bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation and characterization of androgen receptor knockout (ARKO) mice: An
in vivo
model for the study of androgen functions in selective tissues
Authors: Yeh Shuyuan , Tsai Meng-Yin , Xu Qingquan , Mu Xiao-Min , Lardy Henry , Huang Ko-En , Lin Hank , Yeh Shauh-Der , Altuwaijri Saleh , Zhou Xinchang , Xing Lianping , Boyce Brendan F. , Hung Min-Chi , Zhang Su , Gan Lin , Chang Chawnshang
Published: October 2002

By using a cre-lox conditional knockout strategy, we report here the generation of androgen receptor knockout (ARKO) mice. Phenotype analysis shows that ARKO male mice have a female-like appearance and body weight. Their testes are 80% smaller and serum testosterone concentrations are lower than in wild-type (wt) mice. Spermatogenesis is arrested at pachytene spermatocytes. The number and size of adipocytes are also different between the wt and ARKO mice. Cancellous bone volumes of ARKO male mice are reduced compared with wt littermates. In addition, we found the average number of pups per litter in homologous and heterozygous ARKO female mice is lower than in wt female mice, suggesting potential defects in female fertility and/or ovulation. The cre-lox ARKO mouse provides a much-needed
in vivo
animal model to study androgen functions in the selective androgen target tissues in female or male mice.
Amino acid deletions are introduced into the V2 region of gp120 during independent pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV) infections of rhesus monkeys generating variants that are macrophage tropic
Authors: Imamichi Hiromi , Igarashi Tatsuhiko , Imamichi Tomozumi , Donau Olivia K. , Endo Yasuyuki , Nishimura Yoshiaki , Willey Ronald L. , Suffredini Anthony F. , Lane H. Clifford , Martin Malcolm A.
Published: October 2002

Highly pathogenic simian immunodeficiency virus/HIV chimeric viruses (SHIVs) cause extremely rapid, irreversible, and systemic depletions of CD4
+
T lymphocytes in inoculated rhesus monkeys. In the absence of this T cell subset, virus production can be sustained for several months by tissue macrophage. During independent infections of seven animals with uncloned virus stocks, SHIV variants emerged bearing amino acid deletions that affected specific residues of the gp120 V2 loop. Some of these macrophage-phase SHIVs replicated to high levels in alveolar macrophage.
Lipid–protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy
Authors: Fernández César , Hilty Christian , Wider Gerhard , Wüthrich Kurt
Published: October 2002

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from
Escherichia coli
and dihexanoylphosphatidylcholine (DHPC) provided a detailed description of protein–detergent interactions. The NOEs were measured in 3D
15
N- and
13
C-resolved [
1
H,
1
H]-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly
2
H,
13
C,
15
N-labeled OmpX in micelles of DHPC at natural isotope abundance. In these mixed micelles the NMR structure of OmpX consists of an eight-stranded antiparallel β-barrel. The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 Å in height, which is centered about the middle of the long axis through the β-barrel. In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket. The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the β-barrel in a double-layer lipid membrane.
SIRT3, a human SIR2 homologue, is an NAD- dependent deacetylase localized to mitochondria
Authors: Onyango Patrick , Celic Ivana , McCaffery J. Michael , Boeke Jef D. , Feinberg Andrew P.
Published: October 2002

The
SIR2
(silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin
SIRT3
. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NAD
+
-dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of
SIRT3
in mitochondria involves protein deacetylation.
Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo
Authors: Hanna Lynn A. , Foreman Ruth K. , Tarasenko Illya A. , Kessler Daniel S. , Labosky Patricia A.
Published: October 2002

Critical to our understanding of the developmental potential of stem cells and subsequent control of their differentiation in vitro and in vivo is a thorough understanding of the genes that control stem cell fate. Here, we report that Foxd3, a member of the forkhead family of transcriptional regulators, is required for maintenance of embryonic cells of the early mouse embryo. Foxd3−/− embryos die after implantation at approximately 6.5 days postcoitum with a loss of epiblast cells, expansion of proximal extraembryonic tissues, and a distal, mislocalized anterior organizing center. Moreover, it has not been possible to establish Foxd3−/− ES cell lines or to generate Foxd3−/− teratocarcinomas. Chimera analysis reveals that Foxd3 function is required in the epiblast and thatFoxd3−/− embryos can be rescued by a small number of wild-type cells. Foxd3−/− mutant blastocysts appear morphologically normal and express Oct4, Sox2, andFgf4, but when placed in vitro the inner cell mass initially proliferates and then fails to expand even when Fgf4 is added. These results establish Foxd3 as a factor required for the maintenance of progenitor cells in the mammalian embryo.
N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation
Authors: Knoepfler Paul S. , Cheng Pei Feng , Eisenman Robert N.
Published: October 2002

To address the role of N-myc in neurogenesis and in nervous system tumors, it was conditionally disrupted in neuronal progenitor cells (NPCs) with a nestin-Cre transgene. Null mice display ataxia, behavioral abnormalities, and tremors that correlate with a twofold decrease in brain mass that disproportionately affects the cerebellum (sixfold reduced in mass) and the cerebral cortex, both of which show signs of disorganization. In control mice at E12.5, we observe a domain of high N-Myc protein expression in the rapidly proliferating cerebellar primordium. Targeted deletion of N-myc results in severely compromised proliferation as shown by a striking decrease in S phase and mitotic cells as well as in cells expressing the Myc target gene cyclin D2, whereas apoptosis is unaffected. Null progenitor cells also have comparatively high levels of the cdk inhibitors p27Kip1 and p18Ink4c, whereas p15Ink4b, p21Cip1, and p19Ink4d levels are unaffected. Many null progenitors also exhibit altered nuclear morphology and size. In addition, loss of N-myc disrupts neuronal differentiation as evidenced by ectopic staining of the neuron specific marker βTUBIII in the cerebrum. Furthermore, in progenitor cell cultures derived from null embryonic brain, we observe a dramatic increase in neuronal differentiation compared with controls. Thus, N-myc is essential for normal neurogenesis, regulating NPC proliferation, differentiation, and nuclear size. Its effects on proliferation and differentiation appear due, at least in part, to down-regulation of a specific subset of cyclin-dependent kinase inhibitors.
Learning support professionals: the changing role of subject specialists in UK academic libraries
Authors: Biddiscombe Richard
Published: October 2002

This article discusses the changing role of librarians in a higher education sector that is having to adapt to a new social and educational agenda. Many converged library and computing services have been created and this has speeded the process of change for librarians, particularly those who are working as subject specialists. Such librarians, particularly those working in hybrid teams with information professionals from different skill backgrounds are having to acquire expertise and undertake tasks that only tenuously relate to the training they received at the start of their careers. Their work increasingly takes them away from those others in the same profession who undertake more traditional work. A brief list of organisations that can offer training and help for these new professionals is given along with the current UK initiatives for developing support for learning and teaching from the information community.
ELAG 2001: report of a library systems seminar on integrating heterogeneous resources
Authors: Goossens Paula
Published: October 2002

The 25th annual seminar of the European Library Automation Group (ELAG) was held in Prague in June 2001. This report provides an overview of the papers presented, including those describing relevant developments in the Czech Republic, those covering the theme of the seminar (the integration and linking of electronic resources), as well as papers on the functional requirements for bibliographic records (FRBR). Workshops are a key feature of ELAG seminars and the paper also reports on those held during the 2001 meeting.
Competence‐based training, vocational qualifications and learning targets: some lessons for the Learning and Skills Council
Authors: Matlay Harry , Addis Mark
Published: October 2002

Contextualises a number of concerns related to the usage of National and Scottish Vocational Qualifications by both employers and employees in Britain. It provides an analysis of the main factors that are likely to affect the ways in which the newly formed Learning and Skills Council interacts with this well established, but controversial system of vocational qualifications.
Passport to Success
Authors: Tomlinson Nigel
Published: October 2002

Looks at the “Passport to Success” scheme introduced by the Sheffield Chamber of Commerce in a bid to address the employability skills problem among young people. States that the scheme was launched in September 2001 in partnership with a local comprehensive school with the intention of helping pupils make the transition from school into the working world with the relevant skills and abilities companies search for in potential employees. Examines the scheme and its implementation. Concludes that although the Passport to Success is still in its infancy, it has had an influence on the collaborative effort of the schools and local businesses and hopefully will go on to benefit the whole economy.
Emotion at work
Authors: Berman Brown Reva , Brooks Ian
Published: October 2002

This paper introduces the concept of the emotional climate of the workplace and explores how it both shapes and is shaped by the emotions experienced, expressed and redefined by nurses. It extracts emotional aspects of an organizational climate framework developed by Litwin and Stringer and examines these with respect to nurse’s experiences. The primary research was carried out at a general hospital NHS Trust in the East Midlands of the UK using a grounded theory methodology. The research methods included semi‐structured interviews and observation. The themes identified include many of those found by Litwin and Stringer, others which represent variations upon these, and a new set, which, when combined, identify the emotional climate of the organization. The findings have confirmed that the “experiment” of using a 33‐year‐old positivistic framework to investigate aspects of qualitative research has enabled a robust contribution to the conceptual area of emotional climate.
Best dissertation section
Authors: Thomas Cassie
Published: October 2002

Tuberculosis is the cause of a large burden of disease in less developed countries. With the development of drug resistance and the co‐epidemic of HIV, tuberculosis has already started to make a comeback in wealthier countries. The WHO’s solution to this global tuberculosis epidemic is the DOTS strategy, the implementation of which presents many problems. The two issues most common to the majority of locations are delay in presentation for treatment and non‐completion of treatment. This review looks at the reasons for these problems in the less developed world, and addresses some solutions. The main reasons for delayed presentation are: a lack of understanding about TB; the stigma associated with the disease; the inaccessibility of treatment; and a preference for private practitioners. The main reasons for non‐completion of treatment are: the stigma of the disease; a lack of information; dissatisfaction with the treatment and its delivery; and inaccessibility of treatment. Successful implementations of the DOTS strategy need to address all these issues. There is little evidence that DOT enhances treatment completion unless combined with other strategies. Community‐based, patient‐orientated DOTS appears to be an appropriate way of addressing many of these issues. The involvement of volunteers in community‐based strategies is common, but needs more research in order for this strategy to realise its full potential.
How does market orientation contribute to service firm performance?
Authors: Matear Sheelagh , Osborne Phil , Garrett Tony , Gray Brendan J.
Published: October 2002

This study utilises the inter‐relationship between market orientation and innovation in order to examine alternative mechanisms through which market orientation contributes to service firm performance. Three mechanisms (direct, mediated and moderator) are examined using regression analysis and structural equation modelling in a sample of 231 firms which develop new services. Market orientation is found to contribute to performance through a dual mechanism in that it contributes both directly and through innovation, with innovation mediating the contribution. These results emphasise that researchers should consider the inter‐relationships between multiple sources of advantage in seeking explanations of firm performance.
Environmental management of ozone in Beirut urban areas
Authors: El‐Fadel M. , Zein M. , Nuwayhid I. , Jamali D. , Sadek S.
Published: October 2002

Urban tropospheric ozone (O3) concentrations can reach 40 folds its background level due to traffic‐induced emissions and extensive industrial activities. As such, O3 has become a pervasive air pollution problem in urban areas despite major efforts to control its precursors. In this paper, O3 formation mechanisms are briefly described, countrywide emissions with emphasis on O3 precursors are summarized, and field measurements of O3 background levels and its precursors in an urban area are presented. Simulation results using a photochemical air quality model including a sensitivity analysis with respect to design ratios and mixing heights are then presented. Mitigation alternatives and policies to control O3 formation are examined in the context of country‐specific characteristics.
Limiting design criterion analysis of a relay type production line
Authors: Quintana Rolando , Wayne Skelton Gray
Published: October 2002

This research provides an ergonomic analysis of the bucket brigade production line and identifies the potential ergonomic risk factors that may result from the utilization of this production technique. Biomechanical and physiological data of the workers was collected while they worked on the traditional and bucket brigade production lines. The research identifies that while the production level of the bucket brigade system may be higher than the traditional production line, its procedure required significantly less energy expenditure by the operators. However, the difficulty and amount of work experienced by the last worker in the bucket brigade line may cause productivity problems and promote a high turnover rate at that station, and thus is the limiting design criterion of the bucket brigade production methodology from a human factors perspective.
A lotsize reduction model for just‐in‐time manufacturing systems
Authors: Yang Jiaqin , Deane Richard H.
Published: October 2002

The importance of reducing product lotsizes in converting traditional job shops into just‐in‐time (JIT) type manufacturing systems has been addressed in the literature. This paper presents a lotsize reduction model for closed stochastic production systems. The model is formulated based on an M/G/c queuing lotsize model. Product lotsize choice is related to all major components of job flow time: waiting time in queue, batch processing time, batch moving time, and finished goods warehousing time. The research is motivated by the fact that an optimal lotsize solution that minimizes only average job waiting time in the shop may not be optimal when the effects of job batch processing time, batch moving time, and batch warehousing time are also considered. There is no general closed form solution to the model due to the complexity of its nonlinear formulation. Based on the unique properties of the model, heuristic solution procedures are developed. The research demonstrates opportunities for shop managers to significantly reduce product lotsizes while minimizing total operating cost.
Robotic grasping: gripper designs, control methods and grasp configurations – a review of research
Authors: Boubekri N. , Chakraborty Pinaki
Published: October 2002

The application of robots to industrial problems often requires grasping and manipulation of the work piece. The robot is able to perform a task adequately only when it is assigned proper tooling and adequate methods of grasping and handling work pieces. The design of such a task requires an in‐depth knowledge of several interrelated subjects including: gripper design, force, position, stiffness and compliance control and grasp configurations. In this paper, we review the research finding on these subjects in order to present in a concise manner, which can be easily accessed by the designers of robot task, the information reported by the researchers, and identify based on the review, future research directions in these areas.
The corporate governance lessons of Enron
Authors: Vinten Gerald
Published: October 2002

The failure of the Enron Corporation in late 2001, apart from signalling the largest corporate bankruptcy in the USA, has also thrown up a myriad of questions about the effectiveness of contemporary accounting, auditing and corporate governance practices. There are strong historical antecedents for distrust of the corporation, latterly represented in extreme form by the anti‐capitalists. The causes of the Enron failure and the immediate response in the USA are outlined. This is followed by the response in the UK among the accountancy bodies, and the results of a comprehensive survey to assess the impact of Enron. This then leads to a comprehensive series of lessons to be learnt in the form of recommendations under the headings of serving the public interest, accounting and financial reporting, auditing, corporate governance, and education.
Who wants to be a competent director?
Authors: Heracleous Loizos , Luh Luh Lan
Published: October 2002

Awareness of the need for board and director performance evaluation is growing, but implementation has been both partial and slow. It is unusual for boards to undertake evaluations of their own performance, and more so to evaluate individual directors. Directors often feel that individual director evaluation has several drawbacks including undermining collegiality in the boardroom. There is thus a perceived attractiveness of director self‐assessments rather than peer review or outsider assessments. Self‐assessment, even though subject to a higher degree of biases than a combination of self‐assessment and peer assessment, is less likely to give rise to defensive routines and can provide a psychologically safe environment for a director to evaluate their own knowledge. Bearing in mind the above, we developed a 20‐question self‐assessment tool, which aims to assist directors in evaluating their understanding of important governance concepts and principles, as well as their legal duties as directors.
Immunochemical pulsed‐labeling characterization of intermediates during hen lysozyme oxidative folding
Authors: Jarrett Nicole M. , Djavadi‐Ohaniance Lisa , Willson Richard C. , Tachibana Hideki , Goldberg Michel E.
Published: October 2002

Previous studies have shown that reduced hen egg white lysozyme refolds and oxidizes according to a linear model, in which the number of disulfide bonds increases sequentially. In this study, we describe the kinetics of native tertiary structure formation during the oxidative‐renaturation of reduced hen egg white lysozyme, as monitored using an immunochemical pulsed‐labeling method based on enzyme‐linked immunosorbent assay (ELISA) in conjuction with two monoclonal antibodies (mAb). Each of these antibodies recognizes a separate face of the native lysozyme surface and, more importantly, each epitope is composed of discontinuous regions of the polypeptide chain. Renaturation kinetics were studied under the same refolding conditions as previous investigations of the kinetics of the regain of far‐UV CD, fluorescence, enzymatic activity, and disulfide bonds. Comparison of our results with the results from those studies showed that the immunoreactivity (i.e., the native fold) of the α‐domain appeared in intermediates containing two SS bonds only (C6–C127 and C30–C115), while the immunoreactivity of the β‐domain appeared together with the formation of the third SS bond (C64–C80). Thus, the α‐domain folds before the β‐domain during the oxidative folding of reduced lysozyme.
Substance P‐saporin lesion of neurons with NK1 receptors in one chemoreceptor site in rats decreases ventilation and chemosensitivity
Authors: Nattie Eugene E. , Li Aihua
Published: October 2002

All medullary central chemoreceptor sites contain neurokinin‐1 receptor immunoreactivity (NK1R‐ir). We ask if NK1R‐ir neurons and processes are involved in chemoreception. At one site, the retrotrapezoid nucleus/parapyramidal region (RTN/Ppy), we injected a substance P‐saporin conjugate (SP‐SAP; 0.1 pmol in 100 nl) to kill NK1R‐ir neurons specifically, or SAP alone as a control. We made measurements for 15 days after the injections in two groups of rats. In group 1, with unilateral injections made in the awake state via a pre‐implanted guide cannula, we compared responses within rats using initial baseline data. In group 2, with bilateral injections made under anaesthesia at surgery, we compared responses between SP‐SAP‐ and SAP‐treated rats. SP‐SAP treatment reduced the volume of the RTN/Ppy region that contained NK1R‐ir neuronal somata and processes by 44 % (group 1) and by 47 and 40 % on each side, respectively (group 2). Ventilation (V̇E) and tidal volume (VT) were decreased during air breathing in sleep and wakefulness (group 2;P< 0.001; two‐way ANOVA) andPa,CO2was increased (group 2;P< 0.05; Student'sttest). When rats breathed an air mixture containing 7 % CO2during sleep and wakefulness,V̇EandVTwere lower (groups 1 and 2;P< 0.001; ANOVA) and the ΔV̇Ein air containing 7 % CO2compared to air was decreased by 28‐30 % (group 1) and 17‐22 % (group 2). SP‐SAP‐treated rats also slept less during air breathing. We conclude that neurons with NK1R‐ir somata or processes in the RTN/Ppy region are either chemosensitive or they modulate chemosensitivity. They also provide a tonic drive to breathe and may affect arousal.
The acetyl group deficit at the onset of contraction in ischaemic canine skeletal muscle
Authors: Roberts Paul A. , Loxham Susan J. G. , Poucher Simon M. , Constantin‐Teodosiu Dumitru , Greenhaff Paul L.
Published: October 2002

Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant‐flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl‐coenzyme A (acetyl‐CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this ‘acetyl group deficit’. No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl‐CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl‐CoA and acetylcarnitine by ≈20‐fold (P < 0.01), ≈3‐fold (P < 0.01) and ≈4‐fold (P < 0.01), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non‐oxidative ATP resynthesis was reduced by ≈40 % (P < 0.01) and tension development was increased by ≈20 % (P < 0.05) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.
Characterization of K+ currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans
Authors: Jospin Maëlle , Mariol Marie‐Christine , Ségalat Laurent , Allard Bruno
Published: October 2002

The properties of K+ channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole‐cell configuration, depolarizations to potentials positive to −40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage‐dependent K+ currents: a fast activating and inactivating 4‐aminopyridine‐sensitive component and a slowly activating and maintained tetraethylammonium‐sensitive component. In cell‐attached patches, voltage‐dependent K+ channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mm external K+, respectively, activated at membrane potentials positive to −40 mV. Excision revealed that these channels corresponded to Ca2+‐activated K+ channels exhibiting an unusual sensitivity to internal Cl− and whose activity progressively decreased in inside‐out conditions. After complete run‐down of these channels, one third of inside‐out patches displayed activity of another Ca2+‐activated K+ channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mm external K+). In providing a detailed description of native K+ currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K+ channels in this model organism that is highly amenable to molecular and classical genetics.
Hormonal modulation of Na+ transport in rat fetal distal lung epithelial cells
Authors: Ramminger S. J. , Inglis S. K. , Olver R. E. , Wilson S. M.
Published: October 2002

Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (≈48 h) on permeable supports in medium devoid of hormones and growth factors whilst PO2 was maintained at the level found in either the fetal (23 mmHg) or the postnatal (100 mmHg) alveolar regions. The cells became incorporated into epithelial layers that generated a basal short‐circuit current (ISC) attributable to spontaneous Na+ absorption. Cells at neonatal PO2 generated larger currents than did cells at fetal PO2, indicating that this Na+ transport process is oxygen sensitive. Irrespective of PO2, isoprenaline failed to elicit a discernible change in ISC, demonstrating that β‐adrenoceptor agonists do not stimulate Na+ transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled β‐adrenoceptors are present. Further experiments showed that isoprenaline did increase ISC in cells treated (24 h) with a combination of tri‐iodothyronine (T3, 10 nm) and dexamethasone (200 nm). Studies of basolaterally permeabilised cells showed that these hormones are essential for the isoprenaline‐evoked increase in the apical membrane's Na+ conductance (GNa), whereas isoprenaline‐evoked changes in apical Cl− conductance (GCl) can occur in both control and hormone‐treated cells. Irrespective of their hormonal status, FDLE cells thus express β‐adrenoceptors that are functionally coupled to adenylate cyclase, and allow β‐adrenoceptor agonists to modulate the apical membrane's anion conductance. However, T3 and dexamethasone are needed if these receptors are to exert control over GNa. These hormones may thus play an important role in the functional maturation of the lung by allowing β‐adrenoceptor‐mediated control over epithelial Na+ channels in the apical plasma membrane.
GABAB receptor activation desensitizes postsynaptic GABAB and A1 adenosine responses in rat hippocampal neurones
Authors: Wetherington Jonathon P. , Lambert Nevin A.
Published: October 2002

Whole‐cell recordings of EPSCs and G‐protein‐activated inwardly rectifying (GIRK) currents were made from cultured hippocampal neurones to determine the effect of long‐term agonist treatment on the presynaptic and postsynaptic responses mediated by GABAB receptors (GABABRs). GABABR‐mediated presynaptic inhibition was unaffected by agonist (baclofen) treatment for up to 48 h, and was desensitized by about one‐half after 96 h. In contrast, GABABR‐mediated GIRK currents were desensitized by a similar amount after only 2 h of agonist treatment. In addition, presynaptic inhibition mediated by A1 adenosine receptors (A1Rs) was unaffected by prolonged GABABR activation, whereas A1R‐mediated GIRK currents were desensitized. Desensitization of postsynaptic GABABR and A1R responses was blocked by the GABABR antagonist (1‐(S)‐3,4‐dichlorophenylethyl)amino‐2‐(S) hydroxypropyl‐p‐benzyl‐phosphonic acid (CGP 55845A), but not by the A1R antagonist cyclopentyldipropylxanthine (DPCPX). GIRK current amplitude could be partially restored after baclofen treatment by either coapplication of baclofen and adenosine, or intracellular infusion of the non‐hydrolysable GTP analog 5′‐guanylylimidodiphosphate (Gpp(NH)p). Short‐term (4‐24 h) baclofen treatment also significantly desensitized the inhibition of postsynaptic voltage‐gated calcium channels by activation of GABABRs or A1Rs. These results show that responses mediated by GABABRs and A1Rs desensitize differently in presynaptic and postsynaptic compartments, and demonstrate the heterologous desensitization of postsynaptic A1R responses.
Electrophysiological evidence of adult human skeletal muscle fibres with multiple endplates and polyneuronal innervation
Authors: Lateva Zoia C. , McGill Kevin C. , Johanson M. Elise
Published: October 2002

Electromyographic (EMG) signals were recorded using intramuscular electrodes at six different sites in the brachioradialis muscles during voluntary isometric contractions in four subjects. The potential waveforms and discharge patterns of up to 12 simultaneously active motor units were identified from each signal using computer‐aided decomposition. Out of a total of 301 motor unit potentials identified, 23 potentials exhibited behaviour consistent with having been generated by muscle fibres that were innervated by two different motoneurons at widely separated endplates. These potentials discharged in association with two different motor units, but were blocked or delayed whenever the two motor units discharged within a few milliseconds of one another. The blocking was consistent with a collision or refractoriness when one motoneuron tried to excite the fibre while it was already conducting an action potential initiated by the other motoneuron. The delays were consistent with decreased conduction velocity associated with incomplete recovery of the fibre after a preceding action potential. From the temporal separation between the discharges of the two motoneurons that resulted in blocking, the spatial separation between the endplates was estimated to be between 26 and 44 mm. These findings challenge the classical concept of the motor unit as an anatomically distinct and functionally independent entity. It is suggested that the human brachioradialis muscle may contain both long, polyneuronally innervated fibres and short, serially linked, singly innervated fibres.
Cardiovascular and sympathetic neural responses to handgrip and cold pressor stimuli in humans before, during and after spaceflight
Authors: Fu Qi , Levine Benjamin D. , Pawelczyk James A. , Ertl Andrew C. , Diedrich André , Cox James F. , Zuckerman Julie H. , Ray Chester A. , Smith Michael L. , Iwase Satoshi , Saito Mitsuru , Sugiyama Yoshiki , Mano Tadaaki , Zhang Rong , Iwasaki Kenichi , Lane Lynda D. , Buckey Jay C. , Cooke William H. , Robertson Rose Marie , Baisch Friedhelm J. , Blomqvist C. Gunnar , Eckberg Dwain L. , Robertson David , Biaggioni Italo
Published: October 2002

Astronauts returning to Earth have reduced orthostatic tolerance and exercise capacity. Alterations in autonomic nervous system and neuromuscular function after spaceflight might contribute to this problem. In this study, we tested the hypothesis that exposure to microgravity impairs autonomic neural control of sympathetic outflow in response to peripheral afferent stimulation produced by handgrip and a cold pressor test in humans. We studied five astronauts ≈72 and 23 days before, and on landing day after the 16 day Neurolab (STS‐90) space shuttle mission, and four of the astronauts during flight (day 12 or 13). Heart rate, arterial pressure and peroneal muscle sympathetic nerve activity (MSNA) were recorded before and during static handgrip sustained to fatigue at 40 % of maximum voluntary contraction, followed by 2 min of circulatory arrest pre‐, in‐ and post‐flight. The cold pressor test was applied only before (five astronauts) and during flight (day 12 or 13, four astronauts). Mean (±s.e.m.) baseline heart rates and arterial pressures were similar among pre‐, in‐ and post‐flight measurements. At the same relative fatiguing force, the peak systolic pressure and mean arterial pressure during static handgrip were not different before, during and after spaceflight. The peak diastolic pressure tended to be higher post‐ than pre‐flight (112 ± 6 vs. 99 ± 5 mmHg, P= 0.088). Contraction‐induced rises in heart rate were similar pre‐, in‐ and post‐flight. MSNA was higher post‐flight in all subjects before static handgrip (26 ± 4 post‐ vs. 15 ± 4 bursts min−1 pre‐flight, P= 0.017). Contraction‐evoked peak MSNA responses were not different before, during, and after spaceflight (41 ± 4, 38 ± 5 and 46 ± 6 bursts min−1, all P > 0.05). MSNA during post‐handgrip circulatory arrest was higher post‐ than pre‐ or in‐flight (41 ± 1 vs. 33 ± 3 and 30 ± 5 bursts min−1, P= 0.038 and 0.036). Similarly, responses of MSNA and blood pressure to the cold pressor test were well maintained in‐flight. We conclude that modulation of muscle sympathetic neural outflow by muscle metaboreceptors and skin nociceptors is preserved during short duration spaceflight.
Characterization of the A‐type potassium current in murine gastric antrum
Authors: Amberg Gregory C. , Baker Salah A. , Koh Sang Don , Hatton William J. , Murray Keith J. , Horowitz Burton , Sanders Kenton M.
Published: October 2002

A‐type currents are rapidly inactivating potassium currents that operate at subthreshold potentials. A‐type currents have not been reported to occur in the phasic muscles of the stomach. We used conventional voltage‐clamp techniques to identify and characterize A‐type currents in myocytes isolated from the murine antrum. A‐type currents were robust in these cells, with peak current densities averaging 30 pA pF−1 at 0 mV. These currents underwent rapid inactivation with a time constant of 83 ms at 0 mV. Recovery from inactivation at −80 mV was rapid, with a time constant of 252 ms. The A‐type current was blocked by 4‐aminopyridine (4‐AP) and was inhibited by flecainide, with an IC50 of 35 μM. The voltage for half‐activation was −26 mV, while the voltage of half‐inactivation was −65 mV. There was significant activation and incomplete inactivation at potentials positive to −60 mV, which is suggestive of sustained current availability in this voltage range. Under current‐clamp conditions, exposure to 4‐AP or flecainide depolarized the membrane potential by 7‐10 mV. In intact antral tissue preparations, flecainide depolarized the membrane potential between slow waves by 5 mV; changes in slow waves were not evident. The effect of flecainide was not abolished by inhibiting enteric neurotransmission or by blocking delayed rectifier and ATP‐sensitive K+ currents. Transcripts encoding Kv4 channels were detected in isolated antral myocytes by RT‐PCR. Immunocytochemistry revealed intense Kv4.2‐ and Kv4.3‐like immunoreactivity in antral myocytes. These data suggest that the A‐type current in murine antral smooth muscle cells is likely to be due to Kv4 channels. This current contributes to the maintenance of negative resting membrane potentials.
Effect of an N‐terminus deletion on voltage‐dependent gating of the ClC‐2 chloride channel
Authors: Varela Diego , Niemeyer María Isabel , Cid L. Pablo , Sepúlveda Francisco V.
Published: October 2002

ClC‐2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16‐61 in rat ClC‐2 abolishes voltage and pH dependence in two‐electrode voltage‐clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball‐and‐chain type of mechanism in which the N‐terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole‐cell patch‐clamp measurements in mammalian cells showing hyperpolarization‐activation of rClC‐2Δ16‐61 differing only in presenting faster opening and closing kinetics than rClC‐2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin‐perforated patch experiments. The behaviour of wild‐type rClC‐2 did not differ between conventional and nystatin‐perforated patches. Similar results were obtained with ClC‐2 from guinea‐pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild‐type channel but not rClC‐2Δ16‐61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball‐and‐chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC‐2 and rClC‐2Δ16‐61 in either recording configuration, suggesting that protons act independently from possible effects of the N‐terminus on gating. Our observations of voltage‐dependent gating of the N‐terminal deleted ClC‐2 are an argument against a ball‐and‐chain mechanism for this channel.
Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells
Authors: Willoughby Debbie , Schwiening Christof J.
Published: October 2002

Our aim was to test the hypothesis that depolarization‐induced intracellular pH (pHi) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH‐sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole‐cell patch‐clamped rat cerebellar Purkinje cells. In the absence of added CO2‐HCO3−, depolarization to +20 mV for 1 s caused large (≈0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (≈0.06 pH units) and slower. The pHi shifts were smaller in the presence of 5 % CO2‐25 mm HCO3−‐buffered saline (≈0.08 pH units in the dendrites and ≈0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 μM) doubled the size of the dendritic acid shifts in the presence of CO2‐HCO3−, indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mm) inhibited the depolarization‐evoked acid shifts. We investigated more physiological pHi changes by evoking modest bursts of action potentials (≈10 s duration) in CO2‐HCO3−‐buffered saline. Such neuronal firing induced an acidification of ≈0.11 pH units in the fine dendritic regions, but only ≈0.03 pH units in the soma. There was considerable variation in the size of the pHi shifts between cells, with dendritic acid shifts as large as 0.2‐0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pHi changes (pH microdomains) might act as important signals in synaptic function.
Specific Immune Responses and Enhancement of Murine Pulmonary Clearance of
Moraxella catarrhalis
by Intranasal Immunization with a Detoxified Lipooligosaccharide Conjugate Vaccine
Authors: Jiao Xinan , Hirano Takashi , Hou Yingchun , Gu Xin-Xing
Published: October 2002

Moraxella catarrhalis
is an important human mucosal pathogen. This study investigated the effect of intranasal immunization with a detoxified-lipooligosaccharide-cross-reactive mutant of diphtheria toxin (dLOS-CRM) vaccine candidate on pulmonary clearance following an aerosol challenge of mice with
M. catarrhalis
. Intranasal immunization with dLOS-CRM plus cholera toxin induced a significantly dose-dependent increase of immunoglobulin A (IgA) and IgG in the nasal wash, lung lavage fluid, saliva, and fecal extract. In addition, serum IgG, IgM, and IgA against LOS of
M. catarrhalis
were detected. LOS-specific antibody-forming cells were found in the nasal passages, spleens, nasally associated lymphoid tissues, cervical lymph nodes, lungs, and Peyer's patches using an enzyme-linked immunospot assay. The dLOS-CRM vaccine induced a significant bacterial clearance (70 to 90%) of both homologous and heterologous strains in the lungs compared to that observed in the controls (
P
< 0.01). Intriguingly, intranasal immunization with dLOS-CRM showed a higher level of bacterial clearance compared with subcutaneous injections with dLOS-CRM. These data indicate that dLOS-CRM induces specific mucosal and systemic immunity through intranasal immunization and also provides effective bacterial clearance. On the basis of these results, we believe that dLOS-CRM should undergo continued testing to determine whether it would induce protective immune response in humans.
Sequential Immunization with Vesicles Prepared from HeterologousNeisseria meningitidisStrains Elicits Broadly Protective Serum Antibodies to Group B Strains
Authors: Moe Gregory R. , Zuno-Mitchell Patricia , Hammond Samantha N. , Granoff Dan M.
Published: October 2002

The capsular polysaccharide ofNeisseria meningitidisgroup B is an autoantigen, whereas noncapsular antigens are highly variable. These factors present formidable challenges for development of a broadly protective and safe group B vaccine. Mice and guinea pigs were sequentially immunized with three doses of micovesicles or outer membrane vesicles prepared from three meningococcal strains that were each antigenically heterologous with respect to the two major porin proteins, PorA and PorB, and the group capsular polysaccharide. The resulting antisera conferred passive protection against meningococcal group B bacteremia in infant rats and elicited complement-mediated bactericidal activity against genetically diverse group B strains that were either homologous or heterologous with respect to PorA of the strains used to prepare the vaccine. By using knockout strains, a portion of the bactericidal antibody was directed against the highly conserved protein, neisserial surface protein A (NspA). Further, an anti-NspA monoclonal antibody elicited by the sequential immunization was highly bactericidal against strains that were previously shown to be resistant to bacteriolysis by anti-NspA antibodies produced by immunization with recombinant NspA. Sequential immunization with heterologous vesicle preparations offers a novel approach to eliciting broadly protective immunity againstN. meningitidisstrains. An NspA-based vaccine prepared from protein expressed byNeisseriaalso may be more effective than the corresponding recombinant protein made inEscherichia coli.
Toxoplasma gondiiInduces Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Secretion by Human Fibroblasts: Implications for Neutrophil Apoptosis
Authors: Channon Jacqueline Y. , Miselis Kristin A. , Minns Laurie A. , Dutta Chaitali , Kasper Lloyd H.
Published: October 2002

Human neutrophils are rescued from apoptosis following incubation with once-washed, fibroblast-derivedToxoplasma gondiitachyzoites. Both infected and uninfected neutrophils are rescued, implicating a soluble mediator. In this study we investigated the origin and identity of this soluble mediator. Neutrophils were incubated either with purified tachyzoites or with conditioned medium derived fromT. gondii-infected human fibroblasts. Conditioned medium was found to be a potent stimulus that delayed neutrophil apoptosis up to 72 h, whereas purified and extensively washed tachyzoites had no effect. Delayed apoptosis correlated with up-regulation of the neutrophil antiapoptotic protein, Mcl-1, and the neutrophil interleukin 3 receptor α subunit (IL-3Rα), suggesting a role for granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF and granulocyte colony-stimulating factor (G-CSF) were measurable in conditioned medium by enzyme-linked immunosorbent assay. Neutralizing antibodies to GM-CSF and G-CSF were additive in abrogating delayed neutrophil apoptosis induced by conditioned medium. Inhibitors of Src family tyrosine kinases, Giproteins, phosphatidylinositol 3-kinase, p44erk1and p42erk2mitogen-activated protein kinases, and Jak2 kinases partially attenuated the effect of conditioned medium, consistent with a role for G-CSF and/or GM-CSF. Hence, delayed neutrophil apoptosis is mediated by GM-CSF and G-CSF secreted byT. gondii-infected human fibroblasts. This enhanced neutrophil survival may contribute to the robust proinflammatory response elicited in theT. gondii-infected host.
Modulation of a Heterologous Immune Response by the Products ofAscaris suum
Authors: Paterson Jacqueline C. M. , Garside Paul , Kennedy Malcolm W. , Lawrence Catherine E.
Published: October 2002

Helminth infections are among the most potent stimulators of Th2-type immune responses and have been widely demonstrated to modify responsiveness to both nonparasite antigens and other infectious agents in a nonspecific manner in infected animals. We investigated the immunomodulatory properties of pseudocoelomic body fluid from adultAscaris suumgastrointestinal helminths (ABF) and its defined allergen (ABA-1) by examining their effects on the immune response to a heterologous antigen, ovalbumin. Our results indicate that ABF has potent immunomodulatory activity and that the effects observed are consistent with skewing towards a Th2-type response rather than induction of anergy. Our findings show that the immunomodulatory activities of ABF are associated with components other than the major constituent and putative allergen, ABA-1. Furthermore, the allergic responses to ABA-1 are not a result of an intrinsic allergenicity of the protein but are more a reflection of the wider induction of a Th2 response by the infection. Importantly, the induction of interleukin-10 by ABF also suggests that T regulatory cells may play a role in immunomodulation of immune responses by parasitic helminths.