A very-low-calorie ketogenic diet normalises obesity-related enhanced levels of erythropoietin compared with a low-calorie diet or bariatric surgery.

PURPOSE: Nutritional ketosis synergistically with body-weight loss induced by a very-low-calorie ketogenic diet (VLCKD) has proven to be effective in improving obesity-related pathophysiology. Recently, growing attention has been focused on the relation between erythropoietin (EPO) and obesity. Thus, this study aims to investigate whether nutritional ketosis and weight loss induced by a VLCKD modify the circulating levels of EPO in patients with obesity in comparison with the effect of low-calorie diet (LCD) or bariatric surgery (BS). METHODS: EPO levels, iron status and body composition parameters were evaluated in 72 patients with overweight or obesity and 27 normal-weight subjects at baseline and after the three different weight-reduction therapies (VLCKD, LCD and BS) in 69 patients with excess body weight. ß-hydroxybutyrate levels were also measured in the VLCKD group. The follow-up was established at 2-3 months and 4-6 months. RESULTS: It was found that EPO levels were higher in morbid obesity and correlated with higher basal weight, fat mass (FM) and fat-free mass (FFM) in the overall sample. High baseline EPO levels were also correlated with higher impact on the course of weight loss and changes in FM and FFM induced by the three weight-loss interventions. Furthermore, the VLCKD induced a decrease in EPO levels coinciding with maximum ketosis, which was maintained over time, while statistically significant changes were not observed after LCD and BS. CONCLUSION: The obesity-related increased EPO levels are restored after VLCKD intervention at the time of maximum ketosis, suggesting a potential role of the nutritional ketosis induced by the VLCKD. Baseline EPO levels could be a biomarker of response to a weight-loss therapy.

Pituitary surgery in elderly patients: a safe and effective procedure.

PURPOSE: The incidence of pituitary adenoma (PA) increases with age. Transsphenoidal surgery (TSS) in elderly patients is often considered to have greater risk compared to the younger population. The aim of this study is to compare surgical results, evolution and postoperative complications between elderly and young patients undergoing TSS. METHODS: Retrospective review of patients undergoing TSS between 2011 and 2018 in our institution. Patients were divided into two cohorts: elderly (≥65 years) and non-elderly (<65 years). Characteristics and outcomes of both groups were compared at diagnosis, before surgery and for an average of 5.9 years of postoperative follow-up. RESULTS: One hundred and twenty-five patients were included, 53 patients were ≥65 years (42%). The elderly patients were more likely to have non-functioning PA (NFPA) (90.5% vs. 45.8%, p: <0.01), a higher proportion of macroadenomas (92.4% vs. 77.8%, p = 0.029) and greater extrasellar extension (88.7% vs. 68.1%, p = 0.007). The elderly group also had more compressive symptoms (54.7% vs. 34.7%, p = 0.035) and hypopituitarism (66% vs. 47.2%, p = 0.029). Overall, surgical and endocrinological outcomes between the two groups were similar. Inpatient mortality in the elderly group was 1.8%. Regarding long-term outcomes, elderly patients had more postoperative hypopituitarism (67.9% vs. 45.8%, p = 0.03) with no differences in permanent diabetes insipidus, less residual tumours (24.5% vs. 40.3%, p = 0.019) and a higher rate of remission after surgery (71.7% vs. 52.8%, p = 0.034). When only NFPA cases were compared, the only significant difference was a higher frequency of macroadenomas in the elderly group. CONCLUSIONS: Our results support the safety and efficacy of TSS in elderly patients with PA. Age should not be considered an exclusion criterion for TSS given that successful results can be achieved if an experienced pituitary team is available.

Early versus conventional nutritional intervention in head and neck cancer patients before radiotherapy: benefits of a fast-track circuit.

BACKGROUND/OBJECTIVES: Radiotherapy (RT) is a component of therapy for head and neck cancer (HNC) with a negative nutritional impact. Our aim was to compare an early versus a conventional nutritional intervention. SUBJECTS AND METHODS: Retrospective study of HNC patients undergoing RT. Evolution before and after the establishment of a fast-track circuit was evaluated. A conventional group (CG) made up of patients submitted to the nutrition unit during RT after nutritional deterioration, was compared to an early group (EG) represented by patients included in a fast-track circuit, starting nutritional follow-up before the beginning of RT. Only patients with preserved oral intake were involved. Demographic, nutritional and clinical variables were analyzed. Data of hospitalizations and deaths were collected up to three months after RT. RESULTS: 135 subjects constituted the EG and 39 the CG. At baseline, the prevalence of malnutrition was lower in the EG (31.9% vs 69.5%, p = 0.0001), as was the need for nutritional supplements (40% vs 79.5%, p = 0.0001) or nasogastric tube (0% vs 12.8%, p = 0.0001) in comparison to the CG. Three months after RT, there were less patients with oral nutritional support in the EG (79.1% vs 96.9%, p = 0.018), and the number of emergency visits (0.75 vs 1.1 episodes per patient, p = 0.021) and hospitalizations was also lower in this group (29% vs 59%, p = 0.044). CONCLUSIONS: The fast-track approach made early intervention possible. Therefore, patients maintained a better nutritional status, needed less nutritional support and their evolution improved, with a significant decrease in hospitalizations.

Correlates of children's physical activity during the COVID-19 confinement in Portugal.

OBJECTIVES: The aim of the study was to understand the role of household variables on the percentage of physical activity (%PA) during the coronavirus disease 2019 (COVID-19) confinement in Portugal. STUDY DESIGN: A cross-sectional study design using an anonymous online survey was launched to assess how Portuguese families with children aged younger than 13 years adjusted their daily routines to the confinement. METHODS: Separate analyses of variance were performed to investigate how factors such as the number of children, age, sex, the housing characteristics, and the adults' job situation can affect the percentage of time for PA (%PA). RESULTS: Findings, based on data from 2159 children, indicate that (1) boys and girls did not differ in the %PA on any of the age-groups; (2) children with an outdoor space and who had other children in the household were significantly more active (P < .001); (3) children from families with all adults working from home showed lower levels of %PA; and (4) being younger, having a big outdoor space, having other children in the household, and having at least one adult free from working from home were significant positive predictors of children's %PA, explaining 21% of the overall variance. CONCLUSION: Time allocated for PA during this period is reduced compared with what is usually reported on normal days. It is necessary to find strategies to increase children's PA, especially in families in which both parents are working and have no outdoor space.

Vitamin D levels in a pediatric population of a primary care centre: a public health problem?

OBJECTIVE: Vitamin D deficiency is a public health problem that occurs more frequently than expected. The aim of this study is to evaluate the vitamin D levels of children attending the paediatrics unit of the Bertamiráns primary care centre (A Coruña NW Spain). This is an observational study carried out during 1 year on a random sample of the pediatric population aged between 5 and 15 years. The levels of vitamin D (25(OH)D) were determined by immunoassay (ADVIA Centaur Vitamin D®). The results were classified as sufficient (> 20 ng/ml), insufficient (10-20 ng/ml) and deficient (< 10 ng/ml). RESULTS: 153 analyses of vitamin D were carried out (58.2% in girls and 41.8% in boys), distributed in two age groups: 5-10 (62) and 10-15 (91). 66% of the total of the sample presented some degree of vitamin D deficit (60.1% insufficient (92) and 5.9% (11) deficient). In Galicia, there is a high prevalence of vitamin D deficiency/insufficiency in the healthy population, which increases if the patients present some kind of chronic pathology, thus leading to a public health problem. It is advisable to increase the consumption of fortified foods and/or to reconsider the administration of vitamin supplements.

Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy.

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.

Tratamiento del Síndrome de Dolor Miofascial con Toxina Botulínica tipo A / Botulinum toxin type A in the management of Myofascial pain syndrome

Introducción: El síndrome de dolor miofascial (SDM) se caracteriza por áreas dolorosas de la musculatura esquelética y por la evidencia clínica y electromiográfica de contracción de bandas musculares sobre las cuales existe un punto cuya presión desencadena un dolor intenso local y referido (punto gatillo). La fisiopatología es incierta pero una posible explicación sería la lesión del músculo por microtraumatismos, sobreuso o espasmo prolongado. La toxina botulínica la produce el microorganismo Clostridium botulinum en condiciones anaeróbicas y se trata de una de las sustancias más potentes que se conocen. Material y métodos: Se trata de un estudio observacional prospectivo en el que hemos estudiado la aplicación de la toxina botulínica tipo A en el tratamiento del síndrome de dolor miofascial en una serie de 20 pacientes. Todos los pacientes fueron sometidos a una infiltración diagnóstica de la musculatura lumbar o del músculo piramidal con 8 ml de ropivacaína al 0,2% y 6 mg de fosfato sódico de betametasona y 6 mg de acetato de betametasona. Para la localización de los músculos utilizamos referencias anatómicas y administramos de 3 a 5 ml de contraste hidro-soluble para asegurarnos mediante fluoroscopia de la correcta localización de la aguja. La administración de toxina botulínica se realizó siguiendo el mismo método utilizado en las infiltraciones diagnósticas. Decidimos utilizar una dosis de 250 U de Dysport® en cada músculo a infiltrar sin pasar en ningún caso de 1000 U para un mismo paciente. La eficacia del tratamiento se basó en el control del dolor según la Escala Visual Analógica basal (EVA 1), a los 15 días (EVA 15), a los 30 días (EVA 30) y a los 90 días (EVA 90) de las infiltraciones y el test de Lattinen evaluado antes del tratamiento (TLT 1) y al final del estudio (TLT 2). Todos los pacientes fueron preguntados acerca de posibles efectos secundarios. Finalmente se registró el grado de satisfacción del paciente al finalizar el estudio: excelente, buena, regular o mala. Resultados: En todos los pacientes la infiltración diagnóstica fue considerada como positiva con la posterior administración de la toxina botulínica. En todos los casos se produjo una reducción en la EVA de al menos el 50% a los 15 y a los 30 días. A los 90 días, esta reducción mayor o igual al 50% se mantuvo en 13 de los 20 pacientes, mientras que en los 7 pacientes restantes esta reducción fue inferior al 50%. La EVA media inicial fue de 7,7 ± 1,2 de desviación estándar y el TLT medio inicial de 12 ± 2,3, existiendo una alta correspondencia con la EVA. La evolución de la EVA media en los controles posteriores fue: EVA 15 = 3,34 ± 2,5; EVA 30 = 3,54 ± 2,37 y EVA 90 = 4,94 ± 2,83. El TLT medio a los 90 días fue de 7,43 ± 3,49. Tan solo una paciente refirió debilidad muscular ligera en piernas durante las 48 horas posteriores a la infiltración que cedió de forma espontánea. La satisfacción con el tratamiento fue excelente en 10 pacientes (50 %), buena en 7 (35 %) y regular en 3 (15 %). Ningún paciente calificó la experiencia como mala. Conclusión: La infiltración muscular con toxina botulínica tipo A en el tratamiento del SDM se muestra como un tratamiento eficaz y seguro (AU)

Introduction: Myofascial pain syndrome (MPS) is a condition characterised by painful areas of skeletal muscle and by the clinical and electromyographic evidences of contraction of muscle’s band associated with trigger points. Trigger points are locally tender muscle areas when active and refer pain through specific patterns to other areas of the body. The physiopathology is unknown and a possible explication could be to muscle lesion caused by microtrauma, overuse, excessive strain o prolonged spasm.Botulinum toxin is produced by the microorganism Clostridium botulinum in anaerobic conditions and is one of the strongest known substances. Material and methods: It is an observational prospective study. The aim of the study was to evaluate the clinical efficacy and safety of the muscular injection of the botulinum toxin in reducing pain in the MPS in 20 patients. All patients had a diagnostic injection of the lumbar musculature or the piriformis muscle of 8 ml of 0.2% ropi-vacaine and 6 mg of sodium betamethasone phosphate and 6 mg of acetate betamethasone in each muscle. We used anatomical references to localise each muscle and we injected 1 ml of hydrosoluble contrast to confirm by radioscopy guidance the correct localization of the needle in the muscle to treat. The administration of the botulinum toxin was carried out using the same method used for the diagnostic infiltrations. We decided to use one dose of 250 U of Dysport® in each muscle and we exceeded 1000 U in none patient. The assessment of the treatment efficacy was based on the pain reduction according to the visual analog scale (VAS) on the first day of the injection (VAS 1), at day 15 (VAS 15), day 30 (VAS 30) and day 90 (VAS 90) after insertion of the botulinum toxin and the test of Lattinen was evaluated before the treatment (TLT 1) and at the completion of the study (TLT 2). All patients were asked regarding side effects and the grade of satisfaction at the end of the study was defined as excellent, good, regular or bad. Results: In all patients, the diagnostic injection was considerate positive with the posterior administration of the botulinum toxin. There was at least a reduction of 50% of the pain in the EVA in the 20 cases at day 15 and 30. At day 90, the reduction of the pain at 50% was maintained in 13 patients and less than 50% in the rest of the patients. The initial median EVA was 7.7 ± 1.2 standard desvia-tion and the TLT median initial of 12± 2.3. The evolution of the EVA median in the following controls was EVA 15 = 3,34 ± 2,5; EVA 30 = 3,54 ± 2,37 y EVA 90 = 4,94 ± 2,83. The median TLT at day 90 was de 7,43 ± 3,49. Only one patient referred mild muscle weakness of the lower limbs during 48 hours following the injection that improved spontaneously. The satisfaction was excellent in 10 patients (50%), good in 7 (35%) and average in 3 (15%). None of the patient qualified the experience as bad. Conclusion: The muscular injection of botulinum toxin type A in the treatment of MSP is effective and safe (AU)

Fentanilo transdérmico en analgesia postoperatoria / A Transdermal fentanyl in postoperative analgesia

Objetivos: Valorar la efectividad del uso de fentanilo transdérmico en la prevención del dolor postoperatorio, en pacientes intervenidos de artroplastia de cadera o rodilla. Material y métodos: Se han incluido 60 pacientes que iban a ser intervenidos de artroplastia de cadera o rodilla. La noche antes de la intervención, se colocaba a las 12 horas, un parche de fentanilo. El anestesiólogo en la consulta preanestésica, prescribía el tratamiento, en función del peso y estado físico del paciente. Durante el postoperatorio se permitía como medicación de rescate para el dolor paracetamol o metamizol. Se valoraron: control analgésico (escala nominal: ausencia de dolor, dolor leve, moderado, severo o insoportable), uso de medicación de rescate y efectos secundarios. Resultados: De los 60 pacientes incluidos, 28 fueron tratados con fentanilo-TTS 25 µg . h- 1 y 32 con fentanilo-TTS 50 µg . h- 1.No presentaron dolor un total de 41 pacientes, presentaron dolor leve 15 pacientes y tres pacientes experimentaron dolor moderado. De los pacientes que fueron tratados con fentanilo-TTS 25 µg . g- 1, 18 no tuvieron dolor, 6 tuvieron dolor leve y 3 dolor moderado. De los pacientes tratados con fentanilo-TTS 50, ninguno presentó dolor moderado, 9 presentaron dolor leve y 23 no experimentaron dolor. En cuanto a la medicación de rescate, 32 pacientes no necesitaron ninguna y sólo 5 de ellos necesitaron la cantidad pautada o más (3 pacientes del grupo fentanilo-TTS 25 µg . h- 1 y 2 del grupo fentanilo-TTS 50 µg . h- 1). Se presentaron efectos secundarios en 15 pacientes: náuseas y vómitos en 12 pacientes y en tres sedación ligera. Conclusiones: El uso de fentanilo-TTS en la prevención del dolor postoperatorio de pacientes intervenidos de artroplastia de cadera o rodilla, puede ser un tratamiento efectivo y seguro (AU)

Eficacia analgésica de la asociación ketamina+morfina epidural: experiencia en 75 casos / Analgesic effectiveness of combined epidural ketamine+morphine: experience in 75 cases

Objetivo: El objetivo de este estudio fue comprobar la eficacia analgésica de la combinación de morfina y ketamina por vía epidural, y determinar la dosis óptima de ketamina que incrementa la capacidad analgésica de los opiáceos peridurales, en pacientes sometidos a artroplastia total de cadera y rodilla. Material y métodos: Se realizó un estudio prospectivo, de 75 pacientes, a lo largo de un año. La técnica locorregional anestésica realizada fue combinada intradural-epidural, con colocación de catéter epidural para analgesia. Todos los pacientes recibieron 30 mg de ketamina epidural 30 min antes de la intervención. La anestesia intradural se realizó con bupivacaína 0,5 por ciento hiperbara, 2-3 cc, según el paciente tuviera una estatura <170 cm o 170 cm, respectivamente .Los pacientes fueron divididos e 5 grupos: Grupo A: recibieron analgesia i.v. con metamizol (2 g i.v. . 6 h- 1) y tramado l (100 mg i.v./6 h). Grupo B: 10 mg de ketamina epidural, diluidos en 10 ml de suero salino fisiológico (SSF), al llegar al despertar y 12 h después. Grupo C: 2 mg de morfina epidural, diluidos en 10 ml de SSF, a la llegada al despertar y 12 h después. Grupo D: 10 mg de ketamina + 0,5 mg de cloruro mófico epidural, diluidos en 10 ml de SSF, a la llegada al despertar y 12 h después.Grupo E: 5 mg de Ketamina + 0,5 mg de morfina epidural, diluidos en 10 ml de SSF, a la llegada al despertar y 12 h después. Se valoraron la eficacia analgésica (utilizando la Escala Analógica Visual: EVA), la necesidad de analgesia suplementaria y los efectos indeseables (despertar, salida, 12, 24, 48 y 72 h postcirugía).Resultados: Se observó una eficacia analgésica significativamente superior en los grupos C, D y E, respecto a los grupos A y B (p<0,05). La analgesia obtenida en los grupos en los que se administró la asociación ketamina+cloruro mórfico (D,E), fue superior a la del grupo en que se administró sólo cloruro mórfico, pero sin diferencias significativas. El consumo de analgésicos fue significativamente superior (p<0,02) para los grupos A y B, con respecto al resto. Los efectos secundarios fueron significativamente superiores (p<0,03) en el grupo C. Discusión: La administración epidural de ketamina, no se comportó como analgésico eficaz, sin embargo la asociación ketamina+morfina permitió una analgesia eficaz y prolongada, con bajas dosis de ambos fármacos, reduciéndose de forma significativa los efectos secundarios. Ambas dosis de ketamina fueron consideradas como eficaces para la analgesia preventiva (AU)

Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap.

Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803-808, 2001)

Tratamiento del dolor neuropático: estudio preliminar de amitriptilina frente a nefazodona / Management of neuropathic pain: preliminary study of amitriptiline versus nefazodone

Objetivo: Valorar la eficacia analgésica y la tolerancia de la amitriptilina frente a la nefazodona en el tratamiento del dolor neuropático.Material y Métodos: Se realizó un estudio prospectivo en 120 pacientes durante un periodo de 18 meses. Todos los pacientes presentaban dolor neuropático de 1 a 6 meses de evolución. La calidad del dolor fue quemante y cortante en el 62,3 por ciento de los casos, lancinante en el 40 por ciento, y punzante en el 25 por ciento. Se dividieron en dos grupos: a) recibieron amitriptilina a dosis de 25 mg.día- 1 con incrementos semanales de 25 mg, hasta un máximo de 150 mg; b) recibieron nefazodona a dosis de 50 mg.día- 1 con incrementos semanales de 50 mg hasta un máximo de 300 mg. En 48 pacientes se inició tratamiento simultáneo con anticonvulsivantes debido a que presentaban un componente de dolor paroxístico lancinante, estos pacientes fueron valorados aisladamente. Se valoraron el dolor así como los efectos indeseables en la tercera semana de tratamiento, al mes y a los tres meses, mediante la escala analógica visual (EAV ) .Resultados: Los síndromes dolorosos más frecuentes fueron: dolor neuropático por cáncer y neuralgia post-herpética (n = 16), polineuropatía diabética, dolor lumbar y neuralgia del trigémino (n = 14), miembro fantasma doloroso, síndrome de dolor regional complejo tipo I ,y neuralgia post-cirugía (n = 10), fibromialgia y neuralgia de Arnold (n = 6), dolor talámico (n = 4). Las dosis medias de amitriptilina y nefazodona fueron respectivamente: 75 ñ 52 mg y 220 ñ 64 mg.La intensidad global del dolor, medida por EVA fue para el grupo de la amitriptilina de 8,7 ñ 1,2 y para el grupo de la nefazodona 8,5 ñ 0,9. A los tres meses de tratamiento el grupo de la amitriptilina presentaba una intensidad de dolor de 2 ñ 0,9 y el de la nefazodona 3 ñ 1,1. El alivio del dolor fue superior al 75 por ciento (excelente) en 42 pacientes tratados con amitriptilina y en 36 pacientes tratados con nefazodona, entre 50-75 por ciento (bueno) en 18 pacientes tratados con amitriptilina y 12 con nefazodona, y menor del 50 por ciento (malo) en 3 pacientes tratados con amitriptilina y 3 con nefazodona. Los efectos secundarios fueron superiores en la amitriptilina respecto a la nefazodona (p<0,05) excepto para las náuseas y vómitos, que fueron más fre c u e ntes en el grupo de la nefazodona: hipotensión ortostática (10/4), sequedad de boca (20/5), náuseas y vómitos (3/15). Las dosis medias a las que comenzaron a apare c e r los efectos secundarios fueron: 80 ñ 15 mg de amitriptilina y 245 ñ 25 de nefazodona.Conclusiones: Ambos fármacos fueron eficaces en el tratamiento del dolor neuropático. El grupo tratado con nefazodona presentó una menor incidencia de efectos secundarios, excepto en lo re f e rente a las náuseas y vómitos (AU)

Phenotypic analysis of peripheral CD4+ CD8+ T cells in the rat.

Among peripheral T cells, the expression of CD4 and CD8 is almost mutually exclusive. However, here we show, using flow cytometric analysis, that ex vivo approximately 6% of rat T cells stained for both CD4 and CD8. These double positive cells were also detected by confocal microscopy. Only around 50% of double positive cells expressed the CD8beta chain, the remaining cells expressed the CD8alpha chain alone. Double positive cells were blast-like with a phenotype, distinct from that of either CD4 or CD8 single positive cells, suggestive of an activated state. Previous reports of double positive T cells have also suggested that coexpression of CD4 and CD8 is linked to the activation state of the cell. There was an indication that priming animals with a hapten-carrier complex increased the ratio of CD8alphaalpha : alphabeta expressing double positive T cells, although we did not detect an increase in the frequency of double positive T cells following priming. We also show that the frequency of double positive cells was reduced following thymectomy and with age. In conclusion, these studies show that peripheral T cells expressing both CD4 and CD8 can be detected in the rat and that they are phenotypically distinct from CD4 and CD8 single positive T cells.

Specialized transcription factories within mammalian nuclei.

Recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes is reviewed.

The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

The control of RNA synthesis from protein-coding genes is fundamental in determining the various cell types of higher eukaryotes. The activation of these genes is driven by promoter complexes, and RNA synthesis is performed by an enzyme mega-complex-the RNA polymerase II holoenzyme. These two complexes are the fundamental components required to initiate gene expression and generate the primary transcripts that, after processing, yield mRNAs that pass to the cytoplasm where protein synthesis occurs. But although this gene expression pathway has been studied intensively, aspects of RNA metabolism remain difficult to comprehend. In particular, it is unclear why >95% of RNA polymerized by polymerase II remains in the nucleus, where it is recycled. To explain this apparent paradox, this review presents a detailed description of nuclear RNA (nRNA) metabolism in mammalian cells. We evaluate the number of active transcription units, discuss the distribution of polymerases on active genes, and assess the efficiency with which the products mature and pass to the cytoplasm. Differences between the behavior of mRNAs on this productive pathway and primary transcripts that never leave the nucleus lead us to propose that these represent distinct populations. We discuss possible roles for nonproductive RNAs and present a model to describe the metabolism of these RNAs in the nuclei of mammalian cells.-Jackson, D. A., Pombo, A., Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III.

Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved 'physiological' buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br-UTP, before the resulting Br-RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that approximately 10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only approximately 2000 sites (diameter approximately 40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or 'factories', suggesting that different regions of the nucleus specialize in the transcription of different types of gene.