The evolutionary life history of P transposons: from horizontal invaders to domesticated neogenes.

P elements, a family of DNA transposons, are known as aggressive intruders into the hitherto uninfected gene pool of Drosophila melanogaster. Invading through horizontal transmission from an external source they managed to spread rapidly through natural populations within a few decades. Owing to their propensity for rapid propagation within genomes as well as within populations, they are considered as the classic example of selfish DNA, causing havoc in a genomic environment permissive for transpositional activity. Tracing the fate of P transposons on an evolutionary scale we describe different stages in their evolutionary life history. Starting from horizontal transfer events, which now appear to be rather a common phenomenon, the initial transpositional burst in the new host is slowed down by the accumulation of defective copies as well as host-directed epigenetic silencing. This leads to the loss of mobility and, finally, to molecular erosion by random mutations. Possible escape routes from genomic extinction are the reactivation within the original host genome by recombination or suspension of the repressing regime, horizontal emigration to a virgin gene pool, or genomic integration and acquisition of a novel function as a domesticated host gene.

Randomized clinical trial of thalidomide, cyclosporine, and prednisone versus cyclosporine and prednisone as initial therapy for chronic graft-versus-host disease.

Chronic graft-versus-host disease (CGVHD) is a major cause of morbidity following allogeneic bone marrow transplantation. Thalidomide is active in salvage therapy for high-risk or resistant CGVHD. In a prospective randomized trial, we tested initial therapy with thalidomide. Patients with extensive CGVHD were randomized to receive either cyclosporine and alternate-day prednisone (n = 27, no-thalidomide [no-thal] group) or cyclosporine, prednisone, and thalidomide (200-800 mg/day; n = 27, thal group). Although most patients responded, initial therapy with thalidomide did not improve control of CGVHD. Response rates were 83% versus 89% at 2 months (P = .7), 88% versus 84% at 6 months (P > .8) and 85% versus 73% at 1 year (P = .5) in the thal and no-thal groups, respectively. Multivariate analysis revealed related donor transplant (odds ratio [OR] = 11.3; P =.03) and de novo or quiescent onset of CGVHD (OR = 7.7; P =.04) to be significant predictors of good early response, whereas a platelet count of > or =100,000/microL was a significant predictor of good response (OR = 10.4; P =.04) at 1 year. Survival for the thal and no-thal groups was similar at 1 year (66% versus 74%) and 2 years (66% versus 54%, P = .85). Multivariate analysis revealed progressive onset CGVHD (relative risk [RR] = 4.2; P =.01), unrelated donor (RR = 5.7; P < .01), sex mismatch (RR = 7.9; P < .01), and platelet counts of <100,000/microL (RR = 3.8; P = .01) as significant predictors of poorer survival. These data suggest that despite a high response rate (79% response and 53% complete response) and encouraging survival rates (70% at 1 year and 60% at 2 years), thalidomide offers no clinical benefit when incorporated into initial therapy for CGVHD. The value of thalidomide as salvage therapy requires further study.

Evolutionary dynamics of the SGM transposon family in the Drosophila obscura species group.

SGM (Drosophila subobscura, Drosophila guanche, and Drosophila madeirensis) transposons are a family of transposable elements (TEs) in Drosophila with some functional and structural similarities to miniature inverted-repeat transposable elements (MITEs). These elements were recently active in D. subobscura and D. madeirensis (1-2 MYA), but in D. guanche (3-4 MYA), they gave rise to a species-specifically amplified satellite DNA making up approximately 10% of its genome. SGM elements were already active in the common ancestor of all three species, giving rise to the A-type specific promoter section of the P:-related neogene cluster. SGM sequences are similar to elements found in other obscura group species, such as the ISY elements in D. miranda and the ISamb elements in Drosophila ambigua. SGM elements are composed of different sequence modules, and some of them, i.e., LS and LS-core, are found throughout the Drosophila and Sophophora radiation with similarity to more distantly related TEs. The LS-core module is highly enriched in the noncoding sections of the Drosophila melanogaster genome, suggesting potential regulatory host gene functions. The SGM elements can be considered as a model system elucidating the evolutionary dynamics of mobile elements in their arms race with host-directed silencing mechanisms and their evolutionary impact on the structure and composition of their respective host genomes.

Response to thalidomide therapy in refractory chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) refractory to standard immunosuppressive therapy remains a major cause of morbidity and mortality after allogeneic bone marrow transplantation (BMT). Thalidomide may be effective in some patients with high-risk or refractory chronic GVHD. We report a single-institution study of thalidomide in 37 BMT patients with extensive chronic GVHD refractory to standard immunosuppressive therapy. Acute GVHD occurred in 34 (91%) of patients and evolved progressively into chronic GVHD in 23 (62%) patients. Thalidomide was added to standard immunosuppressive therapy a median of 11 months (range 0-105 months) after the diagnosis of chronic GVHD. Fourteen of 37 (38%) patients responded after introduction of thalidomide (one complete, 13 partial). Ten of 21 (46%) children and four of 16 (25%) adults responded. Responses were seen in eight of 17 (47%) recipients of related donor marrow and six of 20 (30%) recipients of unrelated donor marrow. Eight of 23 (34%) patients with progressive onset of chronic GVHD showed a response. There were no deaths among the responders. The remaining 23 patients (62%) did not respond and of these only two survive, one with progressive scleroderma, and the other with bronchiolitis obliterans. Chronic GVHD with associated infection (most commonly disseminated fungal infection) was a major contributor to mortality in all cases. Overall, after initiation of thalidomide, the 2-year Kaplan-Meier survival was 41% (95% C.I. 24%-59%). We conclude that thalidomide is a useful and well-tolerated therapy for patients with previously treated refractory chronic GVHD, including those with progressive onset of chronic GVHD, recipients of unrelated donor marrow, and children. Earlier introduction of thalidomide as an adjunct to standard immunosuppressive therapy may lead to more frequent responses and possible better survival.

Localization of the fragile X mental retardation 2 (FMR2) protein in mammalian brain.

The transcriptional silencing of the FMR2 gene has been implicated in FRAXE mental retardation. FRAXE individuals have been shown to exhibit learning deficits, including speech delay, reading and writing problems. FMR2 encodes a large protein of 1311 amino acids and is a member of a gene family encoding proline-serine-rich proteins that have properties of nuclear transcription factors. To characterize the expression of the fragile X mental retardation 2 (FMR2) protein, polyclonal antibodies were raised against two regions of the human FMR2 protein and used in immunofluorescence experiments on mouse brain cryosections. Our results demonstrate for the first time that the FMR2 protein is localized in neurons of the neocortex, Purkinje cells of the cerebellum and the granule cell layer of the hippocampus. FMR2 staining is shown to colocalize with the nuclear stain 4,6-diamidino-2-phenylindole (DAPI) confirming that FMR2 is a nuclear protein. The localization of FMR2 protein to the mammalian hippocampus and other brain structures involved with cognitive function is consistent with the learning deficits seen in FRAXE individuals.

Molecular domestication--more than a sporadic episode in evolution.

Transposable elements are short but complex pieces of DNA or RNA containing a streamlined minimal-genome with the capacity for its selfish replication in a foreign genomic environment. Cis-regulatory sections within the elements orchestrate tempo and mode of TE expression. Proteins encoded by TEs mainly direct their own propagation within the genome by recruitment of host-encoded factors. On the other hand, TE-encoded proteins harbor a very attractive repertoire of functional abilities for a cell. These proteins mediate excision, replication and integration of defined DNA fragments. Furthermore, some of these proteins are able to manipulate important host factors by altering their original function. Thus, if the host genome succeeds in domesticating such TE-encoded proteins by taming their 'anarchistic behavior,' such an event can be considered as an important evolutionary innovation for its own benefit. In fact, the domestication of TE-derived cis-regulatory modules and protein coding sections took place repeatedly in the course of genome evolution. We will present prominent cases that impressively demonstrate the beneficial impact of TEs on host biology over evolutionary time. Furthermore, we will propose that molecular domestication might be considered as a resumption of the same evolutionary process that drove the transition from 'primitive genomes' to 'modern' ones at the early dawn of life, that is, the adaptive integration of a short piece of autonomous DNA into a complex regulatory network.

Re-organising emergency medical admitting: the Stobhill experience, 1992-1997.

OBJECTIVE: To examine patterns of acute medical emergency admissions and the effect of reorganisation on their management. DESIGN: Examination of statistics for emergency medical admissions from 1992 to 1997, a period that included a major reorganisation of the emergency admitting system within the hospital. SETTING: General hospital in a Scottish conurbation. RESULTS: There was a slow annual increase in numbers of admissions during the period of study with very considerable variations in daily and weekly numbers of admissions. Reorganisation achieved a reduction in average length of stay from seven to 4.5 days permitting reduction of the bed complement from 223 to 161. Following reorganisation, 31% of admissions were discharged home within 48 hours directly from the acute medical receiving ward, 18% of admissions were transferred directly to care of the elderly, and 33% of admissions were transferred to medical wards. Patient and staff satisfaction surveys indicated preference for the new system over the old. Admission peaks over the winter months of the last three years occurred at different weeks in the year. CONCLUSIONS: Reorganisation of the medical admitting system can improve efficiency and allow reductions in staffed beds. The considerable [table: see text] variation in daily demands in the system makes it important to retain flexibility. There may be scope for dealing with the large numbers of short-term admissions in other ways.

Ganciclovir susceptibilities and analysis of UL97 region in cytomegalovirus (CMV) isolates from bone marrow recipients with CMV disease after antiviral prophylaxis.

Ganciclovir susceptibilities and UL97 sequences were analyzed in 20 cytomegalovirus (CMV) isolates recovered from 15 bone marrow transplant recipients with active CMV infection after prophylaxis with acyclovir (group I; 12 isolates) or after acyclovir prophylaxis followed by ganciclovir therapy (group II; 8 isolates). All group I isolates were susceptible to ganciclovir. Five group II isolates were susceptible to ganciclovir, and 3 isolates (all from the same person) were resistant to ganciclovir (IC50 > 12 microM). Ganciclovir resistance UL97 mutations were found in 4 group II isolates, including a ganciclovir-susceptible isolate obtained from 1 patient after 41 days of therapy with ganciclovir and 3 ganciclovir-resistant isolates obtained from another patient after 73, 116, and 132 days of treatment with ganciclovir. Ganciclovir-resistant CMV isolates may emerge rapidly in bone marrow transplant recipients who are treated with ganciclovir after receiving prophylaxis with acyclovir.

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae.

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.

Nested insertions of short mobile sequences in Drosophila P elements.

A potentially full-sized P element isolated from the genome of Drosophila ambigua by polymerase chain reaction amplification was completely sequenced. It has a length of 3329 bp and the termini are formed by 33 bp inverted repeats. Sequence comparisons show that it can be classified as a member of the T-type P element subfamily. The translational reading frames of all four exons are interrupted by stop codons and frameshift mutations. At the 3' end of exon 3 a 687 bp insertion sequence (IS-amb-P) is found that also occurs in the form of dispersed copies (IS-amb) in the genome in D. ambigua. At the interspecific level it shows homology to mobile sequences of other species of the obscura group. Although variable in length, these IS elements are characterized by conserved sections without coding function and by 14 bp inverted repeats, one at a terminal, the other at a subterminal position. In situ hybridization revealed that P elements in D. ambigua are restricted to only two euchromatic sites on chromosome elements A and E. This situation resembles that found in Drosophila guanche and Drosophila subobscura where P homologs are clustered at a single site on chromosome element E and where the section corresponding to exon 3 of P elements carries an IS element. The gene sik-hom, which is located at the 5' side of the D. guanche cluster of P homologs, was used as a marker to examine whether the P element sites on chromosome element E of D. guanche and D. ambigua are homologous. The results suggest that the nested insertions of IS elements into P elements must have occurred independently in the two different lineages.

Relaçäo entre fatores sangüíneos de bovinos em bubalinos / Relationship between cattle blood factors in water buffaloes

Com o objetivo de verificar as relaçöes entre fatores sangüíneos de bovinos e bubalinos, foram tipificados 121 búfalos de rio (Bubalus bubalis bubalis), das raças Mediterrâneo, Murrah, Jafarabadi e também alguns animais sem raça definida (SRD). A tipagem foi realizada em placas de microtitulaçäo segundo técnicas usadas rotineiramente para tipagem de bovinos. Foram empregados 45 reagentes bovino-isoimunes, cobrindo dez sistemas de grupos sangüíneos. Ao final das leituras, os animais da raça Murrah apresentaram reaçöes positivas para os fatores A1, D, H, B, K, Q, T2, Y2, A', B', F', O', Q', C2, R2, X2, F, V, N', J, L e U1; os da raça Mediterrâneo para os fatores A1, D, H, I2, K, P, Q, A', B', F', O', P'2, C2, R2, X2, V, N', J, L e Z; os da raça Jafarabadi para os fatores A1, D, H, Z', K, P, Q, T1, Y2, A', D', E'2, K', O', Q', C2, R2, N', J, M1, S, U1 e R'; os SRD positivos para os fatores A1, D, H, Z', B, K, P, T2, Y2, A', B', D', E'2, F', I', O', Q', C2, R1, R2, N', J, M1, S, U1 e R'. Ressalte-se que os fatores sublinhados foram observados apenas naqueles respectivos grupamentos raciais. Os animais SRD mostraram 13 fatores comuns com os da raça Mediterrâneo, 21 com os da raça Jafarabadi e 15 com os da raça Murrah. Os resultados mostram relaçöes estreitas entre 35 fatores pertencentes a 10 sistemas. Tal fato é indicativo da possível utilizaçäo desses reagentes bovino-isoimunes com reaçöes positivas, em testes de parentesco de bubalinos. Ao mesmo tempo, mostram existir diferenças genéticas quanto aos fatores observados e suas freqüências nas subpopulaçöes analisadas, permitindo, ainda, a detecçäo de mestiçagem

Evaluation of the portal venous system: complementary roles of invasive and noninvasive imaging strategies.

Evaluation of the portal venous system is required in several clinical circumstances, including before and after liver transplantation, before creation of a transjugular intrahepatic portosystemic shunt, in the clinical setting of bowel ischemia, or to evaluate varices. Several noninvasive modalities (magnetic resonance [MR] imaging and MR angiography, computed tomography [CT], and ultrasound [US]) are available for evaluation of the portal venous system in addition to the invasive angiographic methods. In most clinical circumstances, either CT or MR imaging and MR angiography in combination with US of the liver vasculature will allow complete evaluation of the portal venous system. Invasive evaluation of the portal venous system is necessary when results of the noninvasive tests disagree or are inconclusive. Angiography may also be indicated whenever noninvasive tests indicate occlusion of the portal venous system, as this is often a crucial clinical question and false-positive results can occur with the noninvasive tests.

Autologous bone marrow versus non-mobilized peripheral blood stem cell transplantation for lymphoid malignancies: a prospective, comparative trial.

Autologous transplantation using bone marrow stem cells (BMSC) or peripheral blood stem cells (PBSC) is widely used for non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). We report a randomized, comparative trial comparing BMSC vs. non-mobilized PBSC for responsive NHL or HD. Patients randomized to BMSC (n = 13) vs. PBSC (n = 15) had more rapid neutrophil recovery (median 23 vs. 30 days), RBC independence (25 vs. 62 days), platelet independence (24 vs. 54 days), and shorter hospital stay. However, neither relapse, overall survival, nor relapse-free survival were different receiving BMSC vs. PBSC (all P > .7). Concurrently, 54 others (34 BMSC, 20 PBSC) were assigned non-randomly because of resistant disease or marrow unsuitable for harvest and similar patterns of engraftment favoring BMSC over PBSC were observed. In the entire group, BMSC transplantation (n = 47) led to quicker neutrophil recovery (P = .02), RBC (P = .06), and platelet independence (P =.04) and earlier hospital discharge (P = .02) vs. PBSC (n = 35). No difference in relapse, overall, or relapse-free survival were observed using BMSC vs. PBSC. These data suggest that non-mobilized PBSC are a satisfactory alternative to BMSC in patients with unsuitable marrow; however, transplantation with non-mobilized PBSC was associated with slower hematologic recovery, and longer hospital stay. No difference in tumor recurrence rates was observed between the PBSC or BMSC recipients. Unprimed PBSC transplantation offered no clinical advantage to BMSC.

Associaçäo entre marcadores de grupos sangüíneos e peso aos 365 dias de idade em bovinos da raça Nelore / Association between blood groups markers and one year weight in Nelore cattle

Foram levantados dados de tipagem sangüínea e peso ajustado para 365 dias de progênies de três touros da raça Nelore, todos heterozigotos para fenogrupos do Sistema B de grupos sangüíneos. Cada progênie foi agrupada segundo o fenogrupo paterno herdado, no intuito de se verificarem possíveis associaçöes entre esses marcadores de grupos sangüíneos e peso. Em análises de progênies individuais, näo se detectou diferença significativa entre a média de peso aos 365 dias dos animais agrupados segundo o fenogrupo do Sistema B herdado, de modo que näo podem ser eles considerados como marcadores para a característica em questäo. Entretanto, numa análise conjunta de filhos e netos de um touro, houve diferença significativa (P<0,05) entre a média de peso dos dois grupos. Isso caracteriza o fenogrupo do Sistema B, BQY1D'J'O'P, como marcador, embora seu efeito para a variaçäo da característica seja pequeno (coeficiente de determinaçäo do modelo, R ao quadrado = 0,82 por cento). Tais afirmaçöes säo válidas apenas para as linhagens estudadas

LTR retrotransposons and the evolution of eukaryotic enhancers.

Since LTR retrotransposons and retroviruses are especially prone to regional duplications and recombination events, these viral-like systems may be especially conducive to the evolution of closely spaced combinatorial regulatory motifs. Using the Drosophila copia LTR retrotransposon as a model, we show that a regulatory region contained within the element's untranslated leader region (ULR) consists of multiple copies of an 8 bp motif (TTGTGAAA) with similarity to the core sequence of the SV40 enhancer. Naturally occurring variation in the number of these motifs is correlated with the enhancer strength of the ULR. Our results indicate that inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and perhaps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to play a role in regulatory evolution.

Molecular domestication of mobile elements.

Transposable elements are ubiquitous in all organisms and represent a dynamic component of their genomes, causing mutations and thereby genetic variation. Because of their independent and expansive replication strategy, these elements are called selfish and were thought to have no impact on the adaptive evolution of their host organisms. Although most TE-induced mutations seem to exert only negative effects on the fitness of their carrier, recent evidence indicates that in the course of evolution at least some TE-mediated changes have become established features of the host genome. For example, the insertion of TEs may provide novel cis-regulatory regions to preexisting host genes or TE-derived trans-acting factors may undergo a molecular transition into novel host genes through a process described as molecular domestication. The stationary P element related gene clusters of D. guanche, D. madeirensis and D. subobscura provide an excellent model system to study the evolutionary impact of TEs on genome evolution. Each cluster unit consists of a cis-regulating section composed of different insertion sequences followed by the first three exons of a P element that are coding for a 66 kDa 'repressor-like' protein.

Structure and organization of the P element related sequences in Drosophila madeirensis.

The P element homologous sequences of the two closely related species Drosophila guanche and Drosophila subobscura represent a very special case of transposable-element derivatives. Although they have lost the regions known to be essential for P transposition by random mutations, all of them have selectively conserved the coding capacity for "P-repressor-like" proteins during the past few millions years. In both species, they are tandemly amplified in a single euchromatic gene cluster at equivalent chromosomal positions. In contrast, Drosophila madeirensis, an endemic species that is very closely related to both D. subobscura and D. guanche, harbours an additional P homologous site. Several mechanisms can be invoked to explain the generation of the new site in this species. In this work we present several molecular and cytological data in order to elucidate the possible evolutionary origin of the P derivatives of D. madeirensis.

Preferential targeting of oxidative base damage to internucleosomal DNA.

The structure of nuclear chromatin may limit the accessibility of carcinogenic agents to DNA. In the case of oxidative DNA strand cleavage mediated by the physiologically relevant iron chelate, iron-ADP, histone-associated nucleosomal DNA is protected while internucleosomal DNA is susceptible to damage. We now find that the distribution of iron-ADP-generated 8-hydroxydeoxyguanosine, a potentially mutagenic oxidative base change, shows relative targeting to internucleosomal sites (3.5-fold increased oxidative modification of internucleosomal compared with nucleosomal DNA as the minimal degree of enrichment). In contrast, iron-EDTA, which generates hydroxyl radical in the 'fluid phase', does not target internucleosomal DNA. Thus, physiologic iron chelates may promote site-specific damage and thereby be relevant to mechanisms of iron-dependent oxidative mutagenesis and carcinogenesis.

Related donor marrow transplant for chronic myeloid leukemia: patient characteristics predictive of outcome.

Pre-transplant characteristics of 137 consecutive patients (including 103 patients with one or more features suggesting advanced disease) undergoing related donor marrow transplant for chronic myeloid leukemia (CML) were analyzed to determine their association with outcome. Multivariate analysis identified increased recipient age (relative risk (RR) for patients over 30 years of relapse or death 2.37; P = 0.004), and longer interval between diagnosis and transplant (RR 1.20; P = 0.0001) as significant adverse influences on disease-free survival (DFS). The 5-year DFS for patients transplanted within 1 year of diagnosis (¿early transplant', n = 71) was significantly higher (51%) than that for patients transplanted beyond 1 year from diagnosis ('delayed transplant', n = 55) (34%; log rank P = 0.02). For early transplant patients, poor prognostic features included myelofibrosis (RR 3.53; P = 0.018), splenomegaly (RR 2.22; P = 0.029) and the use of a female donor (RR 3.16; P = 0.002). The 5-year DFS for patients transplanted within 1 year of diagnosis and without signs of advanced disease was 67%. The presence of increasing numbers of features suggesting acceleration prior to transplant had a cumulative adverse influence of DFS. The risk of relapse (5 year estimate 20%) was also independently and significantly increased in association with a longer interval from diagnosis to transplant (P = 0.012). Early transplant is an important influence on DFS and relapse after related donor transplant therapy for CML, although additional patient characteristics influencing outcome can be identified and may have cumulative adverse effects.

Autologous bone marrow transplantation for non-transformed low-grade non-Hodgkin's lymphoma.

The purpose of this study was to evaluate autologous bone marrow transplantation (ABMT) for low-grade non-Hodgkin's lymphoma (NHL). Between 1983 and 1994, 34 patients with non-transformed low-grade NHL received high-dose chemoradiotherapy plus stem cell rescue with marrow or peripheral blood. At transplantation (5-182 months (median 32) after diagnosis), 2 patients were in complete remission (CR), 21 in partial remission (PR) or minimal disease; 11 had bulky/resistant disease. Four patients died from transplant-related causes and 10 from lymphoma progression. After 40 months median follow-up, 20 survive between 3+ and 88+ months after ABMT. One-year survival is 81% (95% confidence interval, 65-96%) and 5-year survival 37% (14-60%). The probability of relapse at 1 and 2 years is 53% (34-72%) and 75% (54-96%), respectively. Twenty-nine patients were in CR following ABMT and 11 survive in continuous CR 3 to 54 months later. Disease-free survival at 1 and 2 years is 35% (19-52%) and 18% (2-31%). Resistant disease was the only factor significantly associated with poor overall (relative risk = 3.0, P = 0.04) and disease-free survival (RR = 5.4, P = 0.003), while resistant disease and high LDH were associated with increased relapse. ABMT yields a high CR rate for patients with progressive low-grade NHL, but relapse is frequent. Even longer follow-up is required to determine its effectiveness in extending survival of patients with pre-transplant responsive disease.