bluuebunny/crossref_metadata_2025 huggingface dataset:
Antisepsis for abdominal hysterectomy: a randomised controlled trial of povidone–iodine gel
Authors: Eason E. , Wells G. , Garber G. , Hemmings R. , Luskey G. , Gillett P. , Martin M. ,   for the Vaginal Antisepsis For Abdominal Hysterectomy Study Group (other members listed in the Acknowledgements).
Published: December 2004

Objective  To assess whether infectious morbidity after total abdominal hysterectomy is decreased by the addition of 20 cc povidone–iodine gel at the vaginal apex after the usual vaginal preparation with povidone–iodine solution.

Study design  Randomised controlled trial.

Setting  Fifteen secondary and tertiary hospitals in Canada.

Sample  A total of 1570 women undergoing planned total abdominal hysterectomy.

Methods  Computer‐generated randomisation using a centralised telephone service was stratified by study centre with variable block size. In the operating room, a swab for bacterial vaginosis was taken before vaginal antisepsis. Study group remained concealed until the standard surgical preparation in the operating room was complete. Then povidone‐iodine gel 20 cc was placed at the vaginal apex in the intervention group only. Participants were followed for one month post‐operative.

Main outcome measures  The primary outcome was post‐operative infectious morbidity during the 30 days after surgery, defined as: febrile morbidity with hospital stay greater than five days or antibiotic treatment, or infection requiring readmission to hospital or additional visit. Other outcomes included abdominal wound infection, pelvic abscess and other pelvic infections.

Results  Post‐operative infectious morbidity within 30 days occurred in 128/780 (16%) women receiving povidone–iodine gel preparation and 142/790 (18%) women not receiving gel (RR 0.9, 95% CI 0.7 to 1.1). Pelvic abscess was diagnosed in 0 patients in the gel group and in seven patients in the control group (P < 0.01). No significant difference was found in pelvic cellulitis (eight in each group) or abdominal wound infection (51 in the gel group and 58 in the control group, P= 0.5).

Conclusion  Povidone–iodine vaginal gel antisepsis led to a 9% relative decrease in overall infectious morbidity after abdominal hysterectomy, which was not statistically significant. Povidone–iodine vaginal gel decreased the risk of pelvic abscess after total abdominal hysterectomy.
Pretreatment tumour volume measurement on high‐resolution magnetic resonance imaging as a predictor of survival in cervical cancer
Authors: Soutter W. Patrick , Hanoch Joseph , D'Arcy Tom , Dina Roberto , McIndoe G. Angus , DeSouza Nandita M.
Published: December 2004

Objective  To evaluate pretreatment tumour volume as a predictor of survival in patients with cervical cancer using both endovaginal and external coil magnetic resonance imaging in order to achieve high spatial resolution and delineate small volume disease.

Design  A retrosfection case series.

Setting  A tertiary referral centre for gynaecological oncology.

Population/Sample  One hundred and six consecutive women with invasive carcinoma of the cervix underwent endovaginal and external coil magnetic resonance imaging on a 0.5‐T or 1.5‐T scanner.

Methods  T2‐W FSE images, sagittal and transverse to the cervix, were obtained and tumour volume was calculated on the sagittal images by the standard technique of multiplying the sum of the areas by the slice thickness. Patients were treated in accordance with normal clinical practice and their subsequent outcome was recorded. The relationships between clinical or imaging parameters and survival were assessed with Cox's proportional hazard method.

Main outcome measures  Disease‐free survival.

Results  In 89 of these women, the tumour was Stage I and 88 of the 106 were treated principally by surgery. The median tumour volume was 4.75 cm3 (upper and lower quartiles 22 and 0.6). The median length of follow up of surviving patients was 223 weeks (quartiles 158 and 274 weeks). Stage, treatment type, lymphovascular space involvement, invasion of the parametrium, closeness of the excision margin, lymph node metastases, and magnetic resonance imaging measurements of tumour volume, parametrial invasion and lymph node disease were all significantly associated with survival in univariate analysis. Only magnetic resonance imaging measurement of tumour volume remained consistently and strongly associated with survival after multivariate analysis of parameters available prior to treatment (P= 0.001, Wald statistic 10.74). A receiver operating characteristic curve of tumour volume and disease‐free survival confirmed the utility of this investigation and suggested that a cutoff around 13.0 cm3 would predict survival with a positive predictive value of 0.93 and a negative predictive value of 0.75.

Conclusion  Magnetic resonance imaging assessment of tumour volume using both an endovaginal and an external coil approach provides an accurate prediction of prognosis in cervical cancer and defines a population of women at high risk of recurrence and death. The predictive value of this investigation is superior to the clinical and histological parameters previously used. Use of this technique permits a more accurate choice of treatment options. These results suggest that it is the size of tumour burden that determines the outcome rather than invasion beyond the anatomical margins of the uterus.
Effects of Climate Change on Population Persistence of Desert‐Dwelling Mountain Sheep in California
Authors: EPPS CLINTON W. , McCULLOUGH DALE R. , WEHAUSEN JOHN D. , BLEICH VERNON C. , L. RECHEL JENNIFER
Published: December 2004

Abstract: Metapopulations may be very sensitive to global climate change, particularly if temperature and precipitation change rapidly. We present an analysis of the role of climate and other factors in determining metapopulation structure based on presence and absence data. We compared existing and historical population distributions of desert bighorn sheep (Ovis canadensis) to determine whether regional climate patterns were correlated with local extinction. To examine all mountain ranges known to hold or to have held desert bighorn populations in California and score for variables describing climate, metapopulation dynamics, human impacts, and other environmental factors, we used a geographic information system (GIS) and paper maps. We used logistic regression and hierarchical partitioning to assess the relationship among these variables and the current status of each population (extinct or extant). Parameters related to climate—elevation, precipitation, and presence of dependable springs—were strongly correlated with population persistence in the twentieth century. Populations inhabiting lower, drier mountain ranges were more likely to go extinct. The presence of domestic sheep grazing allotments was negatively correlated with population persistence. We used conditional extinction probabilities generated by the logistic‐regression model to rank native, naturally recolonized, and reintroduced populations by vulnerability to extinction under several climate‐change scenarios. Thus risk of extinction in metapopulations can be evaluated for global‐climate‐change scenarios even when few demographic data are available.
Downstream Effects of Erosion from Small‐Scale Gold Mining on the Instream Habitat and Fish Community of a Small Neotropical Rainforest Stream
Authors: MOL JAN H. , OUBOTER PAUL E.
Published: December 2004

Abstract:  Small‐scale gold mining has caused widespread siltation of South American streams, but little information is available on the effects of an increased load of suspended and deposited sediment on diverse Neotropical fish communities. We used a paired watershed design to compare the instream habitat and fish community structure of an undisturbed rainforest stream and a stream affected by gold mining. In the period 1994–2001, the affected stream showed high turbidity (424–2874 nephelometric turbidity units),
high concentration of suspended sediment (318–2468 mg/L), and elevated concentrations of potassium, aluminum, and iron. Mercury was detected in the low‐water season (0.67 μg/L). The main fractions of P, Al, and Fe were associated with suspended sediment and were not in solution. The sediment load of the polluted stream (3.10 ± 0.77 tonnes/year/ha) was mainly (95.6%) produced by the eroding goldfields. Sedimentation was evident from the accumulation of a 23‐cm‐thick layer of fine sediment (<100 μm) on the streambed. The deposited sediment covered most structural elements of the streambed and thereby reduced instream habitat diversity. The fish community of the stream affected by gold mining showed low species diversity, low proportion of young fishes, high proportion of midchannel surface‐feeding fishes and fishes adapted to low light, low proportion of visually orienting fishes and fishes that hide in leaf‐litter banks and woody debris, and low relative biomass of food fishes. These results suggest that erosion related to small‐scale gold mining reduces fish diversity and shifts community structure because of (1) increased turbidity resulting from an increased load of suspended sediment and (2) a reduction of instream habitat diversity related to sedimentation. Gold mining occurred over 7 years and is still occurring, but the sediment stored in the streambed is expected to pose a pollution problem long after the gold miners have left the area.
Predicting Human‐Carnivore Conflict: a Spatial Model Derived from 25 Years of Data on Wolf Predation on Livestock
Authors: TREVES ADRIAN , NAUGHTON‐TREVES LISA , HARPER ELIZABETH K. , MLADENOFF DAVID J. , ROSE ROBERT A. , SICKLEY THEODORE A. , WYDEVEN ADRIAN P.
Published: December 2004

Abstract:  Many carnivore populations escaped extinction during the twentieth century as a result of legal protections, habitat restoration, and changes in public attitudes. However, encounters between carnivores, livestock, and humans are increasing in some areas, raising concerns about the costs of carnivore conservation. We present a method to predict sites of human‐carnivore conflicts regionally, using as an example the mixed forest‐agriculture landscapes of Wisconsin and Minnesota (U.S.A.). We used a matched‐pair analysis of 17 landscape variables in a geographic information system to discriminate affected areas from unaffected areas at two spatial scales (townships and farms). Wolves (Canis lupus) selectively preyed on livestock in townships with high proportions of pasture and high densities of deer (Odocoileus virginianus) combined with low proportions of crop lands, coniferous forest, herbaceous wetlands, and open water. These variables plus road density and farm size also appeared to predict risk for individual farms when we considered Minnesota alone. In Wisconsin only, farm size, crop lands, and road density were associated with the risk of wolf attack on livestock. At the level of townships, we generated two state‐wide maps to predict the extent and location of future predation on livestock. Our approach can be applied wherever spatial data are available on sites of conflict between wildlife and humans.
Genetic Effects of Habitat Fragmentation on Common Species of Swiss Fen Meadows
Authors: HOOFTMAN DANNY A. P. , BILLETER REGULA C. , SCHMID BERNHARD , DIEMER MATTHIAS
Published: December 2004

Abstract: The area of Caricion davallianae alliance in Switzerland has been considerably reduced and fragmented during the last 150 years. We assessed the genetic variability, inbreeding level, and among‐population differentiation of two common habitat‐specific plant species,Carex davallianaSM. andSuccisa pratensisMoench, in 18 Caricion davallianae fen meadows subjected to fragmentation. We used a spatial field design of fen systems (six systems total), each consisting of one large habitat island and two small habitat islands. We used allozyme electrophoresis to derive standard genetic parameters (A, P, HO, HE,FIS,FST). InCarexwe identified a consistently lower A in isolated habitat islands; furthermore,HEwas lower in small habitat islands than in large habitat islands. In Succisa we identified a lowerHOin small habitat islands than in larger ones. Small habitat islands were marginally significantly differentiated ( FST) from large islands forSuccisa. For both species, no effects were evident forFIS; therefore, we argue that genetic drift rather than inbreeding is the main cause of the observed differences. The genetic structure ofCarexandSuccisain small habitat islands differed from that in large habitat islands, but differences were small. It appears that the observed differences in genetic variability among fen meadows correspond to observed differences in fitness and demographic traits. We show that habitat fragmentation affects not only the rare species in an ecosystem but also reduces the survival probabilities of common species. One of the main goals of conservation should be to mitigate fragmentation of natural habitats in order to increase population sizes and connectivity.
Effects of Forest Management on Amphibians and Reptiles in Missouri Ozark Forests
Authors: RENKEN ROCHELLE B. , GRAM WENDY K. , FANTZ DEBRA K. , RICHTER STEPHEN C. , MILLER TIMOTHY J. , RICKE KEVIN B. , RUSSELL BRADLEY , WANG XIAOYIN
Published: December 2004

Abstract: As part of the Missouri Ozark Forest Ecosystem Project (MOFEP), we experimentally evaluated the impacts of forest management on the relative abundance of amphibians and reptiles in Missouri's Ozark forests (U.S.A.). Using large study sites (average size of 400 ha) as the experimental unit, we tested the effects of uneven‐aged and even‐aged forest management treatments compared with no‐harvest management (i.e., control) on the relative abundance of 13 focal amphibian and reptile species. Within even‐aged management sites, we also focused on the local‐scale effects of clearcutting on these species by comparing relative abundance among plots located within clearcut stands, 50 m away from clearcut stands, and 200 m away from clearcut stands. Pretreatment sampling of species abundance occurred from 1992 through 1995, and post‐treatment sampling occurred from 1997 through 2000. At the landscape scale, treatment significantly affected the abundance of Bufo americanus. This species declined less on even‐aged management sites than on control sites, but the general decline on all sites suggests that other factors may have contributed to this result. Within even‐aged management sites, most amphibian species declined and some reptile species increased relative to pretreatment abundances within clearcut stands. We found significant effects of distance from clearcut for two amphibian species, Ambystoma maculatum and Rana clamitans, and two reptile species, Scincella lateralis and Sceloporus undulatus. In general, we conclude that clearcuts within even‐aged management sites locally affected amphibian and reptile species but, at a larger spatial scale, we did not detect significant effects of even‐aged and uneven‐aged forest management. These findings represent relatively short‐term data but suggest that forest management and maintenance of biodiversity may be compatible when relatively small amounts of the landscape are disturbed.
Land Trusts and Conservation Easements: Who Is Conserving What for Whom?
Authors: MERENLENDER A. M. , HUNTSINGER L. , GUTHEY G. , FAIRFAX S. K.
Published: December 2004

Abstract:  Land trusts, partnered with government agencies or acting alone, are working to conserve habitat, open space, and working landscapes on private land. Spending both public and private funds, such institutions frequently acquire less than full title by purchasing or accepting donations of conservation easements. These title and organizational arrangements are evolving so fast that it is difficult to assess their conservation accomplishments and long‐term viability. To understand the contribution of these arrangements to the preservation and restoration of biodiversity, conservation biologists need to identify the biological resources likely to be conserved and those likely to be left unprotected through easements held by land trusts. We describe land trusts and conservation easements and why they are currently an attractive approach to land protection. Our review of the literature showed that little information is available on (1) the resulting pattern of protected lands and resources being conserved, (2) the emerging institutions that hold conservation easements and the landowners they work with, and (3) the distribution of costs and benefits of land trusts and easements to communities and the general public. The prescriptive literature on how to establish land trusts and negotiate easements is extensive. However, easily available information on protected resources is too aggregated to determine what is actually being conserved, and more detailed data is widely scattered and hence difficult to synthesize. The social science literature provides some insight into the motives of landowners who participate but offers little about the variety of institutions or which type of institution works best in particular ecological and political settings. Equally undeveloped is our understanding of the inherent tension between the public and private benefits of this widely used incentive‐based conservation strategy. Interdisciplinary research is needed to determine the ecological and social consequences of acquiring partial interest in private land for conservation purposes.
Using Decision Modeling with Stakeholders to Reduce Human–Wildlife Conflict: a Raptor–Grouse Case Study
Authors: REDPATH S. M. , ARROYO B. E. , LECKIE F. M. , BACON P. , BAYFIELD N. , GUTIÉRREZ R. J. , THIRGOOD S. J.
Published: December 2004

Abstract:  The successful resolution of human‐wildlife conflicts requires the participation of local communities and other stakeholder groups in formulating management decisions. In the uplands of the United Kingdom, a controversial conservation issue concerns the relationship between the conservation of a legally protected raptor, the Hen Harrier (Circus cyaneus) and the management of a gamebird, the Red Grouse (Lagopus lagopus scoticus). We used multicriteria analysis to evaluate the perspectives of two groups of stakeholders, grouse managers and raptor conservationists, and the acceptability to them of different management solutions to this conflict. Both groups quantified the relative importance of evaluation criteria and used these as a basis for comparing different upland and Hen Harrier management options. In relation to upland management, grouse managers placed more importance on economic criteria than did raptor conservationists, who valued natural‐environment criteria more highly. Intensively managed grouse moors, involving the control of harrier numbers, were ranked most highly by grouse managers and managed nature reserves by raptor conservationists, but both groups also ranked legally managed grouse moors highly. When evaluating Hen Harrier management options, grouse managers considered time scale and cost the most important criteria, whereas raptor conservationists considered the effects on harrier populations to be most important. Harrier quota schemes were the management solution most favored by grouse managers, whereas raptor conservationists preferred allowing harriers to attain natural densities. Notably, however, one technique that has already been partly tested in the field—the use of diversionary feeding was scored highly by both groups and thus holds promise for some form of compromise. This exercise highlighted the value of these objective techniques for developing dialog and trust between stakeholder groups, and it highlighted the need to conduct further research to test the effectiveness of different management options. There was broad agreement that the workshop moved the prior positions of individual stakeholders and was a valuable tool in helping to resolve human‐wildlife conflicts.
Wildfire, Fuel Reduction, and Herpetofaunas across Diverse Landscape Mosaics in Northwestern Forests
Authors: BURY R. BRUCE
Published: December 2004

Abstract:  The herpetofauna (amphibians and reptiles) of northwestern forests (U.S.A.) is diverse, and many species are locally abundant. Most forest amphibians west of the Cascade Mountain crest are associated with cool, cascading streams or coarse woody material on the forest floor, which are characteristics of mature forests. Extensive loss and fragmentation of habitat resulted from logging across approximately 50% of old‐growth forests in northern California and approximately 80% of stands in Oregon and Washington. There is a complex landscape mosaic and overlap of northern and southern biotic elements in the Klamath‐Siskiyou Region along the Oregon and California border, creating a biodiversity hotspot. The region experiences many low‐severity fires annually, punctuated by periodic major fires, including the Biscuit fire, the largest in North America in 2002. In the fire's northern portion, severe fire occurred on >50% of stands of young, managed trees but on only about 25–33% of old‐growth stands. This suggests that the legacy of timber harvest may produce fire‐prone stands. Calls for prescribed fire and thinning to reduce fuel loads will remove large amounts of coarse woody material from forests, which reduces cover for amphibians and alters nutrient inputs to streams. Our preliminary evidence suggests no negative effects of wildfire on terrestrial amphibians, but stream amphibians decrease following wildfire. Most reptiles are adapted to open terrain, so fire usually improves their habitat. Today, the challenge is to maintain biodiversity in western forests in the face of intense political pressures designed to “prevent” catastrophic fires. We need a dedicated research effort to understanding how fire affects biota and to proactively investigate outcomes of fuel‐reduction management on wildlife in western forests.
A Multiscale Landscape Approach to Predicting Bird and Moth Rarity Hotspots in a Threatened Pitch Pine–Scrub Oak Community
Authors: GRAND JOANNA , BUONACCORSI JOHN , CUSHMAN SAMUEL A. , GRIFFIN CURTICE R. , NEEL MAILE C.
Published: December 2004

Abstract:  In the northeastern United States, pitch pine ( Pinus rigida Mill.)–scrub oak (Quercus ilicifolia Wang.) communities are increasingly threatened by development and fire suppression, and prioritization of these habitats for conservation is of critical importance. As a basis for local conservation planning in a pitch pine–scrub oak community in southeastern Massachusetts, we developed logistic‐regression models based on multiscale landscape and patch variables to predict hotspots of rare and declining bird and moth species. We compared predicted moth distributions with observed species‐occurrence records to validate the models. We then quantified the amount of overlap between hotspots to assess the utility of rare birds and moths as indicator taxa. Species representation in hotspots and the current level of hotspot protection were also assessed. Predictive models included variables at all measured scales and resulted in average correct classification rates (optimal cut point) of 85.6% and 89.2% for bird and moth models, respectively. The majority of moth occurrence records were within 100 m of predicted habitat. Only 13% of all bird hotspots and 10% of all moth hotspots overlapped, and only a few small patches in and around Myles Standish State Forest were predicted to be hotspots for both taxa. There was no correlation between the bird and moth species‐richness maps across all levels of richness (r =−0.03, p = 0.62). Species representation in hotspots was high, but most hotspots had limited or no protection. Given the lack of correspondence between bird and moth hotspots, our results suggest that use of species‐richness indicators for conservation planning may be ineffective at local scales. Based on these results, we suggest that local‐level conservation planning in pitch pine–scrub oak communities be based on multitaxa, multiscale approaches.
Contributions of the input signal and prior activation history to the discharge behaviour of rat motoneurones
Authors: Powers R. K. , Dai Y. , Bell B. M. , Percival D. B. , Binder M. D.
Published: December 2004

The principal computational operation of neurones is the transformation of synaptic inputs into spike train outputs. The probability of spike occurrence in neurones is determined by the time course and magnitude of the total current reaching the spike initiation zone. The features of this current that are most effective in evoking spikes can be determined by injecting a Gaussian current waveform into a neurone and using spike‐triggered reverse correlation to calculate the average current trajectory (ACT) preceding spikes. The time course of this ACT (and the related first‐order Wiener kernel) provides a general description of a neurone's response to dynamic stimuli. In many different neurones, the ACT is characterized by a shallow hyperpolarizing trough followed by a more rapid depolarizing peak immediately preceding the spike. The hyperpolarizing phase is thought to reflect an enhancement of excitability by partial removal of sodium inactivation. Alternatively, this feature could simply reflect the fact that interspike intervals that are longer than average can only occur when the current is lower than average toward the end of the interspike interval. Thus, the ACT calculated for the entire spike train displays an attenuated version of the hyperpolarizing trough associated with the long interspike intervals. This alternative explanation for the characteristic shape of the ACT implies that it depends upon the time since the previous spike, i.e. the ACT reflects both previous stimulus history and previous discharge history. The present study presents results based on recordings of noise‐driven discharge in rat hypoglossal motoneurones that support this alternative explanation. First, we show that the hyperpolarizing trough is larger in ACTs calculated from spikes preceded by long interspike intervals, and minimal or absent in those based on short interspike intervals. Second, we show that the trough is present for ACTs calculated from the discharge of a threshold‐crossing neurone model with a postspike afterhyperpolarization (AHP), but absent from those calculated from the discharge of a model without an AHP. We show that it is possible to represent noise‐driven discharge using a two‐component linear model that predicts discharge probability based on the sum of a feedback kernel and a stimulus kernel. The feedback kernel reflects the influence of prior discharge mediated by the AHP, and it increases in amplitude when AHP amplitude is increased by pharmacological manipulations. Finally, we show that the predictions of this model are virtually identical to those based on the first‐order Wiener kernel. This suggests that the Wiener kernels derived from standard white‐noise analysis of noise‐driven discharge in neurones actually reflect the effects of both stimulus and discharge history.
Central 5‐HT7receptors are critical for reflex activation of cardiac vagal drive in anaesthetized rats
Authors: Kellett Daniel O. , Ramage Andrew G. , Jordan David
Published: December 2004

5‐Hydroxytryptamine (5‐HT; serotonin)‐containing neurones contribute to reflex activation of parasympathetic outflow in a number of species, but the 5‐HT receptors mediating these effects have yet to be fully determined. The present experiments demonstrate that central 5‐HT7receptors are involved in the vagal bradycardia evoked during the cardiopulmonary reflex, baroreflexes and the chemoreflex, as well as other autonomic changes caused by these reflexes. The experiments examined the effects of the selective 5‐HT7receptor antagonists SB‐269970 and SB‐656104 on these reflexes. For the cardiopulmonary reflex, when compared to time‐matched vehicle control experiments, intracisternal application of SB‐269970 (30–300 μg kg−1,i.c.) dose‐dependently attenuated the evoked bradycardia. At the highest dose, SB‐269970 also attenuated the reflex hypotension and sympathoinhibition. The structurally different 5‐HT7receptor antagonist SB‐656104 (100 μg kg−1,i.c.) similarly attenuated the reflex bradycardia and hypotension. SB‐269970 (100 μg kg−1,i.c.) also attenuated the bradycardias evoked by electrical stimulation of aortic nerve afferents and the baroreflex evoked by the pressor response to phenylephrine (3–25 μg kg−1,i.v.). The gain of the baroreflex was also significantly attenuated (0.15 ± 0.06versus0.34 ± 0.06 ms mmHg−1). Finally, SB‐269970 (100 μg kg−1,i.c.) significantly attenuated both the bradycardia and sympathoexcitation evoked by the chemoreflex. These data indicate that central 5‐HT7receptors play an important facilitatory role in the reflex activation of vagal outflow to the heart.
Allosteric activation of sodium–calcium exchange by picomolar concentrations of cadmium
Authors: Dinh Le Hoa , Omelchenko Alexander , Hryshko Larry V. , Uliyanova Alexandra , Condrescu Madalina , Reeves John P.
Published: December 2004

Chinese hamster ovary cells expressing the bovine cardiac Na+–Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol. The lag period was greatly reduced in cells expressing a mutant exchanger, Δ(241‐680), that does not require allosteric activation by Ca2+ for activity. Non‐transfected cells did not show Cd2+ uptake under the same conditions. In cells expressing NCX1.1, the lag period was nearly abolished following an elevation of the cytosolic Ca2+ concentration. Cytosolic Cd2+ concentrations estimated at 0.5–2 pm markedly stimulated the subsequent uptake of Ca2+ by Na+–Ca2+ exchange. Outward exchange currents in membrane patches from Xenopus oocytes expressing the canine NCX1.1 were rapidly and reversibly stimulated by 3 pm Cd2+ applied at the cytosolic membrane surface. Exchange currents activated by 3 pm Cd2+ were 40% smaller than currents activated by 1 μm cytosolic Ca2+. Current amplitudes declined by 30% and the rate of current development fell sharply upon repetitive applications of Na+ in the presence of 3 pm Cd2+. Cd2+ mimicked the anomalous inhibitory effects of Ca2+ on outward exchange currents generated by the Drosophila exchanger CALX1.1. We conclude that the regulatory sites responsible for allosteric Ca2+ activation bind Cd2+ with high affinity and that Cd2+ mimics the regulatory effects of Ca2+ at concentrations 5 orders of magnitude lower than Ca2+.
Delayed expression of large conductance K+ channels reshaping agonist‐induced currents in mouse pancreatic acinar cells
Authors: Oshiro Takako , Takahashi Hidenori , Ohsaga Atsushi , Ebihara Satoru , Sasaki Hidetada , Maruyama Yoshio
Published: December 2004

Epithelial secretory cells display cell‐specific mechanisms of fluid secretion and express large conductance voltage‐ and Ca2+‐activated K+ (Maxi‐K) channels that generate the membrane negativity for effective Cl− exit to the lumen. Rat and mouse pancreatic acinar cells had been thought to be peculiar in this sense because of the previously reported lack of Maxi‐K channels. However, this view is not entirely correct as evidenced in the present paper. Searching for their presence in pancreatic acinar cells in mice from 5 to 84 weeks of age with patch‐clamp current measurements, we demonstrated that the expression of Maxi‐K channels is regulated in an age‐associated manner after birth. The expression started at approximately 12 postnatal weeks and increased steadily up to 84 weeks. In support of this, RT‐PCR could not detect mSlo mRNA, the Maxi‐K gene, at either 7 or 8 weeks but could at 58 and 64 postnatal weeks. These results suggest that a key steering element for fluid secretion, the Maxi‐K channel, is progressively re‐organized in rodent pancreas. A pancreatic secretagogue, acetylcholine, evoked Maxi‐K channel current overlapping to various degrees on the previously known current response. This suggests that the rise in internal Ca2+ activates Maxi‐K channels which reshape the mode of secretagogue‐evoked current response and contribute to Cl− driving in fluid secretion in an age‐associated fashion.
Increased glomerular angiotensin II binding in rats exposed to a maternal low protein diet in utero
Authors: Sahajpal Vandana , Ashton Nick
Published: December 2004

In the rat, protein restriction during pregnancy increases offspring blood pressure by 20–30 mmHg. We have shown in an earlier study that this is associated with a reduction in nephron number and increased glomerular sensitivity to angiotensin II (Ang II) in vivo. Hence, we hypothesized that exposure to a maternal low‐protein diet increases glomerular Ang II AT1 receptor expression and decreases AT2 receptor expression. To test this hypothesis, pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (LP) protein from conception until birth. At 4 weeks of age, the kidneys of male offspring were harvested to measure cortical AT1 and AT2 receptor expression, 125I‐Ang II glomerular binding, tissue renin activity, tissue Ang II and plasma aldosterone concentrations. AT1 receptor expression was increased (62%) and AT2 expression was decreased (35%) in LP rats. Maximum 125I‐Ang II (125I‐Ang II) binding (Bmax) was increased in LP rats (control n= 9, 291.6 ± 27.4 versus LP n= 7, 445.7 ± 27.4 fmol (mg glomerular protein)−1, P < 0.01), but affinity (KD) was not statistically different from controls (control 2.87 ± 0.85 versus LP 0.84 ± 0.20 pmol 125I‐Ang II, P= 0.059). Renal renin activity, tissue Ang II and plasma aldosterone concentrations did not differ between control and LP rats. Increased AT1 receptor expression in LP rat kidneys is consistent with greater haemodynamic sensitivity to Ang II in vivo. This may result in an inappropriate reduction in glomerular filtration rate, salt and water retention, and an increase in blood pressure.
Selectivity and interactions of Ba2+ and Cs+ with wild‐type and mutant TASK1 K+ channels expressed in Xenopus oocytes
Authors: O'Connell Anthony D. , Morton Michael J. , Sivaprasadarao Asipu , Hunter Malcolm
Published: December 2004

The acid‐sensitive K+ channel, TASK1 is a member of the K+‐selective tandem‐pore domain (K2P) channel family. Like many of the K2P channels, TASK1 is relatively insensitive to conventional channel blockers such as Ba2+. In this paper we report the impact of mutating the pore‐neighbouring histidine residues, which are involved in pH sensing, on the sensitivity to blockade by Ba2+ and Cs+; additionally we compare the selectivity of these channels to extracellular K+, Na+ and Rb+. H98D and H98N mutants showed reduced selectivity for K+ over both Na+ and Rb+, and significant permeation of Rb+. This enhanced permeability must reflect changes in the structure or flexibility of the selectivity filter. Blockade by Ba2+ and Cs+ was voltage‐dependent, indicating that both ions block within the pore. In 100 mm K+, the KD at 0 mV for Ba2+ was 36 ± 10 mm(n= 6), whilst for Cs+ it was 20 ± 6.0 mm(n= 5). H98D was more sensitive to Ba2+ than the wild‐type (WT); in addition, the site at which Ba2+ appears to bind was altered (WT: δ, 0.64 ± 0.16, n= 6; H98D: δ, 0.16 ± 0.03, n= 5, statistically different from WT; H98N: δ, 0.58 ± 0.09, not statistically different from WT). Thus, the pore‐neighbouring residue H98 contributes not only to the pH sensitivity of TASK1, but also to the structure of the conduction pathway.
Advances in protein complex analysis using mass spectrometry
Authors: Gingras Anne‐Claude , Aebersold Ruedi , Raught Brian
Published: December 2004

Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein–protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)‐based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal‐to‐noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented.
Candidate‐based proteomics in the search for biomarkers of cardiovascular disease
Authors: Anderson Leigh
Published: December 2004

The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers of disease, treatment response and ageing. As the number of proteins that can be detected in plasma or serum (the primary clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease and stroke are presented as a starting point for such a ‘directed proteomics’ approach.
In Vitro and In Vivo Mutational Analysis of the 3′-Terminal Regions of Hepatitis E Virus Genomes and Replicons
Authors: Graff Judith , Nguyen Hanh , Kasorndorkbua Chaiyan , Halbur Patrick G. , St. Claire Marisa , Purcell Robert H. , Emerson Suzanne U.
Published: December 2004

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3′-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3′-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3′-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3′ termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3′ terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3′-terminal mutations in vivo. Incorporation of the 3′-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.
Poliovirus Escape from RNA Interference: Short Interfering RNA-Target Recognition and Implications for Therapeutic Approaches
Authors: Gitlin Leonid , Stone Jeffrey K. , Andino Raul
Published: December 2004

Short interfering RNAs (siRNAs) directed against poliovirus and other viruses effectively inhibit viral replication. Although RNA interference (RNAi) may provide the basis for specific antiviral therapies, the limitations of RNAi antiviral strategies are ill defined. Here, we show that poliovirus readily escapes highly effective siRNAs through unique point mutations within the targeted regions. Competitive analysis of the escape mutants provides insights into the basis of siRNA recognition. The RNAi machinery can tolerate mismatches but is exquisitely sensitive to mutations within the central region and the 3′ end of the target sequence. Indeed, specific mutations in the target sequence resulting in G:U mismatches are sufficient for the virus to escape siRNA inhibition. However, using a pool of siRNAs to simultaneously target multiple sites in the viral genome prevents the emergence of resistant viruses. Our study uncovers the elegant precision of target recognition by the RNAi machinery and provides the basis for the development of effective RNAi-based therapies that prevent viral escape.
Deletion of Penton RGD Motifs Affects the Efficiency of both the Internalization and the Endosome Escape of Viral Particles Containing Adenovirus Serotype 5 or 35 Fiber Knobs
Authors: Shayakhmetov Dmitry M. , Eberly Andrea M. , Li Zong-Yi , Lieber André
Published: December 2004

Adenovirus (Ad) vectors are widely used for gene delivery in vitro and in vivo. A solid understanding of the biology of this virus is imperative for the development of novel, effective, and safe vectors. For the group C adenovirus serotypes 2 and 5 that use CAR as a primary attachment receptor, it is known that the penton base RGD motifs interact with cellular integrins and that this interaction promotes virus internalization. However, the RGD motif's impact on the efficiency of postinternalization steps, such as the escape of the virus particle from the endosome, is less defined. Furthermore, the role of penton-integrin interactions remains unknown for new vectors possessing group B Ad fiber knobs that use CD46 as a primary virus attachment receptor. In this study, we used vectors with the RGD motif deleted that contained Ad5 and B-group Ad35 fiber knobs and long fiber shafts and studied the role of RGD-integrin interactions in virus internalization and endosome escape. The deletion of the RGD motif in the penton base did not affect virus attachment, regardless of the type of cellular receptor used for attachment. RGD motif deletion, however, significantly reduced the rate of virus internalization for both the Ad5 and Ad35 fiber knob-containing vectors. This study also demonstrates the role of penton RGD motifs in facilitating the endosome escape step of virus infection and indicates that penton-integrin interactions are involved in internalization of capsid-chimeric CD46-interacting Ads with long fiber shafts.
Spread of Vaccine-Derived Poliovirus from a Paralytic Case in an Immunodeficient Child: an Insight into the Natural Evolution of Oral Polio Vaccine
Authors: Cherkasova E. A. , Yakovenko M. L. , Rezapkin G. V. , Korotkova E. A. , Ivanova O. E. , Eremeeva T. P. , Krasnoproshina L. I. , Romanenkova N. I. , Rozaeva N. R. , Sirota L. , Agol V. I. , Chumakov K. M.
Published: December 2004

Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.
Vaccinia Virus F1L Protein Is a Tail-Anchored Protein That Functions at the Mitochondria To Inhibit Apoptosis
Authors: Stewart Tara L. , Wasilenko Shawn T. , Barry Michele
Published: December 2004

Members of the poxvirus family encode multiple immune evasion proteins, including proteins that regulate apoptosis. We recently identified one such protein, F1L, encoded by vaccinia virus, the prototypic member of the poxvirus family. F1L localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome
c
, the pivotal commitment step in the apoptotic cascade. Sequence analysis of the F1L open reading frame revealed a C-terminal motif composed of a 12-amino-acid transmembrane domain flanked by positively charged lysines, followed by an 8-amino-acid hydrophilic tail. By generating a series of F1L deletion constructs, we show that the C-terminal domain is necessary and sufficient for localization of F1L to the mitochondria. In addition, mutation of lysines 219 and 222 downstream of the C-terminal transmembrane domain resulted in altered localization of F1L to the endoplasmic reticulum. Using F1L protein generated in an in vitro transcription-translation system, we found that F1L was posttranslationally inserted into mitochondria and tightly associated with mitochondrial membranes as demonstrated by resistance to alkaline extraction. Sensitivity to protease digestion showed that the N terminus of F1L was exposed to the cytoplasm. Utilizing various F1L deletion constructs, we found that F1L localization to the mitochondria was necessary to inhibit apoptosis, since constructs that no longer localized to the mitochondria had reduced antiapoptotic ability. Our studies show that F1L is a new member of the tail-anchored protein family that localizes to mitochondria during virus infection and inhibits apoptosis as a means to enhance virus survival.