Crush injury to motor nerves in the G93A transgenic mouse model of amyotrophic lateral sclerosis promotes muscle reinnervation and survival of functionally intact nerve-muscle contacts.

Selective survival of small motor nerve fibers and their neuromuscular contacts in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS) suggests that smaller regenerated nerve fibers are more able to sustain reformed nerve-muscle connections as functionally intact motor units (MUs). The sciatic nerve was crushed unilaterally in SOD1G93A transgenic mice at 40 days of age and contractile forces of reinnervated muscles and their MUs were recorded at 90 days in order to determine the capacities of the nerves to regenerate and to form and retain functional neuromuscular connections. Reduced MU numbers in fast-twitch tibialis anterior, extensor digitorum longus and medial gastrocnemius muscles and the lesser reductions in slow-twitch soleus muscle of SOD1G93A transgenic mice were reversed in reinnervated muscles: there were more reinnervated MUs and their contractile forces and the muscle forces and weights increased. In line with the contrasting ability of only small not large nerve fibers to sprout to form enlarged MUs in the SOD1G93A transgenic mouse, the smaller regenerating nerve fibers formed enlarged MUs that were better able to survive. Because nerve fibers with and without muscle contacts were severed by the sciatic nerve crush injury, the conditioning lesion is untenable as the explanation for improved maintenance of reinnervated neuromuscular junctions. Elevated neurotrophic factor expression in axotomized motoneurons and/or denervated Schwann cells and the synapse withdrawal from axotomized motoneurons are other factors that, in addition to reduced size of nerve fibers reinnervating muscles, may account for increased survival and size of reinnervated MUs in ALS.

Why do different oceanic archipelagos harbour contrasting levels of species diversity? The macaronesian endemic genus Pericallis (Asteraceae) provides insight into explaining the 'Azores diversity Enigma'.

BACKGROUND: Oceanic archipelagos typically harbour extensive radiations of flowering plants and a high proportion of endemics, many of which are restricted to a single island (Single Island Endemics; SIEs). The Azores represents an anomaly as overall levels of endemism are low; there are few SIEs and few documented cases of intra-archipelago radiations. The distinctiveness of the flora was first recognized by Darwin and has been referred to as the 'Azores Diversity Enigma' (ADE). Diversity patterns in the Macaronesian endemic genus Pericallis (Asteraceae) exemplify the ADE. In this study we used morphometric, Amplified Length Polymorphisms, and bioclimatic data for herbaceous Pericallis lineages endemic to the Azores and the Canaries, to test two key hypotheses proposed to explain the ADE: i) that it is a taxonomic artefact or Linnean shortfall, ie. the under description of taxa in the Azores or the over-splitting of taxa in the Canaries and (ii) that it reflects the greater ecological homogeneity of the Azores, which results in limited opportunity for ecological diversification compared to the Canaries. RESULTS: In both the Azores and the Canaries, morphological patterns were generally consistent with current taxonomic classifications. However, the AFLP data showed no genetic differentiation between the two currently recognized Azorean subspecies that are ecologically differentiated. Instead, genetic diversity in the Azores was structured geographically across the archipelago. In contrast, in the Canaries genetic differentiation was mostly consistent with morphology and current taxonomic treatments. Both Azorean and Canarian lineages exhibited ecological differentiation between currently recognized taxa. CONCLUSIONS: Neither a Linnean shortfall nor the perceived ecological homogeneity of the Azores fully explained the ADE-like pattern observed in Pericallis. Whilst variation in genetic data and morphological data in the Canaries were largely congruent, this was not the case in the Azores, where genetic patterns reflected inter-island geographical isolation, and morphology reflected intra-island bioclimatic variation. The combined effects of differences in (i) the extent of geographical isolation, (ii) population sizes and (iii) geographical occupancy of bioclimatic niche space, coupled with the morphological plasticity of Pericallis, may all have contributed to generating the contrasting patterns observed in the archipelagos.

Axial allometry in a neutrally buoyant environment: effects of the terrestrial-aquatic transition on vertebral scaling.

Ecological diversification into new environments presents new mechanical challenges for locomotion. An extreme example of this is the transition from a terrestrial to an aquatic lifestyle. Here, we examine the implications of life in a neutrally buoyant environment on adaptations of the axial skeleton to evolutionary increases in body size. On land, mammals must use their thoracolumbar vertebral column for body support against gravity and thus exhibit increasing stabilization of the trunk as body size increases. Conversely, in water, the role of the axial skeleton in body support is reduced, and, in aquatic mammals, the vertebral column functions primarily in locomotion. Therefore, we hypothesize that the allometric stabilization associated with increasing body size in terrestrial mammals will be minimized in secondarily aquatic mammals. We test this by comparing the scaling exponent (slope) of vertebral measures from 57 terrestrial species (23 felids, 34 bovids) to 23 semi-aquatic species (pinnipeds), using phylogenetically corrected regressions. Terrestrial taxa meet predictions of allometric stabilization, with posterior vertebral column (lumbar region) shortening, increased vertebral height compared to width, and shorter, more disc-shaped centra. In contrast, pinniped vertebral proportions (e.g. length, width, height) scale with isometry, and in some cases, centra even become more spool-shaped with increasing size, suggesting increased flexibility. Our results demonstrate that evolution of a secondarily aquatic lifestyle has modified the mechanical constraints associated with evolutionary increases in body size, relative to terrestrial taxa.

Estimating motor unit numbers from a CMAP scan: Repeatability study on three muscles at 15 centres.

OBJECTIVE: To assess the repeatability and suitability for multicentre studies of MScanFit motor unit number estimation (MUNE), which involves modelling compound muscle action potential (CMAP) scans. METHODS: Fifteen groups in 9 countries recorded CMAP scans twice, 1-2 weeks apart in healthy subjects from abductor pollicis brevis (APB), abductor digiti minimi (ADM) and tibialis anterior (TA) muscles. The original MScanFit program (MScanFit-1) was compared with a revised version (MScanFit-2), designed to accommodate different muscles and recording conditions by setting the minimal motor unit size as a function of maximum CMAP. RESULTS: Complete sets of 6 recordings were obtained from 148 subjects. CMAP amplitudes differed significantly between centres for all muscles, and the same was true for MScanFit-1 MUNE. With MScanFit-2, MUNE differed less between centres but remained significantly different for APB. Coefficients of variation between repeats were 18.0% for ADM, 16.8% for APB, and 12.1% for TA. CONCLUSIONS: It is recommended for multicentre studies to use MScanFit-2 for analysis. TA provided the least variable MUNE values between subjects and the most repeatable within subjects. SIGNIFICANCE: MScanFit was primarily devised to model the discontinuities in CMAP scans in patients and is less suitable for healthy subjects with smooth scans.

Human responses to nature- and culture-based non-clinical interventions: a systematised review.

AIMS: A wide range of non-clinical nature- and culture-based interventions for the treatment of health issues have been evaluated in evidence and systematic reviews. However, common outcomes of these interventions have not been identified and neuro-bio-psychosocial mechanisms underlying how these interventions impact health are not well understood. We conducted a systematised review and compared the evidence for human responses to nature- and culture-based non-clinical interventions for a range of health issues and assessed the proposed mechanisms and conceptual frameworks underlying these interventions. METHODS: Comprehensive searches were conducted up to May 2018 in six bibliographic databases: Campbell Collaboration, Cochrane Library, Embase, Medline, Scopus and Web of Science. Studies included were evidence reviews or systematic reviews on any nature- or culture-based non-clinical intervention to improve the health of individuals. RESULTS: A total of 60 reviews were included (33 of nature, 26 of culture, 1 of both) covering 1480 individual studies and trials. The most common review types were systematic (32), literature (22) and meta-analyses (6). Positive effects on mental health were reported for the majority of interventions, while other health outcomes such as immunity were not well represented in the review literature. A range of secondary outcomes were common to both nature- and culture-based interventions including psychological and emotional impacts, social interaction and relationship development, skills development, physical health benefits, and positive impact of the intervention environment. Only two reviews proposed conceptual frameworks, and the neuro-bio-psychosocial mechanisms that underpin the health changes were not clarified. CONCLUSION: Future research should focus on reviewing the evidence gaps for non-clinical nature- and culture-based interventions with an emphasis on implementing larger sample sizes, cohort and longitudinal studies, which deploy a wider range of mixed-methods, quasi-experimental and randomised control trials. There should also be agreement on terminology and developing conceptual frameworks to better understand the neuro-bio-psychosocial mechanisms underlying interventions.

AutoBend: An Automated Approach for Estimating Intervertebral Joint Function from Bone-Only Digital Models.

Deciphering the biological function of rare or extinct species is key to understanding evolutionary patterns across the tree of life. While soft tissues are vital determinants of joint function, they are rarely available for study. Therefore, extracting functional signals from skeletons, which are more widely available via museum collections, has become a priority for the field of comparative biomechanics. While most work has focused on the limb skeleton, the axial skeleton plays a critical role in body support, respiration, and locomotion, and is therefore of central importance for understanding broad-scale functional evolution. Here, we describe and experimentally validate AutoBend, an automated approach to estimating intervertebral joint function from bony vertebral columns. AutoBend calculates osteological range of motion (oROM) by automatically manipulating digitally articulated vertebrae while incorporating multiple constraints on motion, including both bony intersection and the role of soft tissues by restricting excessive strain in both centrum and zygapophyseal articulations. Using AutoBend and biomechanical data from cadaveric experiments on cats and tegus, we validate important modeling parameters required for oROM estimation, including the degree of zygapophyseal disarticulation, and the location of the center of rotation. Based on our validation, we apply a model with the center of rotation located within the vertebral disk, no joint translation, around 50% strain permitted in both zygapophyses and disks, and a small amount of vertebral intersection permitted. Our approach successfully reconstructs magnitudes and craniocaudal patterns of motion obtained from ex vivo experiments, supporting its potential utility. It also performs better than more typical methods that rely solely on bony intersection, emphasizing the importance of accounting for soft tissues. We estimated the sensitivity of the analyses to vertebral model construction by varying joint spacing, degree of overlap, and the impact of landmark placement. The effect of these factors was small relative to biological variation craniocaudally and between bending directions. We also present a new approach for estimating joint stiffness directly from oROM and morphometric measurements that can successfully reconstruct the craniocaudal patterns, but not magnitudes, derived from experimental data. Together, this work represents a significant step forward for understanding vertebral function in difficult-to-study (e.g., rare or extinct) species, paving the way for a broader understanding of patterns of functional evolution in the axial skeleton.

Resumo [Portuguese] Decifrar a função biológica de espécies raras ou extintas é fundamental para se compreender os padrões evolutivos na árvore da vida. Embora os tecidos moles sejam determinantes vitais das funções articulares, estes raramente estão disponíveis para estudo. Portanto, extrair dados funcionais provenientes de esqueletos, que são mais amplamente disponíveis por meio de coleções de museus, tornou-se uma prioridade para o campo da biomecânica comparada. Embora a maioria dos trabalhos biomecânicos tenham focado no esqueleto apendicular, o esqueleto axial também desempenha um papel crítico para o suporte corporal, respiração e locomoção e, portanto, é de importância central para a compreensão da evolução funcional em escalas amplas. Nesse trabalho, nós descrevemos e validamos experimentalmente o AutoBend, uma abordagem automatizada para estimar a função da articulação intervertebral de colunas vertebrais ósseas. O AutoBend calcula a amplitude do movimento osteológico (AMO) manipulando automaticamente as vértebras reconstruídas digitalmente e incorporando várias restrições de movimento, incluindo restrições das interseções ósseas e o papel de restrição dos tecidos moles na tensão excessiva sobre as articulações dos centros vertebrais e zigapofisárias. Usando AutoBend e dados biomecânicos de experimentos cadavéricos em gatos e lagartos tegus, validamos parâmetros de modelagem importantes e necessários para as estimativa do AMO, incluindo o grau de desarticulação zigapofisária e a localização do centro de rotação. Com base nessa validação, aplicamos um modelo com o centro de rotação localizado dentro do disco vertebral, sem translação articular, com cerca de 50% de tensão permitida nas zigapófises e discos vertebrais, além de uma pequena quantidade de intersecção vertebral permitida. Nossa abordagem reconstrói com sucesso magnitudes e padrões de movimento craniocaudais obtidos a partir de experimentos ex vivo, corroborando a sua potencial utilidade. Esse modelo também tem um desempenho melhor do que os métodos mais típicos que dependem apenas das interseções ósseas, enfatizando a importância de se levar em conta o papel dos tecidos moles. Estimamos a sensibilidade das análises à reconstrução do modelo vertebral, variando o espaçamento entre articulações, o grau de sobreposição e o impacto da localização dos pontos de referência. O efeito desses fatores foi pequeno em relação à variação biológica craniocaudal e entre as direções de flexão. Apresentamos aqui também uma nova abordagem para se estimar a rigidez articular diretamente à partir do AMO e medidas morfométricas que podem reconstruir com sucesso os padrões craniocaudais (embora não as magnitudes) derivados de dados experimentais. Este trabalho representa um passo significativo para a melhor compreensão da função vertebral em espécies difíceis de estudar (por exemplo, raras ou extintas), abrindo caminho para uma compreensão mais ampla dos padrões de evolução funcional no esqueleto axial.

Resumen [Spanish] Descifrar la función biológica de especies raras o extintas es fundamental para comprender los patrones evolutivos del árbol de la vida. Aunque los tejidos blandos son determinantes vitales de la función articular, raramente están disponibles para su estudio. Por lo tanto, la extracción de datos funcionales de esqueletos, que están más comumente disponibles a través de colecciones de museos, se ha convertido en una prioridad para el campo de la biomecánica comparada. Aunque la mayor parte del trabajo biomecánico se ha centrado en el esqueleto apendicular, el esqueleto axial también desempeña un papel fundamental para el soporte del cuerpo, la respiración y la locomoción y, por lo tanto, es de vital importancia para comprender la evolución funcional a gran escala. En este trabajo, describimos y validamos experimentalmente AutoBend, una herramienta automatizada para estimar la función de la articulación intervertebral en las columnas vertebrales óseas. AutoBend calcula el rango de movimiento osteológico (RMOo) manipulando automáticamente las vértebras reconstruidas digitalmente e incorporando varias restricciones de movimiento, incluidas las restricciones de intersección ósea y el papel de la restricción de tejidos blandos en la tensión excesiva sobre las articulaciones cigapofisarias y centros vertebrales. Utilizando AutoBend y datos biomecánicos de experimentos cadavéricos en gatos y lagartos tegus, validamos importantes parámetros de modelado necesarios para estimar el RMOo, incluido el grado de desarticulación cigapofisaria y la ubicación del centro de rotación. Con base en esta validación, aplicamos un modelo con el centro de rotación ubicado dentro del disco vertebral, sin traslación articular, con 50% de tensión permisible en la cigapófisis y los discos vertebrales, además de una pequeña cantidad de intersección vertebral permitida. Nuestra herramienta reconstruye con éxito magnitudes craneocaudales y patrones de movimiento obtenidos de experimentos ex vivo, corroborando su potencial utilidad. Este modelo también funciona mejor que los métodos más típicos que se basan solo en las intersecciones óseas, enfatizando la importancia de tener en cuenta el papel de los tejidos blandos. Estimamos la sensibilidad de los análisis a la reconstrucción del modelo vertebral, variando el espaciamiento entre articulaciones, el grado de superposición y el impacto de la ubicación de los puntos de referencia. El efecto de estos factores fue pequeño en relación a la variación biológica craneocaudal y entre las direcciones de flexión. También presentamos aquí un nuevo enfoque para estimar la rigidez articular directamente de la RMOo y mediciones morfométricas que pueden reconstruir con éxito los patrones craneocaudales (aunque no las magnitudes) derivados de datos experimentales. Este trabajo representa un paso significativo hacia una mejor comprensión de la función vertebral en especies difíciles de estudiar (por ejemplo, raras o extintas), allanando el camino para una comprensión más amplia de los patrones de evolución funcional en el esqueleto axial.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in pharyngeal and rectal specimens using the BD Probetec ET system, the Gen-Probe Aptima Combo 2 assay and culture.

OBJECTIVES: This study compared the sensitivity and specificity of culture and two nucleic acid amplification tests (NAATs): the BD Probetec ET system (PT) and the Aptima Combo 2 (AC2) in detecting Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) in pharyngeal and rectal specimens. METHODS: Male subjects were prospectively recruited at an MSM clinic in Toronto, Canada. Pharyngeal and rectal specimens were obtained for GC and CT culture, PT and AC2. Urine was also obtained for PT. A true positive was defined as: (1) positive culture, (2) positive PT and AC2 at the same site or (3) a single positive NAAT and detection of the same organism by any method at another site. RESULTS: 248 subjects were recruited. The prevalence of pharyngeal GC was 8.1%, rectal GC 11.7%, pharyngeal CT 2.0% and rectal CT 7.7%. The sensitivity of culture for pharyngeal GC and CT was 0%; 41.4% for rectal GC and 21.1% for rectal CT. The sensitivity of PT for pharyngeal GC, rectal GC, pharyngeal CT and rectal CT was 95.0%, 93.1%, 80.0% and 94.7%, respectively. The sensitivity of AC2 was 95.0% for pharyngeal GC and 100% at all other sites. Specificity was consistently above 98%. CONCLUSIONS: PT and AC2 detected GC and CT with superior sensitivity compared to culture. They detected 73 pharyngeal or rectal GC and CT infections compared to 16 by culture, using a rigorous gold standard. NAATs should be the method of choice for the detection of GC and CT in extragenital sites in men who have sex with men.

Fossils reveal the complex evolutionary history of the mammalian regionalized spine.

A unique characteristic of mammals is a vertebral column with anatomically distinct regions, but when and how this trait evolved remains unknown. We reconstructed vertebral regions and their morphological disparity in the extinct forerunners of mammals, the nonmammalian synapsids, to elucidate the evolution of mammalian axial differentiation. Mapping patterns of regionalization and disparity (heterogeneity) across amniotes reveals that both traits increased during synapsid evolution. However, the onset of regionalization predates increased heterogeneity. On the basis of inferred homology patterns, we propose a "pectoral-first" hypothesis for region acquisition, whereby evolutionary shifts in forelimb function in nonmammalian therapsids drove increasing vertebral modularity prior to differentiation of the vertebral column for specialized functions in mammals.

The fast-slow continuum in mammalian life history: an empirical reevaluation.

Many life-history traits co-vary across species, even when body size differences are controlled for. This phenomenon has led to the concept of a "fast-slow continuum," which has been influential in both empirical and theoretical studies of life-history evolution. We present a comparative analysis of mammalian life histories showing that, for mammals at least, there is not a single fast-slow continuum. Rather, both across and within mammalian clades, the speed of life varies along at least two largely independent axes when body size effects are removed. One axis reflects how species balance offspring size against offspring number, while the other describes the timing of reproductive bouts.

Preliminary data on the effect of osseous anatomy on ex vivo joint mobility in the equine thoracolumbar region.

REASONS FOR PERFORMING STUDY: The thoracolumbar region is clinically important in horses; however, the link between joint mobility and bony joint morphology has not been tested quantitatively. OBJECTIVES: To establish which aspects of vertebral morphology correlate with ex vivo range of motion in the thoracolumbar region of Equus caballus, and demonstrate methodologies for linking vertebral form and function. STUDY DESIGN: Morphometric study of osteological specimens. METHODS: A digital model was created of a disarticulated thoracolumbar region to examine bone-to-bone interactions during in silico bending. Linear measurements and geometric morphometric landmarks were taken from 6 vertebrae per specimen (specimens n = 5, vertebrae n = 30), and compared with experimental range of motion in dorsiflexion, ventroflexion, lateroflexion and axial rotation data using Spearman's rank correlation, to test a priori hypotheses regarding thoracolumbar functional anatomy. RESULTS: Decreased sagittal mobility correlates with a tall, heart-shaped vertebral body, although bony interactions restrict dorsiflexion more than ventroflexion. Lateroflexion correlates with a narrow vertebral body, a short transverse process lever arm, and narrowly placed horizontally oriented zygapophyses. Lateral joints also restrict lateroflexion in the posterior lumbar region. Axial rotation is related to the shape of the zygapophyseal joint. CONCLUSIONS: These preliminary data suggest that vertebral joint morphology does determine experimentally measured range of motion, but patterns depend upon the type of motion. These methods are useful for identifying functionally relevant morphological variation and suggest osteological features are important in determining motion.

Lack of bystander killing in herpes simplex virus thymidine kinase-transduced colon cell lines due to deficient connexin43 gap junction formation.

The efficacy of herpes simplex virus thymidine kinase (HSV-TK) gene therapy for colorectal carcinoma has been investigated in an in vitro system. The magnitude and the mechanism of the HSV-TK bystander effect was determined in three human colon tumor cell lines: HCT-116, HCT-8, and HT-29. Each HSV-TK(+) cell line was generated by stable transduction with a bicistronic retroviral vector containing the HSV-TK and neomycin resistance (neo) genes; each exhibited an IC50 for GCV of < or =4 microM. When GCV was added to HSV-TK(+) cells mixed with parental cells or known bystander-positive cell lines, no bystander killing was evident in the HT-29 or HCT-8 cells. Western blots detected the expression of the gap junction protein connexin43 (Cx43) in HCT-8 and HT-29 cells; however, immunolocalization studies indicated predominantly cytoplasmic staining of Cx43 and no cell surface staining in these cell lines. Stable transfection of HCT-8 and HT-29 cells with Cx43 resulted in increased levels of Cx43 expression with the same subcellular distribution as before, yet there was again no apparent bystander killing. In contrast, Cx43 expression was localized to the cell surface in the bystander-positive colon tumor cell line HCT-116. These results demonstrate that expression and proper surface localization of Cx43 gap junctions are necessary components of the bystander effect in human colon tumor cells. They also indicate that future combination gene therapy approaches using coexpression of HSV-TK and Cx43 genes may not be applicable to all tumor systems.

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone.

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.

Triiodothyronine receptor beta-2 messenger ribonucleic acid expression by somatotropes and thyrotropes: effect of propylthiouracil-induced hypothyroidism in rats.

mRNA for a thyroid hormone receptor isoform that is unique to the pituitary gland (TR beta-2) is down-regulated by T3. Increases in the expression of this mRNA are seen in rats rendered hypothyroid by treatment with propylthiouracil (PTU). This study used dual labeling to determine which pituitary cells expressed TR beta-2 mRNA in normal and PTU-treated rats. In situ hybridization protocols localized the mRNA (with biotinylated complementary oligonucleotide probes detected by avidin-biotin-peroxidase), and immunoperoxidase protocols identified the pituitary hormone proteins. In dispersed pituitary cells, 20 +/- 2% (average +/- SD) of cells from normal rats and 30 +/- 3% of cells from PTU-treated rats were labeled for TR beta-2 mRNA. PTU caused increases in the area of the labeled cells (from 114 +/- 11 to 225 +/- 7 microns 2), the area of the label per cell (from 27 +/- 3 to 71 +/- 11 microns 2), and label density. PTU produced increases in the percentage of TSH cells from 8 +/- 1% to 19 +/- 2%, decreases in the percentage of GH cells from 27 +/- 3% to 11 +/- 2%, and no change in other cell types. After dual labeling, 73% of cells that expressed TR beta-2 mRNA stored either TSH (35 +/- 8) or GH (38 +/- 6). Less than 10% stored other hormones. When each cell type was analyzed, 56 +/- 3% of TSH cells and 43 +/- 4% of GH cells expressed TR beta-2 mRNA. When these percentages were multiplied by the percentages of each cell type in the overall population, TSH and GH cells with TR beta-2 mRNA represented 6.8 +/- 1% and 11.6 +/- 1% of the pituitary cells, respectively. Less than 1% of all pituitary cells expressed TR beta-2 and ACTH (0.9 +/- 0.06), LH (0.8 +/- 0.1), FSH (0.8 +/- 0.1), and PRL (0.9 +/- 0.04). PTU treatment increased the percentage of TSH cells with TR beta-2 mRNA to 72 +/- 4% and decreased the percentage of GH cells with TR beta-2 mRNA to 30 +/- 3%. However, some enlarged putative TSH cells could not be identified by immunolabel because the storage levels were low. Thus, changes in TR beta-2 mRNA in hypothyroid rats may be the net result of the increase in the percentage of TSH cells, the amount of mRNA per cell (measured by area and density of label), and the decrease in the percentage of GH cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that phosphorylation events participate in thyroid hormone action.

Using both a protein phosphatase inhibitor, okadaic acid (OA), and a protein kinase inhibitor, H7, to modify phosphorylation events in the cell, we investigated the effects of these agents on transcriptional activation via exogenous rat thyroid hormone receptor (TR) isoforms in transiently transfected cells and endogenous TRs. CV-1 cells were transiently cotransfected with expression plasmids encoding either the rat TR alpha 1 or TR beta 1, and luciferase reporter plasmids containing either the synthetic DR4 or the chick lysozyme F2 thyroid hormone response elements (TREs). For both receptor isoforms, there was an enhancement of transcriptional activity after incubation with 5 nM T3 for 24 h compared to hypothyroid levels. There was little change in transcriptional activation in the presence of 25 nM OA alone; however, for both TR isoforms and both TREs studied, OA augmented the stimulatory effects of T3. For the F2 TRE, transcriptional activation via TR alpha 1 increased from 19- to 35-fold, and that via TR beta 1 increased from 6- to 10-fold in the presence of T3 and OA compared to that with T3 alone. Similar results were found for the DR4 TRE. OA enhanced transcriptional activation by T3 in a dose-dependent manner. Increasing concentrations of OA (0, 5, 25, and 50 nM) further increased relative luciferase activity from 11-fold in the absence of OA to 45-fold in the presence of 50 nM OA. The protein kinase inhibitor, H7, caused no change in the transcriptional activity of the reporter plasmids via TR alpha 1 in the absence of T3, but completely blocked transcriptional activation by T3 for both the DR4 and the F2 TREs. H7 also blocked stimulation of endogenous GH and inhibition of endogenous TR beta 2 mRNAs by T3 in GH3 cells. These results indicate that phosphorylation events in the cell play an important role in transcriptional activation via both TR isoforms.

Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells.

Rat pituitary GH cells have been used extensively to study the biochemical actions of TRH on lactotropic cells. To investigate the structure and regulation of the rat TRH receptor (rTRHR), we have cloned its cDNA from GH4C1 cells. Using the polymerase chain reaction with degenerate primers and pools of cloned cDNAs from a GH4C1 cDNA library, a fragment sharing high similarity to the mouse thyrotrope TRHR (mTRHR) was identified. Conventional library screening with this fragment was used to isolate a single cDNA. mRNA synthesized in vitro from this cDNA was injected into Xenopus oocytes, and a characteristic conductance response to TRH was detected by voltage clamp recording. DNA sequence analysis revealed a molecule of 412 amino acid residues, with 96% similarity to the mTRHR. However, in contrast to the mTRHR, the rTRHR had an additional 19 amino acid residues at its carboxy-terminus. A mRNA of about 4 kilobases was identified in GH3 cells. Regulation of the rTRHR mRNA concentration was studied in GH3 cells. Steady state rTRHR mRNA levels were decreased to 30% of the control level by incubation with TRH for 48 h and increased 4-fold by incubation with dexamethasone for 12 h. Southern blot analysis of genomic DNA from GH3 cells gave a simple banding pattern consistent with a single copy gene. We conclude that the rTRHR shares high primary sequence similarity to the mTRHR, but the rTRHR has an extension of 19 amino acids at its carboxy-terminus, which is lacking in the mTRHR.

Two-drug combinations that increase apoptosis and modulate bak and bcl-X(L) expression in human colon tumor cell lines transduced with herpes simplex virus thymidine kinase.

Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-hydroxystaurosporine (UCN-01). Over a 2-4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5- to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-X(L), was observed in butyrate-, CPT-, and 7-hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-X(L). In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-X(L) protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.

An alternative splice variant of the mouse TRH receptor mRNA is the major form expressed in the mouse pituitary gland.

The sequences of the mouse and rat TRH receptors (TRH-Rs) show 94% similarity at the protein level. However, they differ significantly at their carboxy terminals, i.e. the mouse TRH-R ends with an asparagine at position 393 while, in the rat, residue 393 is lysine and an additional 19 amino acids are added before the first stop codon. In the mouse cDNA, the sequence encoding these additional amino acids is located 224 bp downstream in the 3' untranslated region (3'UT). As the mouse TRH-R was cloned from thyrotrope-derived TtT97 tissue and the rat TRH-R from lactotrope-derived GH cell lines, we have investigated whether this difference at the carboxy terminus represents a species-specific or cell type-specific pattern of TRH-R expression. Total RNA was isolated from mouse pituitary and TtT97 tissue, and rat pituitary and GH3 cells. Reverse transcription PCR analysis was performed using primers that would generate DNA fragments including the stop codon in either the mouse or the rat TRH-R and, in the mouse form, the extra 224 bp of 3'UT. This would generate a product of 234 bp from the rat sequence and 441 bp from the mouse sequence. In rat pituitary and GH3 cDNA, PCR generated the expected 234 bp product but not a band representing the mouse sequence. In both mouse pituitary and TtT97 cDNA, neither the expected 441 bp nor the 234 bp fragments were amplified; instead a larger, 829 bp, product was generated. Sequence analysis revealed a 388 bp insertion at position 1663 in the 3'UT compared with the published mouse TRH-R sequence. Ribonuclease protection analysis using this 829 bp fragment as a probe showed that this sequence represented the major TRH-R mRNA species in mouse pituitary and TtT97 RNA. A genomic clone containing this region of the mouse TRH-R gene was isolated and analysis of the sequence in this region revealed that this longer form of the mouse TRH-R could be generated by alternative splicing. In summary, we have shown that the carboxyterminal differences between the mouse and rat TRH-Rs are species-specific rather than cell type-specific, and that the major TRH-R mRNA expressed in mouse pituitary contains an additional 388 bp of 3'UT compared with the published sequence. As a region in the 3'UT of the published mTRH-R sequence has been shown to be important for stability of this mRNA, this additional 3'UT sequence could have major effects on the regulation and stability of the mouse TRH-R mRNA.

Regulation of thyroid hormone receptor beta-2 mRNA levels by retinoic acid.

The thyroid hormone receptor, TR beta-2, whose expression is limited to the pituitary and parts of the central nervous system, is strongly negatively regulated at the pre-translational level by thyroid hormone (T3). We have investigated whether retinoic acid (RA), whose receptors (RARs) share a high degree of homology with the thyroid hormone receptors (TRs), can regulate this gene in a manner similar to T3, as has been shown for the growth hormone (GH) gene. GH3 cells were incubated with 10 nM T3, 1 microM RA or both for 48 h and then TR beta-2 mRNA levels determined by RNA blot hybridization analysis. We observed a 73% decrease in TR beta-2 mRNA levels after incubation with T3 and a two-fold increase in TR beta-2 mRNA levels after incubation with RA alone. In the presence of RA, the T3 effect on TR beta-2 mRNA levels was blunted with mRNA levels decreasing by only 20%. We investigated the mechanism by which retinoic acid increases and opposes the effects of T3 on levels of TR beta-2 mRNA. In transient transfection experiments using a reporter plasmid containing the TR beta-2 promoter and in nuclear run on assays, we found no effect of RA on TR beta-2 gene transcription. We then investigated whether the effects of RA were mediated at the post-transcriptional level. Determination of the apparent half-life of TR beta-2 mRNA using the transcriptional inhibitor, actinomycin D, showed that RA had no effect on TR beta-2 mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a cDNA encoding human Rev-ErbA alpha: transcription from the noncoding DNA strand of a thyroid hormone receptor gene results in a related protein that does not bind thyroid hormone.

A cDNA encoding Rev-ErbA alpha, a member of the thyroid/steroid hormone receptor superfamily, has been isolated from a human fetal skeletal muscle library. This cDNA contains 269 consecutive base pairs identical to a region of a human c-erbA alpha-2 cDNA, but the respective long open reading frames utilize this nucleotide sequence in opposite orientations. Thus, human Rev-ErbA alpha (hRev) is derived from opposite-strand transcription of the c-erbA alpha genomic locus. mRNA encoding hRev was detected in human skeletal muscle by Northern analysis. Comparison of hRev and Rev-ErbA (rRev) reveals 99% identity in the putative DNA-binding and "ligand-binding" (carboxy-terminal) domains. hRev does not bind thyroid hormone (T3), as has also been found for its rat homolog. Interestingly, the human and rat Rev-ErbA alpha cDNAs are dissimilar at their 5' ends, corresponding to the first exon that we have identified in the rat gene. The conservation of the bidirectionally transcribed regions of c-erbA alpha-2 and Rev-ErbA alpha in human and rat suggests that this unusual genomic arrangement has an important function, perhaps related to the regulation of gene expression.