Honeybee dietary neonicotinoid exposure is associated with pollen collection from agricultural weeds.

Neonicotinoid insecticides have been linked to bee declines. However, tracking the primary exposure route for bees in the field has proven to be a major logistical challenge, impeding efforts to restore pollinator health in agricultural landscapes. We quantified neonicotinoid concentrations and botanical species composition in 357 pollen samples collected from 114 commercial honeybee colonies placed along a gradient of agricultural intensity between June and September. Neonicotinoid concentrations increased through the season, peaking at the end of August. As a result, concentrations in pollen were negatively associated with collection from woody and crop plants that flower early-mid season, and positively associated with collection from herbaceous plants that flower mid-late season. Higher clothianidin and thiamethoxam residues were correlated with samples containing a greater proportion of pollen collected from agricultural weeds. The percentage of agricultural land within 1500 m was positively correlated with thiamethoxam concentration; however, this spatial relationship was far weaker than the relationship with the proportion of pollen collected from herbaceous plants. These results indicate that both plant species identity and agricultural dominance are important in determining honeybee neonicotinoid exposure through the pollen diet, but that uncultivated plants associated with agriculture are the source of the greatest acute exposure.

Limited phenological and dietary overlap between bee communities in spring flowering crops and herbaceous enhancements.

Wild bee populations have undergone declines in recent years across much of the Western world, and these declines have the potential to limit yield in pollination-dependent crops. Highbush blueberry, Vaccinium corymbosum, and tart cherry, Prunus cerasus, are spring-blooming crops that rely on the movement of pollen by bees and other insects for pollination. Wild bee populations can be increased on farmland by providing floral resources, but whether the addition of these plants translates into increased pollinator density on crop flowers has not been documented in most cropping systems. To determine the importance of providing additional floral resources for wild bee pollinator communities, we selected blueberry fields and tart cherry orchards with and without herbaceous floral enhancements in western Michigan, USA. The bee communities visiting crop flowers, enhancements and control grassy field margins were sampled over a 5-yr period. In addition, the pollen diets of the most abundant wild bee crop pollinators were quantified across Michigan to better understand their foraging niches and to identify potentially important alternative host plants. The presence of floral enhancements did not increase the abundance of wild bees on either blueberry or cherry flowers during bloom. The bee community visiting blueberry was evenly composed of short-season bees that fly only during the spring and long-season bees that fly in both spring and summer. In contrast, the bee community visiting cherry was dominated by short-season spring bees. The majority of pollen collected by the wild bee communities visiting blueberry and cherry was from spring-flowering woody plants, with limited use of the herbaceous enhancements. Enhancements attracted greater abundance and species richness of bees compared to control areas, including twice as many floral specialists. Conserving summer-flying, grassland-associated bees is an appropriate goal for pollinator conservation programs. However, herbaceous enhancements may not provide adequate resources for the wild bees that pollinate spring-flowering crops. This study demonstrates that an examination of the pollen collected by wild bees across their flight periods can identify plant species to help them persist in intensively managed landscapes.

A comparison of techniques for assessing farmland bumblebee populations.

Agri-environment schemes have been implemented across the European Union in order to reverse declines in farmland biodiversity. To assess the impact of these schemes for bumblebees, accurate measures of their populations are required. Here, we compared bumblebee population estimates on 16 farms using three commonly used techniques: standardised line transects, coloured pan traps and molecular estimates of nest abundance. There was no significant correlation between the estimates obtained by the three techniques, suggesting that each technique captured a different aspect of local bumblebee population size and distribution in the landscape. Bumblebee abundance as observed on the transects was positively influenced by the number of flowers present on the transect. The number of bumblebees caught in pan traps was positively influenced by the density of flowers surrounding the trapping location and negatively influenced by wider landscape heterogeneity. Molecular estimates of the number of nests of Bombus terrestris and B. hortorum were positively associated with the proportion of the landscape covered in oilseed rape and field beans. Both direct survey techniques are strongly affected by floral abundance immediately around the survey site, potentially leading to misleading results if attempting to infer overall abundance in an area or on a farm. In contrast, whilst the molecular method suffers from an inability to detect sister pairs at low sample sizes, it appears to be unaffected by the abundance of forage and thus is the preferred survey technique.

Validation of a technique for estimating organ doses for kilovoltage cone-beam CT of the prostate using the PCXMC 2.0 patient dose calculator.

The use of cone beam CT in common radiotherapy treatments is increasing with the growth of image guided radiotherapy. Whilst the benefits that this technology offers are clear, such as improved patient positioning prior to treatment, it is always important to consider the implications of such intensive imaging regimes on the patient, especially when considering the fundamental radiation protection requirements for justification and optimisation.The purpose of this study was to develop a technique that uses readily available dose calculation software (PCXMC 2.0) to estimate the organ and effective doses that result from these types of examination in prostate treatments on the Varian OBI system. It has been shown that by separating these types of examinations into 28 different projections, with a range of x-ray beam qualities, it is possible to reproduce the complex geometry that is used on these imaging systems in PCXMC i.e. asymmetric radiation field with a half bowtie filter rotating 360° around the patient.This new technique has been validated with thermo-luminescent dosimeter measurements in the Rando anthropomorphic phantom, and has been shown to give excellent agreement with this established method (R(2) = 0.995). This technique will prove to be valuable to radiotherapy departments that are looking to optimise their CBCT imaging protocols as it allows a rapid evaluation of the impact of any changes on patient dose. It also serves to further highlight the levels of dose that these types of patient are subject to when having daily CBCT scans as part of the treatment, which further reinforces the need for optimisation of both patient dose and image quality on these systems.

A practical method to standardise and optimise the Philips DoseRight 2.0 CT automatic exposure control system.

Given the increasing use of computed tomography (CT) in the UK over the last 30 years, it is essential to ensure that all imaging protocols are optimised to keep radiation doses as low as reasonably practicable, consistent with the intended clinical task. However, the complexity of modern CT equipment can make this task difficult to achieve in practice. Recent results of local patient dose audits have shown discrepancies between two Philips CT scanners that use the DoseRight 2.0 automatic exposure control (AEC) system in the 'automatic' mode of operation. The use of this system can result in drifting dose and image quality performance over time as it is designed to evolve based on operator technique. The purpose of this study was to develop a practical technique for configuring examination protocols on four CT scanners that use the DoseRight 2.0 AEC system in the 'manual' mode of operation. This method used a uniform phantom to generate reference images which form the basis for how the AEC system calculates exposure factors for any given patient. The results of this study have demonstrated excellent agreement in the configuration of the CT scanners in terms of average patient dose and image quality when using this technique. This work highlights the importance of CT protocol harmonisation in a modern Radiology department to ensure both consistent image quality and radiation dose. Following this study, the average radiation dose for a range of CT examinations has been reduced without any negative impact on clinical image quality.

New Asian Andrena species, with notes on the subgenus Cnemidandrena (Hymenoptera: Andrenidae).

Asia is the worlds largest continent and comprises most of the Holarctic biogeographic region. The genus Andrena is principally distributed through the Holarctic, but it remains understudied in Asia. The subgenus Cnemidandrena Hedicke, 1933 is distributed across Asia with some species reaching south to the Tibetan Plateau. However, some southern members display an unusual morphology, including A. (Cnemidandrena) kishidai chagyabensis Wu, 1982 (newly recorded for Bhutan, India, and Nepal) and A. (Cnemidandrena) granulitergorum Tadauchi & Xu, 2002 (newly recorded for Nepal). The male of A. granulitergorum is described for the first time, A. (Cnemidandrena) rufina Morawitz, 1876 is reported from Kyrgyzstan, Afghanistan, and Kashmir for the first time, and possible senior synonyms for Asian A. (Cnemidandrena) are suggested. To contribute to an improved understanding of localised Asian Andrena diversity, the following new species are described: A. (Cnemidandrena) textrix sp. nov. from China, A. (incertae sedis) liebigi sp. nov. and A. (Troandrena) monfaredi sp. nov. from Iran, and A. (incertae sedis) quercorum sp. nov. from Turkey. These results further confirm that additional taxonomic work is needed to harmonise Andrena species concepts across the different biogeographic regions of Asia.

The effects of chlorhexidine, povidone-iodine and vancomycin on growth and biofilms of pathogens that cause prosthetic joint infections: an in-vitro model.

BACKGROUND: Chlorhexidine gluconate (CHG) and povidone-iodine (PI) are commonly used to prevent prosthetic joint infection (PJI) during total joint replacement; however, their effective concentrations and impact on biofilms are not well defined. AIM: To determine: (1) the in-vitro minimum inhibitory concentration of CHG and PI against model PJI-causing organisms and clinical isolates; (2) their impact on biofilm formation; (3) whether there is a synergistic benefit to combining the two solutions; and (4) whether adding the antibiotic vancomycin impacts antiseptic activity. METHODS: We measured in-vitro growth and biofilm formation of Staphylococcus epidermidis, meticillin-sensitive and meticillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans, as well as recent clinical isolates, in the presence of increasing concentrations of CHG and/or PI. Checkerboard assays were used to measure potential synergy of the solutions together and with vancomycin. FINDINGS: CHG and PI inhibited growth and biofilm formation of all model organisms tested at concentrations of 0.0004% and 0.33% or lower, respectively; highly dilute concentrations paradoxically increased biofilm formation. The solutions did not synergize with one another and acted independently of vancomycin. CONCLUSION: CHG and PI are effective at lower concentrations than typically used, establishing baselines to support further clinical trials aimed at optimizing wound disinfection. There is no synergistic advantage to using both in combination. Vancomycin is effective at inhibiting the growth of S. epidermidis and S. aureus; however, it stimulates P. aeruginosa biofilm production, suggesting in the rare case of P. aeruginosa PJI, it could exacerbate infection.

Elevated rates of dietary generalization in eusocial lineages of the secondarily herbivorous bees.

BACKGROUND: Within the Hymenoptera, bees are notable for their relationship with flowering plants, being almost entirely dependent on plant pollen and nectar. Though functionally herbivorous, as a result of their role as pollinators, bees have received comparatively little attention as models for insect herbivory. Bees often display dietary specialization, but quantitative comparison against other herbivorous insects has not previously been conducted. RESULTS: In the most comprehensive analysis to date for 860 bee species, dietary specialization amounted to 50.1% of studied species collecting pollen from between 1 and 2 botanical families with a relatively long tail of dietary generalists, with 11.1% of species collecting from more than 10 botanical families. This distribution deviated from the truncated Pareto distribution of dietary breadth seen in other herbivorous insect lineages. However, this deviation was predominantly due to eusocial bee lineages, which show a range of dietary breadths that conformed to a normal distribution, while solitary bees show a typical truncated distribution not strongly different from other herbivorous insects. We hypothesize that the relatively low level of dietary specialization in bees as a whole reflects the relaxation of the constraints typically observed in herbivorous insects with a comparatively reduced importance of plant chemistry and comparatively increased importance of phenology and foraging efficiency. The long flight periods of eusocial bees that are necessary to allow overlapping generations both allows and necessitates the use of multiple flowering resources, whereas solitary bees with short flight periods have more limited access to varied resources within a constrained activity period. CONCLUSIONS: Collectively, solitary bees show slightly lower specialization compared to other herbivorous insects, possibly due to their balanced relationship with plants, rather than direct antagonism such as seen in the direct consumption of plant tissues. An additional factor may be the mediocre diversity of bees at low latitudes combined with low levels of dietary specialization, whereas these areas typically display a high rate of specialization by herbivorous insects in general. Though the most important factors structuring dietary specialization in bees appear to differ from many other herbivorous insects, solitary bees show a surprisingly similar overall pattern of dietary specialization.

Superhydrophobic hierarchical honeycomb surfaces.

Two-dimensional hexagonally ordered honeycomb surfaces have been created by solvent casting polybutadiene films under controlled humidity. Subsequent CF(4) plasmachemical fluorination introduces cross-linking and surface texturing, leading to hierarchical surfaces with roughness on both the 10 µm (honeycomb) and micrometer (texturing) length scales. For microliter droplets, these display high water contact angle values (>170°) in combination with low contact angle hysteresis (i.e., superhydrophobicity) while displaying bouncing of picoliter water droplets. In the case of picoliter droplets, it is found that surfaces which exhibit similar static contact angles can give rise to different droplet impact dynamics, governed by the underlying surface topography. These studies are of relevance to technological processes such as rapid cooling, delayed freezing, crop spraying, and inkjet printing.

Impact of picoliter droplets on superhydrophobic surfaces with ultralow spreading ratios.

The impact of picoliter-sized water droplets on superhydrophobic CF(4) plasma fluorinated polybutadiene surfaces is investigated with high-speed imaging. Variation of the surface topography by plasmachemical modification enables the dynamics of wetting to be precisely controlled. Final spreading ratios as low as 0.63 can be achieved. A comparison of the maximum spreading ratio and droplet oscillation frequencies to models described in the literature shows that both are found to be much lower than theoretically predicted.

An international survey of imaging practices in radiotherapy.

Improvements in delivery of radiation dose to target tissues in radiotherapy have increased the need for better image quality and led to a higher frequency of imaging patients. Imaging for treatment planning extends to function and motion assessment and devices are incorporated into medical linear accelerators (linacs) so that regions of tissue can be imaged at time of treatment delivery to ensure dose distributions are delivered as accurately as possible. A survey of imaging in 97 radiotherapy centres in nine countries on six continents has been undertaken with an on-line questionnaire administered through the International Commission on Radiological Protection mentorship programme to provide a snapshot of imaging practices. Responses show that all centres use CT for planning treatments and many utilise additional information from magnetic resonance imaging and positron emission tomography scans. Most centres have kV cone beam CT attached to at least some linacs and use this for the majority of treatment fractions. The imaging options available declined with the human development index (HDI) of the country, and the frequency of imaging during treatment depended more on country than treatment site with countries having lower HDIs imaging less frequently. The country with the lowest HDI had few kV imaging facilities and relied on MV planar imaging intermittently during treatment. Imaging protocols supplied by vendors are used in most centres and under half adapt exposure conditions to individual patients. Recording of patient doses, a knowledge of which is important in optimisation of imaging protocols, was limited primarily to European countries.

Narrow pollen diets are associated with declining Midwestern bumble bee species.

Many species of bumble bee (Bombus) have declined in range and abundance across Europe, the Americas, and Asia, whereas other species have persisted and remain common and widespread. One explanation as to why some species have declined, based primarily on studies of the European bumble bee fauna, is that declining species have relatively narrow pollen-foraging niches and are less able to use alternative host plants in the absence of their preferred hosts. Though extensively explored in Europe, this hypothesis has not been investigated in North America, in part due to incomplete information on the foraging niche of many species. We selected 12 bumble bee species found in Michigan and quantified their pollen diets using museum specimens. We also extensively resurveyed the state to understand their contemporary status and distribution. Compared to a pre-2000 baseline, six species remain relatively common and widespread, whereas six species show range contractions of over 50%. There was a significant relationship between dietary breadth and distributional range change, with declined or declining species collecting around one-third fewer pollen types than stable species. Though there were significant compositional differences, we found no differences in the number of pollen types collected by species with differing tongue lengths. Overall, these results support the hypothesis that species with narrower dietary niches are at greater risk of decline. However, it is not clear if narrow dietary niches are a cause of declines, or if both are driven by an underlying factor such as proximity to the edge of climatic niches. Further research is needed to improve our understanding of dietary niche in bumble bees, and how it interacts with other factors to influence population trajectories of stable and at-risk species.

Diet characterisation of solitary bees on farmland: dietary specialisation predicts rarity.

Changes in agricultural practice across Europe and North America have been associated with declines in wild bee populations. Bee diet breadth has been associated with sensitivity to agricultural intensification, but much of this analysis has been conducted at the categorical level of generalist or specialist, and it is not clear to what extent the level of generalisation within generalist species is also associated with species persistence. We used pollen load analysis to quantify the pollen diets of wild solitary bees on 19 farms across southern England, UK. A total of 72 species of solitary bees were recorded, but only 31 species were abundant enough to allow for formal diet characterisation. The results broadly conformed to existing literature with the majority of species polylectic and collecting pollen from a wide range of plants. Pollen load analysis consistently identified pollens from more plant species and families from each bee species than direct observation of their foraging behaviour. After rarefaction to standardise pollen load sample sizes, diet breadth was significantly associated with frequency of occurrence, with more generalist bees present on more farms than less generalist bees. Our results show that the majority of bee species present on farmland in reasonable numbers are widely variable in their pollen choices, but that those with the broadest diet were present on the greatest number of farms. Increasing the diversity of plants included in agri-environment schemes may be necessary to provide a wider range of pollen resources in order to support a diverse bee community on farmland.

Automatic exposure control calibration and optimisation for abdomen, pelvis and lumbar spine imaging with an Agfa computed radiography system.

The use of three physical image quality metrics, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and mean effective noise equivalent quanta (eNEQm) have recently been examined by our group for their appropriateness in the calibration of an automatic exposure control (AEC) device for chest radiography with an Agfa computed radiography (CR) imaging system. This study uses the same methodology but investigates AEC calibration for abdomen, pelvis and spine CR imaging. AEC calibration curves were derived using a simple uniform phantom (equivalent to 20 cm water) to ensure each metric was held constant across the tube voltage range. Each curve was assessed for its clinical appropriateness by generating computer simulated abdomen, pelvis and spine images (created from real patient CT datasets) with appropriate detector air kermas for each tube voltage, and grading these against reference images which were reconstructed at detector air kermas correct for the constant detector dose indicator (DDI) curve currently programmed into the AEC device. All simulated images contained clinically realistic projected anatomy and were scored by experienced image evaluators. Constant DDI and CNR curves did not provide optimized performance but constant eNEQm and SNR did, with the latter being the preferred calibration metric given that it is easier to measure in practice. This result was consistent with the previous investigation for chest imaging with AEC devices. Medical physicists may therefore use a simple and easily accessible uniform water equivalent phantom to measure the SNR image quality metric described here when calibrating AEC devices for abdomen, pelvis and spine imaging with Agfa CR systems, in the confidence that clinical image quality will be sufficient for the required clinical task. However, to ensure appropriate levels of detector air kerma the advice of expert image evaluators must be sought.

Growth hormone pretranslationally regulates the sexually dimorphic expression of the prolactin receptor gene in rat liver.

The female-specific expression of the rat liver PRL receptor (PRL-R) gene was investigated by Northern analysis of hypophysectomized rats after two alternative human GH treatments that were to mimic either 1) the continuous female-specific or 2) the discontinuous male-specific serum GH patterns. The former (female-specific) pattern was shown to result in a dramatic increase in PRL-R mRNA in both males and females, while the latter (male-specific) pattern failed to evoke this response. A similar inductive effect in hypophysectomized females was shown after continuous administration of bovine GH and was found to constitute an approximately 60-fold increase in PRL-R mRNA levels. This effect by bovine GH, which, unlike the human isoform, is devoid of lactogenic properties, thus indicates the somatogenic origin of the signal resulting in this inductive response. These observations in conjunction with previous data obtained for other GH-regulated nonreceptor genes are interpreted to support the proposal of GH serum patterns being an early signal in a more general mechanism for pretranslational regulation of sex-specific gene expression. In contrast to GH, only a slight elevation of PRL-R mRNA was evoked by the ligand ovine PRL, while coadministration of ovine PRL with bovine GH failed to enhance the mRNA level found with bovine GH alone. The detection of previously unreported PRL-R mRNAs in liver of approximately 3.0, 3.8, and 5 kilobases in addition to the major 2.2-kilobase form was also evident after continuous GH administration.

Ionising radiation exposure in patients with circular frame treatment of distal tibial fractures.

Total radiation exposure accumulated during circular frame treatment of distal tibial fractures was quantified in 47 patients treated by a single surgeon from February 2007 until Oct 2010. The radiation exposures for all relevant radiology procedures for the distal tibial injury were included to estimate the radiation risk to the patient. The median time of treatment in the frame was 169 days (range 105-368 days). Patients underwent a median of 13 sets of plain radiographs; at least one intra operative exposure and 16 patients underwent CT scanning. The median total effective dose per patient from time of injury to discharge was 0.025mSv (interquartile range 0.013-0.162 and minimum to maximum 0.01-0.53). The only variable shown to be an independent predictor of cumulative radiation dose on multivariate analysis was the use of CT scanning. This was associated with a 13-fold increase in overall exposure. Radiation exposure during treatment of distal tibial fractures with a circular frame in this group was well within accepted safe limits. The fact that use of CT was the only significant predictor of overall exposure serves as a reminder to individually assess the risk and utility of radiological investigations on an individual basis. This is consistent with the UK legal requirements for justification of all X-ray imaging, as set out in the Ionising Radiation (Medical Exposure) Regulations 2000 [1].

Correlation between the signal-to-noise ratio improvement factor (KSNR) and clinical image quality for chest imaging with a computed radiography system.

This work assessed the appropriateness of the signal-to-noise ratio improvement factor (KSNR) as a metric for the optimisation of computed radiography (CR) of the chest. The results of a previous study in which four experienced image evaluators graded computer simulated chest images using a visual grading analysis scoring (VGAS) scheme to quantify the benefit of using an anti-scatter grid were used for the clinical image quality measurement (number of simulated patients = 80). The KSNR was used to calculate the improvement in physical image quality measured in a physical chest phantom. KSNR correlation with VGAS was assessed as a function of chest region (lung, spine and diaphragm/retrodiaphragm), and as a function of x-ray tube voltage in a given chest region. The correlation of the latter was determined by the Pearson correlation coefficient. VGAS and KSNR image quality metrics demonstrated no correlation in the lung region but did show correlation in the spine and diaphragm/retrodiaphragmatic regions. However, there was no correlation as a function of tube voltage in any region; a Pearson correlation coefficient (R) of -0.93 (p = 0.015) was found for lung, a coefficient (R) of -0.95 (p = 0.46) was found for spine, and a coefficient (R) of -0.85 (p = 0.015) was found for diaphragm. All demonstrate strong negative correlations indicating conflicting results, i.e. KSNR increases with tube voltage but VGAS decreases. Medical physicists should use the KSNR metric with caution when assessing any potential improvement in clinical chest image quality when introducing an anti-scatter grid for CR imaging, especially in the lung region. This metric may also be a limited descriptor of clinical chest image quality as a function of tube voltage when a grid is used routinely.

Investigating the use of an antiscatter grid in chest radiography for average adults with a computed radiography imaging system.

OBJECTIVE: The aim of this study was to investigate via simulation a proposed change to clinical practice for chest radiography. The validity of using a scatter rejection grid across the diagnostic energy range (60-125 kVp), in conjunction with appropriate tube current-time product (mAs) for imaging with a computed radiography (CR) system was investigated. METHODS: A digitally reconstructed radiograph algorithm was used, which was capable of simulating CR chest radiographs with various tube voltages, receptor doses and scatter rejection methods. Four experienced image evaluators graded images with a grid (n = 80) at tube voltages across the diagnostic energy range and varying detector air kermas. These were scored against corresponding images reconstructed without a grid, as per current clinical protocol. RESULTS: For all patients, diagnostic image quality improved with the use of a grid, without the need to increase tube mAs (and therefore patient dose), irrespective of the tube voltage used. Increasing tube mAs by an amount determined by the Bucky factor made little difference to image quality. CONCLUSION: A virtual clinical trial has been performed with simulated chest CR images. RESULTS indicate that the use of a grid improves diagnostic image quality for average adults, without the need to increase tube mAs, even at low tube voltages. ADVANCES IN KNOWLEDGE: Validated with images containing realistic anatomical noise, it is possible to improve image quality by utilizing grids for chest radiography with CR systems without increasing patient exposure. Increasing tube mAs by an amount determined by the Bucky factor is not justified.

Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription.

We have investigated the effect of GH on the organization of the actin cytoskeleton within the cell. Human GH (hGH) treatment (50 nM) of Chinese hamster ovary (CHO) cells stably transfected with the complementary DNA for the rat GH receptor (CHO-GHR(1-638)) resulted in a reorganization of actin filaments in the cell that was not observed upon GH treatment of the untransfected parental CHO cell line. hGH initially induced depolymerization of actin stress fibers similar in magnitude to that induced by treatment of the cells with 100 nM human insulin-like growth factor I. This loss of stress fibers was observed as early as 30 sec after addition of hGH to the medium, and maximal depolymerization of stress fibers was observed between 1-4 min after addition of hGH. This was followed by a slow, but submaximal, repolymerization of the stress fibers and the formation of localized focal filamentous actin containing complexes. Similar cytoskeletal changes were observed after hGH treatment in Swiss 3T3 fibroblasts and BRL cells stably transfected with rat GH receptor complementary DNA (BRL-GHR(1-6381)). Pretreatment of CHO-GHR(1-638) cells with wortmannin (a phosphatidylinositol 3-kinase inhibitor) and verapamil (a calcium channel antagonist) both inhibited the hGH-induced actin reorganization. The integrity of the actin cytoskeleton was not required for GH-induced STAT5 (signal transducer and activator of transcription-5)-mediated transcription, as treatment of cells with cytochalasins B and D did not alter the fold stimulation of the STAT5-mediated transcriptional response to GH. We conclude that GH induces a rapid reorganization of the actin cytoskeleton by a process requiring phosphatidylinositol 3-kinase activation and calcium influx, but this cytoskeletal reorganization is not required for the STAT5-mediated transcriptional response to GH.

Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter.

The growth hormone regulated serine protease inhibitor (SPI) 2.1 and 2.2 gene promoters have been shown to contain a response element similar to the gamma-interferon activated sequence (GAS) family of signal transducer and activator of transcription (STAT) response elements. We have investigated the STAT and cytokine specificity of the SPI 2.1 STAT responsive element using a luciferase (LUC) reporter construct and a cDNA complementation strategy in the COS 7 cell line. Growth hormone was found to stimulate SPI-LUC reporter gene expression via activation of STAT 5, but not STATs 1 or 3, which indicates that the SPI 2.1 STAT responsive element is STAT 5 specific. In addition to the growth hormone receptor, the receptors for prolactin and erythropoietin enhanced gene transcription via the SPI 2.1 STAT responsive element, which indicates that this element is, on the other hand, not cytokine specific. Activation of STAT 5 was also observed after growth hormone treatment of cells transfected with cDNA expression plasmids for several different truncated growth hormone receptor mutants, although this activation was less efficient than with the wild type receptor. Point mutation of individual tyrosines in the growth hormone receptor intracellular domain to phenylalanines had no significant effect on signal transduction via STAT 5. These data, taken together with results from experiments using the phosphatase inhibitor sodium orthovanadate, suggest that STAT 5 may not have an absolute requirement for specific phosphorylated receptor tyrosine docking sites. That receptor tyrosine residues in a variety of amino acid contexts, or phosphorylated Janus kinase (JAK) 2 alone, can facilitate STAT 5 activation could explain the observed lack of cytokine specificity in STAT 5 activation.