DNA Methylation-Guided Prediction of Clinical Failure in High-Risk Prostate Cancer.

BACKGROUND Prostate cancer (PCa) is a very heterogeneous disease with respect to clinical outcome. This study explored differential DNA methylation in a priori selected genes to diagnose PCa and predict clinical failure (CF) in high-risk patients. METHODS A quantitative multiplex, methylation-specific PCR assay was developed to assess promoter methylation of the APC, CCND2, GSTP1, PTGS2 and RARB genes in formalin-fixed, paraffin-embedded tissue samples from 42 patients with benign prostatic hyperplasia and radical prostatectomy specimens of patients with high-risk PCa, encompassing training and validation cohorts of 147 and 71 patients, respectively. Log-rank tests, univariate and multivariate Cox models were used to investigate the prognostic value of the DNA methylation. RESULTS Hypermethylation of APC, CCND2, GSTP1, PTGS2 and RARB was highly cancer-specific. However, only GSTP1 methylation was significantly associated with CF in both independent high-risk PCa cohorts. Importantly, trichotomization into low, moderate and high GSTP1 methylation level subgroups was highly predictive for CF. Patients with either a low or high GSTP1 methylation level, as compared to the moderate methylation groups, were at a higher risk for CF in both the training (Hazard ratio [HR], 3.65; 95% CI, 1.65 to 8.07) and validation sets (HR, 4.27; 95% CI, 1.03 to 17.72) as well as in the combined cohort (HR, 2.74; 95% CI, 1.42 to 5.27) in multivariate analysis. CONCLUSIONS Classification of primary high-risk tumors into three subtypes based on DNA methylation can be combined with clinico-pathological parameters for a more informative risk-stratification of these PCa patients.

Genetic diversity of bovine Neospora caninum determined by microsatellite markers

Neospora caninum is one of the most significant parasitic organisms causing bovine abortion worldwide. Despite the economic impact of this infection, relatively little is known about the genetic diversity of this parasite. In this study, using Nc5 and ITS1 nested PCR, N. caninum has been detected in 12 brain samples of aborted fetuses from 298 seropositive dairy cattle collected from four different regions in Tehran, Iran. These specimen (Nc-Iran) were genotyped in multilocus using 9 different microsatellite markers previously described (MS4, MS5, MS6A, MS6B, MS7, MS8, MS10, MS12 and MS21). Microsatellite amplification was completely feasible in 2 samples, semi-completely in 8 samples, and failed in 2 samples. Within the two completely performed allelic profiles of Nc-Iran strains, unique multilocus profiles were obtained for both and novel allelic patterns were found in the MS8 and MS10 microsatellite markers. The Jaccard's similarity index showed significant difference between these two strains and from other standard isolates derived from GenBank such as Nc-Liv, Nc-SweB1, Nc-GER1, KBA1, and KBA2. All samples originating from the same area showed identical allelic numbers and a correlation between the number of repeats and geographic districts was observed.

Longitudinal Dichelobacter nodosus status in 9 sheep flocks free from clinical footrot

Footrot is a widespread problem in Swiss sheep farming. The objectives of this study were to determine whether flocks which were clinically free from footrot carry virulent strains of Dichelobacter nodosus, and to describe the infection dynamics for flocks and individual sheep. To this purpose, a new PCR-diagnostic tool was used, which is able to distinguish benign from virulent D. nodosus. Nine farms were examined three times at intervals of 6 months. Cotton swabs were used to collect samples from the interdigital skin to analyze for the presence of virulent and benign strains of D. nodosus. Additionally, epidemiological data of the farms were collected with the aid of a standardized questionnaire. On four farms, benign strains were diagnosed at each visit; in one farm, benign strains were detected once only. Two flocks revealed sheep infected with virulent D. nodosus throughout the study but without clinical evidence of footrot. In two flocks, the virulent strains of D. nodosus were introduced into the flock during the study period. In one farm, clinical symptoms of virulent footrot were evident only two weeks after the positive finding by PCR. Only individual sheep with previously negative status, but none with previously benign status became infected with virulent strains during the study. The newly developed competitive RT PCR proved to be more sensitive than clinical diagnosis for detecting footrot infection in herds, as it unequivocally classified the four flocks as infected with virulent D. nodosus, even though they did not show clinical signs at the times of sampling. This early detection may be crucial to the success of any control program. Both new infections with virulent strains could be explained by contact with sheep from herds with virulent D. nodosus as evaluated from the questionnaires. These results show that the within-herd eradication of footrot becomes possible using the competitive PCR assay to specifically diagnose virulent D. nodosus.

One-Step Identification of Five Prominent Chicken Salmonella Serovars and Biotypes.

Based on bacterial genomic data, we developed a one-step multiplex PCR assay to identify Salmonella and simultaneously differentiate the two invasive avian-adapted S. enterica serovar Gallinarum biotypes Gallinarum and Pullorum, and the most frequent, specific, and asymptomatic colonizers of chickens, serovars Enteritidis, Heidelberg, and Kentucky.

Influenza A (H1N1) con alteración de enzimas hepáticas en paciente vacunado

El compromiso hepático en la infección por virus Influenza A (H1N1) es muy infrecuente. Presentamos un caso de un paciente varón de 21 años, sano, vacunado, que consulta con un cuadro clásico de gripe. En el examen físico no se palpa hepatoesplenomegalia ni se observa ictericia. Se diagnostica gripe por virus de la influenza A (H1N1) y se inicia tratamiento con oseltamivir oral. Los exámenes de laboratorio revelaron elevación moderada de enzimas hepáticas. Los anticuerpos para virus de la hepatitis A, B y C, virus Epstein-Barr y citomegalovirus (CMV) fueron negativos. El hisopado nasofaríngeo fue positivo para influenza A (H1N1) con prueba de reacción en cadena de polimerasa en tiempo real (PCRRT). Se detectó hepatomegalia homogénea por ecografía abdominal. El cuadro clínico se resolvió en una semana, permaneciendo elevadas las enzimas hepáticas por 21 días. Discutimos los probables mecanismos de la injuria hepática en este caso.

Compatibilidad de Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) y Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), importantes enemigos naturales de la mosca blanca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) en cultivos hortícolas, con nuevas barreras físicas selectivas y modernos plaguicidas

Los programas de Gestión Integrada de Plagas (GIP) promueven el uso de estrategias de control que sean respetuosas con el medio ambiente, sin embargo el uso de insecticidas en los cultivos hortícolas sigue siendo necesario para el control de determinadas plagas, como es el caso de la mosca blanca Bemisia tabaci (Gennadius). Por ello, el objetivo de esta tesis es el estudio de la integración de las tres estrategias de control más empleadas hoy en día para el control de plagas: el control biológico, el físico y el químico. Una primera parte de este trabajo ha consistido en el estudio de los efectos letales y subletales de once insecticidas, aplicados a la dosis máxima de campo, sobre los enemigos naturales Eretmocerus mundus Mercet y Amblyseius swirskii Athias-Henriot, mediante ensayos de laboratorio y persistencia (laboratorio extendido). Para la evaluación de la toxicidad de los insecticidas sobre los estados de vida más protegidos de estos enemigos naturales, se trataron bajo la Torre de Potter las pupas de E. mundus y los huevos de A. swirskii. Además, se llevaron a cabo ensayos de contacto residual para determinar los efectos letales y subletales de estos insecticidas sobre el estado adulto de ambas especies de enemigos naturales. Para ello, los pesticidas se aplicaron sobre placas de cristal (laboratorio) o sobre plantas (laboratorio extendido: persistencia). Los resultados mostraron que los insecticidas flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen y spirotetramat eran compatibles con el estado de pupa de E. mundus (OILB 1: Inocuos). Sin embargo, abamectina, deltametrina y emamectina fueron categorizadas como ligeramente tóxicas (OILB 2) al causar efectos deletéreos. Los dos pesticidas más tóxicos fueron spinosad y sulfoxaflor, los cuales redujeron significativamente la emergencia de las pupas tratadas (OILB 4: Tóxicos). Flonicamida, flubendiamida, metoxifenocida y spiromesifen fueron compatibles con el estado adulto de E. mundus (OILB 1: Inocuos). Abamectina, deltametrina, emamectina, metaflumizona y spiromesifen pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Al contrario, spinosad y sulfoxaflor no resultaron ser compatibles (OILB D: Persistentes), aunque la realización de ensayos adicionales es necesaria para ver los efectos de los mismos en campo. Todos los insecticidas estudiados, excepto el spirotetramat (OILB 2: Ligeramente tóxico), fueron selectivos para el estado de huevo de A. swirskii (OILB 1: Inocuos). Flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen, spirotetramat y sulfoxaflor, fueron compatibles con el estado adulto de A. swirskii (OILB 1: Inocuos). Abamectina, deltametrina, emamectina y spinosad pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Entre las nuevas estrategias de la GIP, los plásticos y mallas fotoselectivas han demostrado ser una herramienta importante para el control de plagas y enfermedades en cultivos hortícolas protegidos. Por ello, en una segunda parte de este trabajo, se estudiaron tanto los efectos directos, como la combinación de efectos directos y mediados por planta y plaga de ambientes pobres en luz UV, en presencia o ausencia del Virus del rizado amarillo del tomate (TYLCV), sobre E. mundus. En primer lugar, se realizó un ensayo al aire libre para la evaluación de la capacidad de vuelo de E. mundus en cajas tipo túnel (1 x 0,6 x 0,6 m) cubiertas con distintas barreras absorbentes de luz UV. Se detectó un efecto directo en la capacidad de orientación de E. mundus, debido a que este parasitoide utiliza estímulos visuales para localizar a sus huéspedes, únicamente en las barreras que bloqueaban más del 65% de la luz UV (malla G). En segundo lugar, bajo condiciones de invernadero, se evaluó la combinación de efectos directos y mediados por planta y plaga sobre E. mundus, usando plantas de tomate sanas o infectadas con el TYLCV y cajas (30 x 30 x 60 cm) cubiertas con los distintos plásticos fotoselectivos. En este caso, no se observó ningún efecto en la capacidad benéfica del parasitoide cuando este estaba en contacto con plantas de tomate infestadas con ninfas de B. tabaci, lo que demuestra que este insecto usa estímulos táctiles para encontrar a sus huéspedes a cortas distancias. Además, las diferentes condiciones de radiación UV estudiadas tuvieron cierto impacto en la morfología, fisiología y bioquímica de las plantas de tomate, infestadas o no con el virus de la cuchara, detectándose pequeñas alteraciones en alguno de los parámetros estudiados, como el peso fresco y seco, el contenido en H y el espesor de las cutículas y de las paredes celulares de la epidermis foliar. Por último, no se observaron efectos de la radiación UV mediados por planta, ni en B. tabaci ni en su parasitoide, E. mundus. En una tercera parte, se evaluaron los efectos de una malla tratada con bifentrin sobre ambos enemigos naturales, en ensayos de laboratorio, semicampo y campo. Las mallas tratadas fueron diseñadas originariamente para el control de mosquitos vectores de la malaria, y actualmente se está trabajando para su uso en agricultura, como una nueva estrategia de control de plagas. En ensayos de laboratorio, cuando adultos de E. mundus y A. swirskii se expusieron por contacto durante 72 horas con la malla tratada (cajas de 6 cm diámetro), se registró una alta mortalidad. Sin embargo, en el ensayo de preferencia, estos enemigos naturales no fueron capaces de detectar la presencia de bifentrin y, en aquellos individuos forzados a atravesar la malla tratada, no se observó mortalidad a corto plazo (72 horas). En estudios de semicampo, llevados a cabo bajo condiciones de invernadero en cajas de 25 x 25 x 60 cm de altura, la capacidad benéfica de E. mundus no se vio afectada. Finalmente, en ensayos de campo llevados a cabo en invernaderos comerciales (4000m2) en Almería, A. swirskii no se vio afectado por la presencia en el cultivo de la malla tratada con bifentrin y los niveles de infestación de B. tabaci y F. occidentalis detectados bajo dicha malla, fueron inferiores a los del control. Por último, se ha evaluado la composición de la microflora bacteriana de tres especies de parasitoides, E. mundus, Eretmocerus eremicus Rose & Zolnerowich y Encarsia formosa Gahan, y la influencia de la misma en su susceptibilidad a insecticidas. Se llevó a cabo una extracción total de ADN de los insectos y la región variable V4 del ARNr se amplificó usando cebadores universales bacterianos. Para identificar las secuencias de los géneros bacterianos presentes en los parasitoides, se realizó una Next Generation sequencing (Illumina sequencing). Una vez identificados los géneros bacterianos, el gen ADNr 16S de las Actinobacterias se amplificó del ADN extraído de los insectos, usando cebadores universales bacterianos y específicos de Actinobacterias, y los productos de la Nested PCR fueron clonados para identificar todas las especies del género Arthrobacter. Tres bacterias (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi y A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), próximas a las especies de Arthrobacter presentes en los parasitoides, se obtuvieron de la colección bacteriana del BCCMTM/LMG y se midió su actividad esterasa. Finalmente, se realizaron ensayos con antibióticos (tetraciclina) y de contacto residual con insecticidas (abamectina) para determinar la influencia de las especies de Arthrobacter en la susceptibilidad de E. mundus a insecticidas. Los resultados muestran que este género bacteriano puede afectar a la toxicidad de E. mundus a abamectina, mostrando la importancia de la comunidad microbiana en enemigos naturales, factor que debe ser considerado en los estudios de evaluación de los riesgos de los insecticidas. ABSTRACT Integrated Pest Management (IPM) programs promote the use of control strategies more respectful with the environment; however the use of insecticides in vegetable crops is still needed to control certain pests, such as the whitefly Bemisia tabaci (Gennadius). Therefore, the objective of this work is to study the integration of the three most commonly used pest control strategies nowadays: biological, physical and chemical control. Firstly, the lethal and sublethal effects of eleven insecticides, applied at their maximum field recommended concentration, on the parasitic wasp Eretmocerus mundus Mercet and the predator Amblyseius swirskii Athias-Henriot has been assessed in the laboratory and in persistence tests (extended laboratory). To test the effects of pesticides on the most protected life stage of these natural enemies, E. mundus pupae and A. swirskii eggs were sprayed under a Potter precision spray tower. Laboratory contact tests were therefore conducted to determine the lethal and sublethal effects of these pesticides on the adult stage of these natural enemies. In the residual contact tests the pesticides were applied on glass plates (laboratory) or plants (extended laboratory: persistence). The study showed that the insecticides flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen and spirotetramat were selective for E. mundus pupae (IOBC 1: Harmless). Nevertheless, abamectin, deltamethrin and emamectin were categorized as slightly harmful (IOBC 2) due to the deleterious effects caused. The two most harmful pesticides were spinosad and sulfoxaflor, which significantly reduced the adult emergence from treated pupae (IOBC 4: Harmful). Flonicamid, flubendiamide, methoxyfenozide and spiromesifen were compatible with E. mundus adults (IOBC 1: Harmless). Base on the duration of the harmful activity, abamectin, deltamethrin, emamectin, metaflumizone and spirotetramat could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. On the contrary, spinosad and sulfoxaflor were not compatible (IOBC D: persistent), although additional studies are required to determine their effects under field conditions. All the pesticides tested, except spirotetramat (IOBC 2: Slightly harmful), were selective for A. swirskii eggs (IOBC 1: Harmless). Flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen, spirotetramat and sulfoxaflor were compatible with A. swirskii adults (IOBC 1: Harmless). However, abamectin, deltamethrin, emamectin and spinosad could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. Among new IPM strategies, UV-absorbing photoselective plastic films and nets have been shown to be an important tool for the control of pests and diseases in horticultural protected crops. Because of that, we secondly studied the plant and pest insect-mediated and/or the direct effects on E. mundus under different UV radiation conditions, in presence or absence of the Tomato Yellow Leaf Curl Virus (TYLCV). In the first experiment, performed outdoors, the flight activity of E. mundus was studied in one-chamber tunnels (1 x 0.6 x 0.6 m) covered with different photoselective barriers. Because E. mundus uses visual cues for host location at a long distance, a direct effect on its host location ability was detected, but only in the UV-absorbing barriers blocking more than 65% of the UV light (G net). In a second experiment, the direct and plant and pest insect-mediated effects of different UV radiation conditions on E. mundus were studied, inside cages (30 x 30 x 60 cm) covered with the different UVplastic films and under greenhouse conditions, using healthy or TYLCV-virus infected tomato plants. In this case, not any effect on the beneficial capacity of this parasitoid was detected, proving that he uses tactile cues at a short distance of the host. Moreover, the different UV radiation conditions studied had a certain direct impact in the morphology, physiology and biochemistry of tomato plants infested or not with the TYLCV, and small alterations in some parameters such as fresh and dry weight, H percentage and cuticle and cell wall thickness of epidermal cells of the leaves, were detected. Finally, none plant-mediated UV effects neither in the whitefly B. tabaci nor in their parasitic wasp were found. Thirdly, the effects of a bifenthrin treated net were evaluated in different laboratory, semi-field and field experiments on the natural enemies studied. Treated nets were developed long time ago aiming at the control of the mosquitoes vectors of malaria, and nowadays, there is a great interest on assessing the possibility of their use in agriculture. In laboratory assays, a high mortality was recorded when E. mundus and A. swirskii adults were exposed by contact to the bifenthrin treated net for 72 hours in small cages (12 cm diameter). However, these natural enemies were not able to detect the presence of bifenthrin in a dual-choice test and no short-term mortality (72 hours) was recorded in those individuals that went through the treated net. In semi-field assays, performed under greenhouse conditions with cages of 25 x 25 x 60 cm high, the beneficial capacity of E. mundus was not affected. Finally, in field assays carried out in commercial multispan greenhouses (4000 m2) in Almería, A. swirskii was not affected by the presence of the bifenthrin treated net in the crop and the B. tabaci and F. occidentalis infestation levels were significantly lower than in the control. Finally, the composition of the microflora present in three species of parasitoids, E. mundus, Eretmocerus eremicus Rose & Zolnerowich and Encarsia formosa Gahan, and its influence in their susceptibility to insecticides, have been assessed. A total DNA extraction was performed on insects and universal bacterial primers were used to amplify the variable V4 region of the rRNA. A Next Generation sequencing (Illumina sequencing) was performed to identify the sequences of the bacterial genera present in the parasitic wasps. Once, the bacterial genera were identified, 16S rDNA gene of Actinobacteria were amplified from insects DNA extracts using the universal bacterial and actinobacterial primers, and the nested PCR products, were cloned to identify the Arthrobacter species. Three bacteria (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi and A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), having the closest match with the Arthrobacter species present in the parasitic wasps, were obtained from the BCCMTM/LMG bacteria collection and its esterase activity was measured. Finally, antibiotic and residual contact tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides (abamectin). The results suggest that this bacterial genus can affect the toxicity of E. mundus to abamectin, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides.

The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer

The non-Mendelian inheritance of organelle genes is a phenomenon common to almost all eukaryotes, and in the isogamous alga Chlamydomonas reinhardtii, chloroplast (cp) genes are transmitted from the mating type positive (mt+) parent. In this study, the preferential disappearance of the fluorescent cp nucleoids of the mating type negative (mt−) parent was observed in living young zygotes. To study the change in cpDNA molecules during the preferential disappearance, the cpDNA of mt+ or mt− origin was labeled separately with bacterial aadA gene sequences. Then, a single zygote with or without cp nucleoids was isolated under direct observation by using optical tweezers and investigated by nested PCR analysis of the aadA sequences. This demonstrated that cpDNA molecules are digested completely during the preferential disappearance of mt− cp nucleoids within 10 min, whereas mt+ cpDNA and mitochondrial DNA are protected from the digestion. These results indicate that the non-Mendelian transmission pattern of organelle genes is determined immediately after zygote formation.

Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.

Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea

The voltage-gated Ca2+ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca2+ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca2+ channels, we investigated the expression of L-type α1 subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α1D products, but only once an α1C product and never an α1S product. A full-length α1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α1D subunit was about 90% identical to those of the mammalian α1D subunits. In situ hybridization confirmed that the α1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α1D mRNA is 100–500 times more abundant than the α1C mRNA. We conclude that most, if not all, voltage-gated Ca2+ channels in hair cells contain an α1D subunit. Furthermore, we propose that the α1D subunit plays a hitherto undocumented role at tonic synapses.

Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: “suspension transduction” and “stromal growth factor transduction.” However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.

Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8- to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation.

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

The genomic and physical organization of Ty1-copia-like sequences as a component of large genomes in Pinus elliottii var. elliottii and other gymnosperms.

A DNA sequence, TPE1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliottii (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8 kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE1 represents a large subgroup of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE1 transcription could be detected, it is most likely an inactive element--at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three- and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.

Structural variation of the pseudoautosomal region between and within inbred mouse strains.

The pseudoautosomal region (PAR) is a segment of shared homology between the sex chromosomes. Here we report additional probes for this region of the mouse genome. Genetic and fluorescence in situ hybridization analyses indicate that one probe, PAR-4, hybridizes to the pseudoautosomal telomere and a minor locus at the telomere of chromosome 9 and that a PCR assay based on the PAR-4 sequence amplifies only the pseudoautosomal locus (DXYHgu1). The region detected by PAR-4 is structurally unstable; it shows polymorphism both between mouse strains and between animals of the same inbred strain, which implies an unusually high mutation rate. Variation occurs in the region adjacent to a (TTAGGG)n array. Two pseudoautosomal probes can also hybridize to the distal telomeres of chromosomes 9 and 13, and all three telomeres contain DXYMov15. The similarity between these telomeres may reflect ancestral telomere-telomere exchange.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.