT has a detrimental effect on the probability of recovering EF after BNSRP. This should be taken into account when balancing the potential benefits and side effects of multimodal therapies in PCa patients.

Preservation of the internal vesical sphincter and proximal urethra during retropubic radical prostatectomy may improve earlier recovery of continence in selected patients.

OBJECTIVES: To evaluate the influence of preservation of the muscular internal sphincter and proximal urethra on continence recovery after radical prostatectomy (RP). MATERIAL AND METHODS: Fifty-five consecutive patients with organ confined prostate cancer were submitted to RP with the preservation of muscular internal sphincter and the proximal urethra (group 1) and compared to 55 patients submitted to standard procedure (group 2). Continence rates were assessed using a self-administrated questionnaire at 3, 7, 30 days and 3, 12 months after removal of the catheter. RESULTS: Group 1 had a faster recovery of continence than group 2 at 3 days (50.9% vs. 25.5%; P=.005), at 7 days (78.2% vs. 58.2%; P=.020), at 30 days (80.0% vs. 61.8%; P=.029) and at 3 months (81.8% vs. 61.8%; P=.017); there were no statistically difference in terms of continence at 12 months among the two groups. Multivariate logistic regression analysis of continence showed that surgical technique was significantly associated with earlier time to continence at 3 and 7 days. The two groups had no significant differences in terms of surgical margins. CONCLUSIONS: Our modified technique of RP with preservation of smooth muscular internal sphincter as well as of the proximal urethra during bladder neck dissection resulted in significant increased early urinary continence at 3, 7, 30 days and 3 months after catheter removal. The technique does not increase the rate of positive margins and the duration of the procedure.

Decreased whole-blood global DNA methylation is related to serum hormones in anorexia nervosa adolescents.

OBJECTIVES: The one-carbon metabolism, also known as methionine-homocysteine cycle, governs the dynamics of DNA methylation, epigenetically regulating gene expression, and has been reported altered in anorexia nervosa (AN) adult patients. The aim of this study consisted in assessing whole-blood DNA methylation in adolescent AN patients, assessing its significance in relationship to clinical and hormonal variables. METHODS: Whole-blood global DNA methylation was measured as incorporation of [(3)H]dCTP following HpaII cut in 32 adolescent females affected by restrictive type AN and compared to 13 healthy controls. Homocysteine, vitamin B12 and folate plasma levels were assessed as well as fasting plasma levels of leptin and steroid hormones. Clinical variables, including severity and associate states and traits, were assessed by means of the EDI-3, CDI and STAI-Y scales. RESULTS: We confirm that whole-blood global DNA methylation is modestly albeit significantly reduced in AN adolescents with respect to controls, correlating with plasma leptin and steroid hormone levels. Conversely, clinical traits did not correlate with the outcome variable. CONCLUSIONS: A better definition of the epigenetic dysregulation underlying AN pathology or vulnerability might lead to develop useful markers for diagnosis, prognostic classification and tailored therapeutic interventions in these vulnerable patients since the earliest phases of their disease.

Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis.

Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the Y. pestis CO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of Serratia marcescens. This locus consisted of the hasR(yp) receptor gene, the hasA(yp) hemophore gene, and genes encoding components of the HasA(yp) dedicated ABC transporter factor (hasDE(yp)), as well as a tonB homologue (hasB(yp)). By using a reconstituted secretion system in Escherichia coli, we showed that HasA(yp) is a secreted heme-binding protein and that expression of HasA(yp) is iron regulated in E. coli. The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur regulated and has increased activity at 37 degrees C. Hemoglobin utilization via the Has(yp) system was studied with both E. coli and Y. pestis, for which has and has hmu mutant strains were used. No contribution of the Has system to heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu(-) Has(-) double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity.

Recurrent amplifications and deletions of satellite DNA accompanied chromosomal diversification in South American tuco-tucos (genus Ctenomys, Rodentia: Octodontidae): a phylogenetic approach.

We investigated the relationship between satellite copy number and chromosomal evolution in tuco-tucos (genus Ctenomys), a karyotypically diverse clade of rodents. To explore phylogenetic relationships among 23 species and 5 undescribed forms, we sequenced the complete mitochondrial cytochrome b genes of 27 specimens and incorporated 27 previously published sequences. We then used quantitative dot-blot techniques to assess changes in the copy number of the major Ctenomys satellite DNA (satDNA), named RPCS. Our analysis of the relationship between variation in copy number of RPCS and chromosomal changes employed a maximum-likelihood approach to infer the copy number of the satellite RPCS in the ancestors of each clade. We found that amplifications and deletions of RPCS were associated with extensive chromosomal rearrangements even among closely related species. In contrast, RPCS copy number stability was observed within clades characterized by chromosomal stability. This example reinforces the suspected role of amplification, deletion, and intragenomic movement of satDNA in promoting extensive chromosomal evolution.

MRNA encoding a putative RNA helicase of the DEAD-box gene family is up-regulated in trypomastigotes of Trypanosoma cruzi.

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.

[Comparison of various parameters (pitch 1 and 2) in the study of the lung with spiral computerized tomography]. / Confronto tra diversi parametri (pitch 1 e 2) nello studio del polmone con tomografia computerizzata spirale.

INTRODUCTION: In Spiral CT, the pitch is the ratio of the distance the tabletop travels per 360 degrees rotation to nominal slice width, expressed in mm. Performing Spiral CT examinations with pitch 2 allows to reduce examination time, exposure and contrast dose, and X-ray tube overload. We investigated the yield of pitch 2 in lung parenchyma studies, particularly relative to diagnostic image quality. MATERIAL AND METHODS: Thirty patients were submitted to Spiral CT with pitch 1 [10 mm slice thickness, 10 mm/s table feed; 10 mm (a') and 5 mm (a") reconstruction index: protocol A] and with pitch 2 [10 mm slice thickness, 20 mm/s table feed; 10 mm (b') and 5 mm (b") reconstruction index: protocol B]. Five expert radiologists evaluated the images separately and blindly, grading noise, bronchial wall resolution and diagnostic yield on a 0-5 point scale. The results of protocol A versus protocol B images were analyzed statistically using the Mann-Whitney U-test. RESULTS: The mean scores for each parameter ranged 4.13 (.70 standard deviation) for protocol B with 5 mm reconstruction index (b") to 4.81 (.44 standard deviation) for protocol A with 10 mm reconstruction index (a'). These values (max: 5) indicate very positive results on both protocol A and B images. There were no statistically significant interprotocol differences, except for bronchial wall resolution, in favor of protocol A with 5 mm reconstruction index (a") (p = .025), and for diagnostic yield, in favor of protocol A with 10 mm reconstruction index (a') (p = .018). CONCLUSIONS: Spiral CT with pitch 2 is a reliable tool for lung parenchyma studies which permits to reduce examination time and contrast dose, as well as X-ray tube overload and exposure dose.

Novel processing in a mammalian nuclear 28S pre-rRNA: tissue-specific elimination of an 'intron' bearing a hidden break site.

Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp 'intron' located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis-specific processing eliminates the intron and concomitantly the break site, producing non-split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.

The evolutionary history of Drosophila buzzatii. XXX. Mitochondrial DNA polymorphism in original and colonizing populations.

Both original and colonizer populations of Drosophila buzzatii have been analyzed for mtDNA restriction polymorphisms. Most of the mtDNA nucleotide variation in original populations of NW Argentina can be explained by intrapopulation diversity and only a small fraction can be accounted for by between-population diversity. Similar results are obtained using either the estimated number of nucleotide substitutions per site or considering each restriction site as a locus. Colonizer populations of the Iberian Peninsula are monomorphic and show only the most common haplotype from the original populations. Under the infinite island model and assuming that populations are in equilibrium, fixation indices indicate enough gene flow to explain why the populations are not structured. Yet, the possibility exists that populations have not reached an equilibrium after a founder event at the end of the last Pleistocene glaciation. Tajima's test suggests that directional selection and/or a recent bottleneck could explain the present mtDNA differentiation. Considering the significant population structure found for the chromosomal and some allozyme polymorphisms, the among-population uniformity for mtDNA variability argues in favor of the chromosomal and some allozyme polymorphisms being adaptive.

The octogenarian cardiac surgery patient.

As advances are made in medical-surgical technology and overall life expectancy increases, cardiac surgery previously done only in younger populations is now becoming common in older adult age groups. Most of the nursing literature regarding elderly cardiac surgery patients focuses on the 65- to 75-year-old age group; little has been written about the 80- to 90-year-old age group. Very elderly patients present unique and complex challenges to the interdisciplinary teams involved in their care. Nurses must recognize and anticipate the specialized needs of these frail individuals to optimally manage their care. Aortic valve replacement is the most common cardiac valve surgical procedure performed in very elderly persons. A case study and an integrated care plan for the aortic valve surgery patient are described.

Chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys.

The chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys was analyzed in eight species by in situ hybridization, using a probe isolated from C. porteousi. The hybridization patterns showed different numbers of chromosomes with positive pericentromeric regions and/or complete short arms. In some species, a positive signal was scarce (or not detectable, as in C. opimus), and was usually located in the pericentromeric areas (C. occultus and C. latro). In those species where the satellite was highly amplified, its chromosomal localization tended to encompass the entire length of the short arms. These patterns were compared with C-band distribution patterns in the same species. We discuss the putative evolutionary trend of this satellite DNA in the genus Ctenomys and suggest that it evolved from a strictly pericentromeric localization to comprising the whole short arms of some chromosomes.

Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

Retroviral-like features in the monomer of the major satellite DNA from the South American rodents of the genus Ctenomys.

It is well known that uninfected mammalian cells contain DNA sequences which are closely related to retroviral genomic segments. However, these sequences seldom (if ever) have been found associated to highly repetitive (satellite) DNA. RPCS is a 348 bp monomer of a major satellite DNA from the South American rodents of the genus Ctenomys. It was found that RPCS contains several elements which are typical of the U3 region of retroviral LTRs. These elements are: a) a polypurine tract; b) two enhancer core sequences; c) two NF1 binding sites; d) two C/EBP binding sites; e) two CCAAT-motifs; f) a TATA box, and g) two putative polyadenylation motifs. Furthermore, the relative positions of these elements are as in the U3 retroviral regions.

Evidence for rolling-circle replication in a major satellite DNA from the South American rodents of the genus Ctenomys.

A major PvuII satellite DNA has been cloned from a South American octodontid rodent of the genus Ctenomys (C. porteousi). The satellite monomer, termed RPCS, is 337 bp in size and 42% G + C. Analysis of the nucleotide sequence demonstrates that RPCS is not composed of a series of shorter repeats. RPCS-related sequences were found in 11 of 12 Ctenomys species analyzed by hybridization under high-stringency conditions. The only negative species, C. opimus, was reactive under low-stringency conditions. RPCS-related sequences were not found under high- or low-stringency conditions in Calomys musculinus and Mus musculus. However, under low-stringency conditions, RPCS-related sequences were found in the octodontid Octodontomys gliroides, which is thought to have diverged from the genus Ctenomys more than 10 Myr ago. The pattern of periodicities observed, by restriction analysis, between Ctenomys species in the satellite array can be mainly accounted for by a rolling-circle amplification mechanism but cannot be solely accounted for by unequal crossing-over.

Detection of foot-and-mouth disease virus with DNA probes in bovine esophageal-pharyngeal fluids.

Infectivity and dot-blot hybridization techniques were compared for the detection of FMDV in esophageal-pharyngeal fluids from experimentally infected cows. The probe used includes the viral polymerase sequence which allows the detection of the three types of virus (A, O, and C) with equivalent sensitivity. Virus was detected by dot-blot hybridization as well as by infectivity, according to sample analysis of esophageal-pharyngeal fluids extracted seven days post-infection. It was not possible to recover infective virus from some samples extracted at 180 and 560 days post-infection, although specific viral RNA was detected by dot-blot hybridization. This could indicate the presence of a high ratio of non-infective viral mutants in FMDV carrier cattle. These results emphasize the usefulness of molecular hybridization techniques for FMDV carrier-state detection.

Molecular cloning of rotavirus genome

Los rotavirus son los principales agentes responsables de las gastroenteritis virales humanas y animales. La identificación y caracterización de su genoma es necesaria para la comprensión de esta patología así como para el desarrollo de nuevos métodos de diagnóstico y, eventualmente, para la preparación de antígenos virales utilizando técnicas de DNA recombinante. Estos virus poseen un genoma formado por once fragmentos de RNA doble cadena (cd). Aquí se describe la construcción de bancos de cDNA para rotavirus bovino y humano, ambos purificados de materia fecal. Los cDNA fueron preparados por síntesis in vitro utilizando transcriptasa reversa sobre los RNAs genómicos virales, previamente poliadenilados en sus extremos 3. Los cDNAs fueron ligados a un vector plasmídico y propagados en E. coli. Se obtuvieron genotecas correspondientes a los virus bovino y humano con 500 y 100 recombinantes respectivamente. Análisis de restricción de algunos clones permitieron establecer el tamaño de los insertos correspondientes a los distintos segmentos genómicos virales. Dos de estos clones fueron caracterizados, determinándose que contienen las secuencias completas de los fragmentos 10 y 8 del virus bovino. La utilización de estos clones como sondas radioactivas nos permitió diagnosticar la presencia de rotavirus en muestras de materia fecal mediante la detección de los correspondientes RNAs. Este ensayo pudo ser utilizado para la detección viral en muestras infectadas provenientes de distintas especies