bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacillus subtilisDuring Feast and Famine: Visualization of the Overall Regulation of Protein Synthesis During Glucose Starvation by Proteome Analysis
Authors: Bernhardt Jörg , Weibezahn Jimena , Scharf Christian , Hecker Michael
Published: February 2003

Dual channel imaging and warping of two-dimensional (2D) protein gels were used to visualize global changes of the gene expression patterns in growingBacillus subtiliscells during entry into the stationary phase as triggered by glucose exhaustion. The 2D gels only depict single moments during the cells' growth cycle, but a sequential series of overlays obtained at specific points of the growth curve facilitates visualization of the developmental processes at the proteomics scale. During glucose starvation a substantial reprogramming of the protein synthesis pattern was found, with 150 proteins synthesized de novo and cessation of the synthesis of almost 400 proteins. Proteins induced following glucose starvation belong to two main regulation groups: general stress/starvation responses induced by different stresses or starvation stimuli (ςB-dependent general stress regulon, stringent response, sporulation), and glucose-starvation-specific responses (drop in glycolysis, utilization of alternative carbon sources, gluconeogenesis). Using the dual channel approach, it was not only possible to identify those regulons or stimulons, but also to follow the fate of each single protein by the three-color code: red, newly induced but not yet accumulated; yellow, synthesized and accumulated; and green, still present, but no longer being synthesized. These green proteins, which represent a substantial part of the protein pool in the nongrowing cell, are not accessible by using DNA arrays. The combination of 2D gel electrophoresis and MALDI TOF mass spectrometry with the dual channel imaging technique provides a new and comprehensive view of the physiology of growing or starving bacterial cell populations, here for the case of the glucose-starvation response.

[This is presented as a movie ofB. subtilis's growth/glucose-starvation response, available atwww.genome.organd also athttp://microbio1.biologie.uni-greifswald.de/starv/movie.htm.]
Global RNA Half-Life Analysis inEscherichia coliReveals Positional Patterns of Transcript Degradation
Authors: Selinger Douglas W. , Saxena Rini Mukherjee , Cheung Kevin J. , Church George M. , Rosenow Carsten
Published: February 2003

Subgenic-resolution oligonucleotide microarrays were used to study global RNA degradation in wild-typeEscherichia coliMG1655. RNA chemical half-lives were measured for 1036 open reading frames (ORFs) and for 329 known and predicted operons. The half-life of total mRNA was 6.8 min under the conditions tested. We also observed significant relationships between gene functional assignments and transcript stability. Unexpectedly, transcription of a single operon (tdcABCDEFG) was relatively rifampicin-insensitive and showed significant increases 2.5 min after rifampicin addition. This supports a novel mechanism of transcription for thetdcoperon, whose promoter lacks any recognizable ς binding sites. Probe by probe analysis of all known and predicted operons showed that the 5′ ends of operons degrade, on average, more quickly than the rest of the transcript, with stability increasing in a 3′ direction, supporting and further generalizing the current model of a net 5′ to 3′ directionality of degradation. Hierarchical clustering analysis of operon degradation patterns revealed that this pattern predominates but is not exclusive. We found a weak but highly significant correlation between the degradation of adjacent operon regions, suggesting that stability is determined by a combination of local and operon-wide stability determinants. The 16 ORFdcwgene cluster, which has a complex promoter structure and a partially characterized degradation pattern, was studied at high resolution, allowing a detailed and integrated description of its abundance and degradation. We discuss the application of subgenic resolution DNA microarray analysis to study global mechanisms of RNA transcription and processing.
The β-Subunit of the Arabidopsis G Protein Negatively Regulates Auxin-Induced Cell Division and Affects Multiple Developmental Processes[W]
Authors: Ullah Hemayet , Chen Jin-Gui , Temple Brenda , Boyes Douglas C. , Alonso José M. , Davis Keith R. , Ecker Joseph R. , Jones Alan M.
Published: February 2003

Plant cells respond to low concentrations of auxin by cell expansion, and at a slightly higher concentration, these cells divide. Previous work revealed that null mutants of the α-subunit of a putative heterotrimeric G protein (GPA1) have reduced cell division. Here, we show that this prototypical G protein complex acts mechanistically by controlling auxin sensitivity toward cell division. Loss-of-function G protein mutants have altered auxin-mediated cell division throughout development, especially during the auxin-induced formation of lateral and adventitious root primordia. Ectopic expression of the wild-type Gα-subunit phenocopies the Gβ mutants (auxin hypersensitivity), probably by sequestering the Gβγ-subunits, whereas overexpression of Gβ reduces auxin sensitivity and a constitutively active (Q222L) mutant Gα behaves like the wild type. These data are consistent with a model in which Gβγ acts as a negative regulator of auxin-induced cell division. Accordingly, basal repression of approximately one-third of the identified auxin-regulated genes (47 of 150 upregulated genes among 8300 quantitated) is lost in the Gβ transcript-null mutant. Included among these are genes that encode proteins proposed to control cell division in root primordia formation as well as several novel genes. These results suggest that although auxin-regulated cell division is not coupled directly by a G protein, the Gβ-subunit attenuates this auxin pathway upstream of the control of mRNA steady state levels.
HLM1, an Essential Signaling Component in the Hypersensitive Response, Is a Member of the Cyclic Nucleotide–Gated Channel Ion Channel Family[W]
Authors: Balagué Claudine , Lin Baiqing , Alcon Carine , Flottes Guylaine , Malmström Susanna , Köhler Claudia , Neuhaus Gunther , Pelletier Georges , Gaymard Frédéric , Roby Dominique
Published: February 2003

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide–gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K+ and Na+ and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.
Quantitative Nature of Arabidopsis Responses during Compatible and Incompatible Interactions with the Bacterial PathogenPseudomonas syringae [W]
Authors: Tao Yi , Xie Zhiyi , Chen Wenqiong , Glazebrook Jane , Chang Hur-Song , Han Bin , Zhu Tong , Zou Guangzhou , Katagiri Fumiaki
Published: February 2003

We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae. The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene–resistance (R) gene combinations. Approximately 2000 of the ∼8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions. Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not. A large part of the difference between incompatible and compatible interactions can be explained quantitatively. Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not. This finding is consistent with the resistance phenotypes of these plants. We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.
Genomic Analysis of the Unfolded Protein Response in Arabidopsis Shows Its Connection to Important Cellular Processes[W]
Authors: Martínez Immaculada M. , Chrispeels Maarten J.
Published: February 2003

We analyzed the breadth of the unfolded protein response (UPR) in Arabidopsis using gene expression analysis with Affymetrix GeneChips. With tunicamycin and DTT as endoplasmic reticulum (ER) stress–inducing agents, we identified sets of UPR genes that were induced or repressed by both stresses. The proteins encoded by most of the upregulated genes function as part of the secretory system and comprise chaperones, vesicle transport proteins, and ER-associated degradation proteins. Most of the downregulated genes encode extracellular proteins. Therefore, the UPR may constitute a triple effort by the cell: to improve protein folding and transport, to degrade unwanted proteins, and to allow fewer secretory proteins to enter the ER. No single consensus response element was found in the promoters of the 53 UPR upregulated genes, but half of the genes contained response elements also found in mammalian UPR regulated genes. These elements are enriched from 4.5- to 15-fold in this upregulated gene set.
Macroeconomic variables and the Malaysian equity market
Authors: Ibrahim Mansor H. , Aziz Hassanuddeen
Published: February 2003

Analyzes dynamic linkages between stock prices and four macroeconomic variables for the case of Malaysia using standard and well‐accepted methods of cointegration and vector autoregression. Empirical results suggest the presence of a long‐run relationship between these variables and the stock prices and substantial short‐run interactions among them. In particular, documents positive short‐run and long‐run relationships between the stock prices and two macroeconomic variables. The exchange rate, however, is negatively associated with the stock prices. For the money supply, documents immediate positive liquidity effects and negative long‐run effects of money supply expansion on the stock prices. Also notes the predictive role of the stock prices for the macroeconomic variables. However, there seems to be irregularity in the data when observations from the recent crisis are included. Finally, documents the disappearance of the immediate positive liquidity effects of the money supply shocks and unstable interactions between the stock prices and the exchange rate over time.
Increasing returns and economic progress revisited
Authors: Fischer Jörg
Published: February 2003

It is hypothesised that the phenomenon of increasing returns and economic progress may only be consistently explained by employing the epistemological paradox, that where knowledge is part of the system, knowledge about the system necessarily and inescapably changes the system itself. Accordingly, one expects to find the principle of increasing returns embodied in the theoretical writing about increasing returns. This is shown by an exemplary reading of the works of Young and Penrose.
A decision model for self‐assessment of business process based on the EFQM excellence model
Authors: Li M. , Yang J.B.
Published: February 2003

In response to the criticism on the measurement system of self‐assessment against the European Foundation for Quality Management (EFQM) model, this paper reports the development of a more scientific and accurate scoring method. The decision model constructed has focused on the “processes” criterion of the EFQM model and can perform three main tasks: to score the self‐assessment submission document; to identify strengths and areas for improvement; and to simulate different scenarios for improvement planning. The model was implemented and tested using two award simulation documents from an electricity distribution utility and a water‐supply company. The results for one of the companies are reported and analysed in this paper. The analysis of the results has proved the reliability and accuracy of the new model. Using the decision model, two systematic methods were developed to identify strengths and areas for improvement and the findings are reported. The model's ability to link self‐assessment with the strategic‐planning process is also commented upon.
Inside modular production networks: “soft operational issues” in building factory competitiveness
Authors: Guzmán Gustavo A.C.
Published: February 2003

This paper examines a new model of production in a developing nation, the Electronics Contract Manufacturing Industry. It explores issues related to manufacturing management, competence building, organisation and implementation of manufacturing best practice. It argues that the understanding of both contextual institutional factors and soft operational issues is crucial for implementing manufacturing practices that support competitiveness building.
On an adaptive product development system: a hybrid database approach
Authors: Lee K.M. , Lau H.C.W. , Hui I.‐K. , Yu K.‐M.
Published: February 2003

Getting the right information at the right time is one of the success factors of product development. However, there exist a great variety of data formats which make the bi‐directional data interchange among various development groups inefficient and costly. Due to the lack of adequate product and engineering data, design practitioners may find it difficult to transform the customer’s requirements to a value‐added product. This paper attempts to propose a hybrid database approach (HDA) to support effective data exchange among a case based reasoning knowledge repository and a corporate relational database model, presenting an integrated view of a company’s knowledge sources so as to enable better decision‐making by providing immediate access to key business information. To introduce machine learning capability to the whole system, case based reasoning (CBR) is embraced to capture past experience and knowledge for case matching in terms of product development issues. The significance of this research is the provision of a cross‐platform data exchange system, which is able to facilitate the creation of a conducive product development environment.
Issues of security in hotels
Authors: Groenenboom Karen , Jones Peter
Published: February 2003

This article reports the findings of in‐depth interviews with security managers from some of London’s top hotels. What emerges is a range of different approaches to executing security policy. Key issues are identified in relation to the balance between providing hospitality and security, the degree of overt security, and the contribution that security staff make to the success of the business. Whilst there is a shift away from a focus on security against criminal threat towards broader concerns with health and safety, these hotels have not yet adopted the loss prevention strategies developed in the USA.
Revolution to revolution: why is tourism booming in Cuba?
Authors: Jayawardena Chandana
Published: February 2003

Tourism in Cuba is a relatively unexplored area in terms of research. Therefore, the main purpose of this article is to shed some light on the past, present and future potential of tourism in Cuba. The author has monitored trends of tourism in Cuba since 1994. He visited Cuba for the first time in 1997. He met President Fidel Castro in 1998, and had a brief but friendly conversation. The research methodology included a combination of personal observations, e´lite interviews and desk research. The article analyses tourism in Cuba during three different phases between 1945 and 2002. Based on his research, the author predicts that by 2010. Cuba will become the number one tourist destination in the Caribbean, a position it enjoyed for a long time until the revolution in 1959. In conclusion, the key arguments to justify this prediction for 2010 are presented.
A case study in organisational change: implications for theory
Authors: Nelson Lindsay
Published: February 2003

Organisational change is typically conceptualised as moving from the status quo to a new, desired, configuration to better match the environment. Change could, therefore, be seen as a departure from the norm, or alternatively as normal and simply a natural response to environmental and internal conditions. Static models of organisations are being displaced by dynamic models, which reflect the discontinuous nature of organisational change. Developments in theory suggest limitations to contingency approaches, which carry the assumptions of static models of change. Analysis of this case at PowerCo in Australia reveals a number of issues related to changes aimed at achieving a more commercial, profit‐oriented, focus. Points out that the contextualist approach is holistic, in which these aspects interact with each other as change unfolds temporally. A contextualist framework permits models of change to be visualised as dynamic rather than static, having a temporal setting which has multiple causes acting as loops rather than simple lines. This enables change to be understood as a discontinuous phenomenon having the benefits, without the limitations, of rational contingency models.
Cognition, culture and competition: an empirical test of the learning organization
Authors: Jashapara Ashok
Published: February 2003

This research examines the principal assumption underlying the learning organization literature that organizational learning leads to increased organizational performance and explores the role of organizational learning, culture and focused learning on organizational performance. The study is based on a stratified sample of 181 UK construction firms and adopts a structural equation methodology. As no scales exist from prior research, a new instrument is developed for a learning organization. The results suggest that double‐loop learning and cooperative cultures have a positive effect on organizational performance. The effect of competitive forces means that organizational learning focused on efficiency and proficiency leads to competitive advantage in the UK construction industry.
Fetal lamb cerebral blood flow (CBF) and oxygen tensions during hypoxia: a comparison of laser Doppler and microsphere measurements of CBF
Authors: Bishai John M. , Blood Arlin B. , Hunter Christian J. , Longo Lawrence D. , Power Gordon G.
Published: February 2003

This study was undertaken to compare microsphere and laser Doppler flowmetry techniques for the measurement of cerebral blood flow, to assess the effect of probe implantation at the tip of the sensing probe and to measure brain tissue PO2 (tPO2) in response to acute hypoxia. Fetal sheep of ≈131 days gestation (n= 8) were chronically instrumented with bilateral laser Doppler probes in the parietal cortices and catheters for injection of fluorescent microspheres. Five days after surgery fetuses were subjected to 1 h periods of baseline control breathing, hypoxia and recovery. Microspheres were injected 10 min prior to and 10, 30, 50 and 120 min after initiation of hypoxia. Microspheres were counted in four 12 mm3 tissue samples from each hemisphere, the tip of the laser Doppler probe being positioned in the centre of one of the cubes. The cube containing the probe tip was also subdivided into 4 mm3 pieces of tissue. In response to hypoxia, fetal arterial PO2 declined from 21 ± 2 to 12 ± 1 Torr and brain tissue PO2 fell from 10 ± 1 to a nadir of 1 ± 1 Torr. Each method detected a significant increase in CBF that reached a maximum after 30‐45 min, although the increase of flow measured by laser Doppler flowmetry was less than that measured by spheres after 10 and 30 min (P < 0.05). Microspheres did not detect altered flow at the probe tip or heterogeneity of flow in surrounding volumes of cortical tissue. In summary, laser Doppler flowmetry is a useful measure of continuous relative changes of CBF in the chronically instrumented fetal sheep. Flow compensations in acute hypoxia are not adequate to sustain O2 delivery, and other compensations, including reduced metabolic rate, are possible.
Evolution of regional performance after an acute anterior myocardial infarction in humans using magnetic resonance tagging
Authors: Rademakers Frank , Van de Werf Frans , Mortelmans Luc , Marchal Guy , Bogaert Jan
Published: February 2003

Regional remodelling after a left ventricular myocardial infarction is the first step in a cascade that may lead to heart failure and death. To understand better the mechanisms underlying this process, it is important to study not only the evolution in local deformation parameters but also the corresponding loading conditions. Using magnetic resonance (MR) myocardial tagging, we measured the regional contribution to ejection (regional ejection fraction) and loading (systolic blood pressure × radius of curvature (mean of short and long axes)/wall thickness) in 32 regions throughout the left ventricle (LV) in patients 1 week (1W) and 3 months (3M) after a first anterior infarction. Using positron emission tomography (PET), the LV was divided into infarct, adjacent and remote regions. In the remote regions the average deformation decreased between 1W and 3M (from 59.3 ± 5.6 to 57.9 ± 6.7 %, P < 0.05) due to an increase in loading conditions only (from 730 ± 290 to 837 ± 299 mmHg, P < 0.05). In the adjacent myocardium, no change in function was observed (49.0 ± 10.8 to 49.0 ± 6.5 %, P= n.s.), although loading increased (806 ± 297 to 978 ± 287 mmHg, P < 0.05). In the infarct region only, an increase in deformation was seen (30.7 ± 14.2 to 37 ± 6.9 %, P < 0.05), together with a higher loading level (1229 ± 422 to 1466 ± 284 mmHg, P < 0.05), which indicates a true improvement in function. The fact that MR tagging can identify both regional deformation and loading permits us to differentiate between changes due to alterations in regional loading conditions and true changes in function. After an acute myocardial infarction (MI), an improvement can be observed in the deformation‐loading relation in the adjacent and infarct regions, but the improvement is mainly in the infarct region. Using this technique, types of intervention leading to even more functional gain could be evaluated.
Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles
Authors: Pellegrino M. A. , Canepari M. , Rossi R. , D'Antona G. , Reggiani C. , Bottinelli R.
Published: February 2003

Maximum shortening velocity (V0) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V0 were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V0 were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V0 decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V0 of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation (Vf) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V0 and Vf determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres.
Centre and surround responses of marmoset lateral geniculate neurones at different temporal frequencies
Authors: Kilavik Bjørg Elisabeth , Silveira Luiz Carlos L. , Kremers Jan
Published: February 2003

The responses of marmoset lateral geniculate neurones to stimuli that were composed of a sinusoidally modulating centre stimulus and a surround that was modulated in counterphase were measured. The size of the stimulus centre was varied. These measurements were repeated at different temporal frequencies between 1 and 30 Hz. The response amplitudes and phases depended in a characteristic manner on the stimulus centre size. The response behaviour could be modelled by assuming Gaussian responsivity profiles of the cells' receptive field (RF) centres and surrounds and a phase delay in the RF surround responses, relative to the centre, enabling the description of RF centre and surround response characteristics. We found that the RF centre‐to‐surround phase difference increased linearly with increasing temporal frequency, indicating a constant delay difference of about 4.5 to 6 ms. A linear model, including low‐pass filters, a lead lag stage and a delay, was used to describe the mean RF centre and surround responses. The separate RF centre and surround responses were less band pass than the full receptive field responses of the cells. The linear model provided less satisfactory fits to M‐cell responses than to those of P‐cells, indicating additional nonlinearities.
Distribution of inspiratory drive to the external intercostal muscles in humans
Authors: De Troyer André , Gorman Robert B. , Gandevia Simon C.
Published: February 2003

The external intercostal muscles in humans show marked regional differences in respiratory effect, and this implies that their action on the lung during breathing is primarily determined by the spatial distribution of neural drive among them. To assess this distribution, monopolar electrodes were implanted under ultrasound guidance in different muscle areas in six healthy individuals and electromyographic recordings were made during resting breathing. The muscles in the dorsal portion of the third and fifth interspace showed phasic inspiratory activity with each breath in every subject. However, the muscle in the ventral portion of the third interspace showed inspiratory activity in only three subjects, and the muscle in the dorsal portion of the seventh interspace was almost invariably silent. Also, activity in the ventral portion of the third interspace, when present, and activity in the dorsal portion of the fifth interspace were delayed relative to the onset of activity in the dorsal portion of the third interspace. In addition, the discharge frequency of the motor units identified in the dorsal portion of the third interspace averaged (mean ±s.e.m.) 11.9 ± 0.3 Hz and was significantly greater than the discharge frequency of the motor units in both the ventral portion of the third interspace (6.0 ± 0.5 Hz) and the dorsal portion of the fifth interspace (6.7 ± 0.4 Hz). The muscle in the dorsal portion of the third interspace started firing simultaneously with the parasternal intercostal in the same interspace, and the discharge frequency of its motor units was even significantly greater (11.4 ± 0.3 vs. 8.9 ± 0.2 Hz). These observations indicate that the distribution of neural inspiratory drive to the external intercostals in humans takes place along dorsoventral and rostrocaudal gradients and mirrors the spatial distribution of inspiratory mechanical advantage.
Oscillations in ciliary beat frequency and intracellular calcium concentration in rabbit tracheal epithelial cells induced by ATP
Authors: Zhang Luo , Sanderson Michael J.
Published: February 2003

To investigate how Ca2+ regulates airway ciliary activity, changes in ciliary beat frequency (CBF) and intracellular calcium concentration ([Ca2+]i) of rabbit tracheal ciliated cells, in response to ATP, were simultaneously quantified with high‐speed phase‐contrast and fast fluorescence imaging. [ATP]⩽ 1 μm induced an increase in [Ca2+]i and CBF that declined to the initial basal levels and was followed by irregular brief increases in [Ca2+]i and CBF. [ATP] > 1 but < 16 μm induced a similar increase in [Ca2+]i and CBF but this was followed by oscillations in CBF and [Ca2+]i. The minimum CBF of the oscillations in CBF remained elevated above the basal rate while the minimum concentration of the [Ca2+]i oscillations returned to the basal level. The minimum and maximum CBF of the oscillations in CBF were independent of the [ATP], whereas the frequency of the oscillations in CBF was dependent on the [ATP]. Similar oscillations in CBF and [Ca2+]i were induced by ATP‐ γ ‐S. Although ADP, AMP and adenosine induced a Ca2+‐independent increase in CBF, neither ATP nor ATP‐ γ ‐S induced an increase in CBF when the Ca2+ increases were abolished by 20 μm BAPTA AM, a result suggesting that ATP hydrolysis was minimal. [ATP]≥16 μm induced a sustained elevation in CBF and only a temporary, non‐oscillating increase in [Ca2+]i. A similar response was induced by thapsigargin (2 μm). Flash photolysis of caged Ca2+ (NP‐EGTA) produced both transient and prolonged increases in [Ca2+]i which were accompanied by transient and sustained increases in CBF, respectively. From these results, we propose that CBF can be increased by a direct Ca2+ ‐dependent mechanism that generates the rapid increases in CBF associated with the oscillations or by an indirect Ca2+‐dependent mechanism that is responsible for the sustained minimum increase in CBF.
Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca2+ Sensitization in Rabbit Smooth Muscle
Authors: Kitazawa Toshio , Eto Masumi , Woodsome Terence P. , Khalequzzaman Md
Published: February 2003

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca2+ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr695 of the MLCP regulatory subunit (MYPT1) and at Thr38 of the MLCP inhibitor protein CPI‐17 results in inhibition of MLCP activity. We have previously demonstrated that CPI‐17 Thr38 phosphorylation plays an important role in G‐protein‐mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site‐ and phospho‐specific antibodies, phosphorylation of MYPT1 Thr695 and CPI‐17 Thr38 was examined along with MYPT1 Thr850, which is a non‐inhibitory Rho‐kinase site. We found that both CPI‐17 Thr38 and MYPT1 Thr850 were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α‐toxin‐permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr695 did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho‐kinase inhibitor Y‐27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr850 and CPI‐17 Thr38, respectively, in intact VD while MYPT1 Thr695 phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr695 is independent of stimulation of G‐proteins, Rho‐kinase or PKC. In the phasic PV, phosphorylation of CPI‐17 Thr38 may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI‐17 concentration, probably utilizes other Ca2+ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI‐17.
The effect of diazepam on motor cortical oscillations and corticomuscular coherence studied in man
Authors: Baker Mark R. , Baker Stuart N.
Published: February 2003

EEG recordings from sensorimotor cortex show oscillations around 10 and 20 Hz. These modulate with task performance, and are strongest during periods of steady contraction. The 20 Hz oscillations are coherent with contralateral EMG. Computer modelling suggests that oscillations arising within the cortex may be especially dependent on inhibitory systems. The benzodiazepine diazepam enhances the size of GABAA IPSPs; its effects are reversed by the antagonist flumazenil. We tested the effect of these drugs on spectral measures of EEG and EMG, whilst eight healthy human subjects performed a precision grip task containing both holding and movement phases. Either an auxotonic or isometric load was used. EEG changes following electrical stimulation of the contralateral median nerve were also assessed. The EEG power showed similar changes in all task/stimulation protocols used. Power around 20 Hz doubled at the highest dose of diazepam used (5 mg), and returned to control levels following flumazenil. EEG power at 10 Hz was by contrast little altered. The peak frequency of EEG power in both bands was not changed by diazepam. Corticomuscular coherence at ca 20 Hz was reduced following diazepam injection, but the magnitude of this effect was small (mean coherence during steady holding in the auxotonic task was 0.062 in control recordings, 0.051 after 2.5 mg and 5 mg doses of diazepam). These results imply that 20 Hz oscillations in the sensorimotor cortex are at least partially produced by local cortical circuits reliant on GABAA‐mediated intracortical inhibition, whereas 10 Hz rhythms arise by a different mechanism. Rhythms generated during different tasks, or following nerve stimulation, are likely to arise from similar mechanisms. By examining the formulae used to calculate coherence, we show that if cortical oscillations are simply transmitted to the periphery, corticomuscular coherence should increase in parallel with the ratio of EEG to EMG power. The relative constancy of coherence even when the amplitude of cortical oscillations is perturbed suggests that corticomuscular coherence itself may have a functional role in motor control.
Autonomic nervous control of myoepithelial cells and secretion in submandibular gland of anaesthetized dogs
Authors: Lung Mary A.
Published: February 2003

In dog submandibular gland, the activity of myoepithelial cells was assessed by simultaneous measurement of intraductal pressure (Pdu) and subcapsular pressure (Pca) using catheter‐tip pressure transducers; their resting values were 2.5 ± 0.21 and 3.0 ± 0.19 mmHg, respectively (n= 40). Retrograde infusion of saliva (collected from preceding parasympathetic nerve stimulation) increased Pdu (coefficient of 50 mmHg ml−1 for rates < 1 ml min−1 and 85 mmHg ml−1 for higher rates) and Pca (coefficient of 0.47 mmHg ml−1 for all rates). Blood flow changes did not affect Pdu but increased Pca (coefficient of 0.04 mmHg ml−1). Parasympathetic nerve stimulation increased Pdu but decreased Pca abruptly; the response threshold was 0.1 Hz, with maximal responses at 16 Hz. The coefficients for Pdu and Pca on salivary secretion to parasympathetic nerve stimulation in glands with spontaneous blood flow (5.3 × 10−3 and 4.87 × 10−2 ml min−1 g−1 mmHg−1) were close to their values in glands with constant‐flow vascular perfusion (4.9 × 10−3 and 3.68 × 10−2 ml min−1 g−1 mmHg−1). The finding that Pca fell despite concomitant increased blood flow suggests contraction of myoepithelial cells. Additional ductal occlusion further increased Pdu and enhanced the fall in Pca, suggesting that the myoepithelial cells can contract when distended. Atropine blocked salivary secretion and responses of Pdu and Pca to parasympathetic nerve stimulation. ACh elicited responses similar to that of parasympathetic nerve stimulation. VIP caused very scanty salivary secretion and gradual slight increases in Pdu and Pca; the change in Pca was abolished in glands with constant‐flow vascular perfusion. Hence, contraction of myoepithelial cells to parasympathetic nerve stimulation is via muscarinic receptors. Sympathetic nerve stimulation increased Pdu and decreased Pca abruptly; the response threshold was 0.1 Hz, with maximal responses at 16 Hz. The coefficients for Pdu and Pca on salivary secretion to sympathetic nerve stimulation in glands with spontaneous blood flow (3.0 × 10−3 and 3.2 × 10−3 ml min−1 g−1 mmHg−1) were similar to their values in glands with constant‐flow vascular perfusion (3.2 × 10−3 and 3.1 × 10−3 ml min−1 g−1 mmHg−1). The finding that Pca fell even in glands with constant‐flow vascular perfusion suggests contraction of myoepithelial cells. Superimposed sympathetic nerve stimulation immediately enhanced the pressure changes and secretory response to parasympathetic nerve stimulation, indicating that the two autonomic nerves act synergistically to evoke myoepithelial cell contraction. Phentolamine and prazosin but not propranolol and yohimbine blocked the sympathetic enhancement. The finding that phenylephrine, but not clonidine and isoproterenol, abruptly decreased Pca in glands with constant‐flow vascular perfusion suggests that the sympathetic activation of myoepithelial cells is via the α1‐adrenoceptors.
The effect of chronic skeletal muscle stimulation on capillary growth in the rat: are sensory nerve fibres involved?
Authors: Hudlicka Olga , Graciotti Laura , Fulgenzi Gianluca , Brown Margaret D. , Egginton S. , Milkiewicz Malgorzata , Granata Anna‐Luisa
Published: February 2003

Indirect chronic electrical stimulation of skeletal muscle activates not only efferent but also afferent nerve fibres. To investigate effects specific to this on capillary growth, one of the earliest changes, cell proliferation and capillary ultrastructure were studied in ankle flexors of rats with and without deafferentation of the stimulated side. Two weeks after preganglionic section of dorsal roots L4‐L6, the peroneal nerve was stimulated (10 Hz, 8 h day−1) for 2 or 7 days. Proliferating nuclei labelled by bromodeoxyuridine or proliferating cell nuclear antigen staining were colocalized to alkaline phosphatase‐stained capillaries (Lc) or other interstitial nuclei (Li) in frozen sections of extensor digitorum longus. Capillary fine structure was examined in extensor hallucis proprius by transmission electron microscopy. The stimulation‐induced increase in capillary and interstitial proliferation (Lc 9.9 ± 1.9 %, Li 8.8 ± 2.1 % vs. Lc 2.6 ± 0.4 %, Li 1.9 ± 0.3 % in controls, P < 0.05) was depressed at 2 days by dorsal root section (Lc 4.8 ± 0.7 %, Li 3.2 ± 0.9 %, P < 0.05), an effect likely to be mainly on fibroblasts; no depression was seen at 7 days. Dorsal root section reduced stimulation‐induced capillary endothelial swelling at both time points. In contralateral muscles of intact rats, stimulation increased interstitial cell proliferation and capillary swelling, both effects being eliminated by dorsal root section. Capillary growth induced by stimulation (24 % increase in capillary : fibre ratio at 7 days) was unaffected by deafferentation. The reduction in capillary ultrastructural changes and interstitial proliferation in both stimulated and contralateral muscles implies that stimulation of afferent fibres leads directly to release of humoral factors and/or activation via dorsal roots of fibres that release humoral substances. Contralateral muscles are an inadequate control for the effects of chronic stimulation in the intact animal.
Essential role of rho kinase in the ca2+ Sensitization of Prostaglandin F2α‐Induced Contraction of Rabbit Aortae
Authors: Ito Katsuaki , Shimomura Erika , Iwanaga Takahiro , Shiraishi Mitsuya , Shindo Kazutoshi , Nakamura Junji , Nagumo Hiromitsu , Seto Minoru , Sasaki Yasuharu , Takuwa Yoh
Published: February 2003

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC20) is an important mechanism for the Ca2+‐induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F2α (PGF2α)‐induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho‐associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF2α (10 μm) increased the phosphorylation of MLC20 and developed tension. The rho‐kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol‐ester‐induced MLC20 phosphorylation. After treatment with verapamil or chelation of external Ca2+ with EGTA, PGF2α increased the MLC20 phosphorylation and tension without an increase in [Ca2+]i, all of which were sensitive to fasudil and hydroxyfasudil. ML‐9, a MLC kinase inhibitor, quickly reversed the KCl‐induced MLC20 phosphorylation and contraction to the resting level. However, fractions of PGF2α‐induced contraction and MLC20 phosphorylation were resistant to ML‐9 but were sensitive to fasudil. Ro31‐8220 (10 μm), a PKC inhibitor, did not affect the phosphorylation of MLC20 and the tension caused by PGF2α, thus excluding the possibility of the involvement of PKC in the PGF2α‐induced MLC20 phosphorylation. PGF2α increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF2α‐induced contraction is accompanied by the inhibition of MLC20 dephosphorylation through rho kinase‐induced MBS phosphorylation, leading to Ca2+ sensitization of contraction. An actin‐associated mechanism may also be involved in the PGF2α‐induced sensitization.
Functional analysis of neurotransmission at β2‐laminin deficient terminals
Authors: Knight David , Tolley Lynn K. , Kim David K. , Lavidis Nick A. , Noakes Peter G.
Published: February 2003

β2‐Laminin is important for the formation of neuromuscular junctions in vertebrates. Previously, we have inactivated the gene that encodes for β2‐laminin in mice and observed predominantly prejunctional structural defects. In this study, we have used both intra‐ and extracellular recording methods to investigate evoked neurotransmission in β2‐laminin‐deficient mice, from postnatal day 8 (P8) through to day 18 (P18). Our results confirmed that there was a decrease in the frequency of spontaneous release, but no change in the postjunctional response to such release. Analysis of evoked neurotransmission showed an increase in the frequency of stimuli that failed to elicit an evoked postjunctional response in the mutants compared to litter mate controls, resulting in a 50 % reduction in mean quantal content at mutant terminals. Compared to littermate controls, β2‐laminin‐deficient terminals showed greater synaptic depression when subjected to high frequency stimulation. Furthermore, the paired pulse ratio of the first two stimuli was significantly lower in β2‐laminin mutant terminals. Statistical analysis of the binomial parameters of release showed that the decrease in quantal content was due to a decrease in the number of release sites without any significant change in the average probability of release. This suggestion was supported by the observation of fewer synaptic vesicle protein 2 (SV2)‐positive varicosities in β2‐laminin‐deficient terminals and by ultrastructural observations showing smaller terminal profiles and increased Schwann cell invasion in β2‐laminin mutants; the differences between β2‐laminin mutants and wild‐type mice were the same at both P8 and P18. From these results we conclude that β2‐laminin plays a role in the early structural development of the neuromuscular junction. We also suggest that transmitter release activity may act as a deterrent to Schwann cell invasion in the absence of β2‐laminin.
PSD‐95 regulates synaptic transmission and plasticity in rat cerebral cortex
Authors: Béïque Jean‐Claude , Andrade Rodrigo
Published: February 2003

PSD‐95 is one of the most abundant proteins found in the postsynaptic density of excitatory synapses. However, the precise functional role played by PSD‐95 in regulating synaptic transmission and plasticity remains undefined. To address this issue, we have overexpressed PSD‐95 in cortical pyramidal neurons in organotypic brain slices using particle‐mediated gene transfer and assessed the consequences on synaptic transmission and plasticity. The AMPA receptor/NMDA receptor (AMPAR/NMDAR) ratio of evoked EPSCs recorded at +40 mV was greater in PSD‐95‐transfected pyramidal neurons than in controls. This difference could not be accounted for by a change in rectification of AMPAR‐mediated synaptic currents since the current‐voltage curves obtained in controls and in PSD‐95‐transfected neurons were indistinguishable. However, the amplitude of AMPAR‐mediated evoked EPSCs was larger in PSD‐95‐transfected neurons compared to matched controls. Paired‐pulse ratio analysis suggested that overexpression of PSD‐95 did not alter presynaptic release probability. Transfection of PSD‐95 was further accompanied by an increase in the frequency, but not amplitude, of AMPAR‐mediated mEPSCs. Together, these results indicate that transfection of PSD‐95 increased AMPAR‐mediated synaptic transmission. Furthermore, they suggest that this phenomenon reflects an increased number of synapses expressing AMPARs rather than an increased number or function of these receptors at individual synapses. We tested the consequences of these changes on synaptic plasticity and found that PSD‐95 transfection greatly enhanced the probability of observing long‐term depression. These results thus identify a physiological role for PSD‐95 and demonstrate that this protein can play a decisive role in controlling synaptic strength and activity‐dependent synaptic plasticity.
Sympathetic neural overactivity in healthy humans after prolonged exposure to hypobaric hypoxia
Authors: Hansen Jim , Sander Mikael
Published: February 2003

Acute exposure to hypoxia causes chemoreflex activation of the sympathetic nervous system. During acclimatization to high altitude hypoxia, arterial oxygen content recovers, but it is unknown to what degree sympathetic activation is maintained or normalized during prolonged exposure to hypoxia. We therefore measured sympathetic nerve activity directly by peroneal microneurography in eight healthy volunteers (24 ± 2 years of age) after 4 weeks at an altitude of 5260 m (Chacaltaya, Bolivian Andes) and at sea level (Copenhagen). The subjects acclimatized well to altitude, but in every subject sympathetic nerve activity was highly elevated at altitude vs. sea level (48 ± 5 vs. 16 ± 3 bursts min−1, respectively, P < 0.05), coinciding with increased mean arterial blood pressure (87 ± 3 vs. 77 ± 2 mmHg, respectively, P < 0.05). To examine the underlying mechanisms, we administered oxygen (to eliminate chemoreflex activation) and saline (to reduce cardiopulmonary baroreflex deactivation). These interventions had minor effects on sympathetic activity (48 ± 5 vs. 38 ± 4 bursts min−1, control vs. oxygen + saline, respectively, P < 0.05). Moreover, sympathetic activity was still markedly elevated (37 ± 5 bursts min−1) when subjects were re‐studied under normobaric, normoxic and hypervolaemic conditions 3 days after return to sea level. In conclusion, acclimatization to high altitude hypoxia is accompanied by a striking and long‐lasting sympathetic overactivity. Surprisingly, chemoreflex activation by hypoxia and baroreflex deactivation by dehydration together could account for only a small part of this response, leaving the major underlying mechanisms unexplained.