Search

Introduction: High-rate non-penetrating blunt impacts to the thorax, such as from impacts to protective equipment, can lead to a wide range of thoracic injuries. These injuries can include rib fractures, lung contusions, and abdominal organ contusions. Ovine animals have been used to study such impacts, in a variety of ways, including in silico. To properly model these impacts in silico, it is imperative that the tissues impacted are properly characterized. The objective of this study is to characterize and validate two tissues impacted that are adjacent to the point of impact-costal cartilage and hide. Heretofore, these materials have not been characterized for use in computational models despite their nearly immediate engagement in the high-rate, non-penetrating loading environment., Materials and Methods: Ovine costal cartilage and hide samples were procured from a local abattoir following USDA regulations. Costal cartilage samples were then cut into ASTM D638 Type V tensile coupons and compressive disks for testing. The cartilage tensile coupons were tested at 150 ε/s, and the compressive samples were tested at -150 ε/s. Identical coupons and disks were then simulated in LS-Dyna using a hyperelastic material model based on test data and experimental boundary conditions. Hide samples were shaved and cut into ASTM D638 Type V tensile coupons and validated in silico using identical boundary conditions and an Ogden rubber model based on test data., Results: The structural responses of costal cartilage and hide are presented and exhibit typical behavior for biological specimens. The respective model fits in LS-Dyna were a hyperelastic- based "simplified rubber" for the costal cartilage and an Ogden rubber for the hide. The costal cartilage had a mean failure strain of 0.094 ± 0.040 in tension and -0.1755 ± 0.0642 in compression. The costal cartilage was also noted to have an order-of-magnitude difference in the stresses observed experimentally between the tensile and compressive experiments. Hide had a mean failure strain of 0.2358 ± 0.1362. The energies for all three simulations showed material stability., Conclusions: Overall, we successfully characterized the mechanical behavior of the hide and costal cartilage in an ovine model. The data are intended for use in computational analogs of the ovine model for testing non-penetrating blunt impact in silico. To improve upon these models, rate sensitivity should be included, which will require additional mechanical testing., (© The Association of Military Surgeons of the United States 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

The use of small ruminants, mainly sheep and goats, is increasing in biomedical research. Small ruminants are a desirable animal model due to their human-like anatomy and physiology. However, the large variability between studies and lack of baseline data on these animals creates a barrier to further research. This knowledge gap includes a lack of computed tomography (CT) scans for healthy subjects. Full body, contrast enhanced CT scans of caprine and ovine subjects were acquired for subsequent modeling studies. Scans were acquired from an ovine specimen (male, Khatadin, 30-35 kg) and caprine specimen (female, Nubian 30-35 kg). Scans were acquired with and without contrast. Contrast enhanced scans utilized 1.7 mL/kg of contrast administered at 2 mL/s and scans were acquired 20 seconds, 80 seconds, and 5 minutes post-contrast. Scans were taken at 100 kV and 400 mA. Each scan was reconstructed using a bone window and a soft tissue window. Sixteen full body image data sets are presented (2 specimens by 4 contrast levels by 2 reconstruction windows) and are available for download through the form located at: https://redcap.link/COScanData. Scans showed that the post-contrast timing and scan reconstruction method affected structural visualization. The data are intended for further biomedical research on ruminants related to computational model development, device prototyping, comparative diagnostics, intervention planning, and other forms of translational research., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Collaborative nonregulatory programs can benefit the long-term sustainability of environmental resources. Such programs benefit from extensive planning and assessment relative to ecological systems as well as public participation. While many programs use adaptive management as a guiding programmatic framework, few programs successfully integrate social and human context into their adaptive management frameworks. While this adaptive governance framework can be successful, many potential challenges arise when incorporating public stakeholders into the adaptive management framework. To reduce those challenges, programs need participation from diverse stakeholder groups that represent multiple communities of interest, place, and identity. The participatory process benefits from a diverse group of stakeholders and can result in successful management of environmental resources. We highlight the participatory co-management process of three newly developing nonregulatory programs that are modeled after the United States EPA's National Estuary Program in the Perdido and Pensacola Bay systems, Choctawhatchee Bay, and the St. Andrew and St. Joseph Bay systems (Florida USA). This case study illustrates how collaborative nonregulatory programs can be implemented not only in the United States, but also in other regions of the world., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Suppression of established populations of invasive alien species can be a complex and expensive process, which is frequently unsuccessful. The Asian clam, Corbicula fluminea (Müller, 1774), is considered a high impact invader that can adversely alter freshwater ecosystems and decrease their socioeconomic value. To date, C. fluminea continues to spread and persist within freshwater environments worldwide, despite repeated management attempts to prevent dispersal and suppress established populations. As extensive C. fluminea beds can often become exposed during low-water conditions, the direct application of hot or cold thermal shock treatments has been proposed as suitable mechanism for their control. Further, mechanical substrate disturbance may enhance the efficacy of thermal shock treatments by facilitating exposures to multiple layers of buried clams. In the present study, we advanced these methods by assessing combined applications of both hot and cold thermal shock treatments for control of C. fluminea, using steam spray (≥100 °C; 350 kPa), low- or high-intensity open-flame burns (~1000 °C) and dry ice (-78 °C). In a direct comparison of raking combined with hot thermal shock applications, both steam and high-intensity open-flame treatments tended to be most effective, especially following multiple applications. In addition, when hot thermal treatments are followed by a final cold shock (i.e. dry ice), steam treatments tended to be most effective. Further, when dry ice was applied either alone or prior to an application of a hot shock treatment, substantial if not complete C. fluminea mortality was observed. Overall, this study demonstrated that combined applications of hot and cold thermal shock treatments, applied following the disruption of the substrate, can substantially increase C. fluminea mortality compared to separate hot or cold treatments.

Control strategies for established populations of invasive alien species can be costly and complex endeavours, which are frequently unsuccessful. Therefore, rapid-reaction techniques that are capable of maximising efficacy whilst minimising environmental damage are urgently required. The Asian clam (Corbicula fluminea Müller, 1774), and the zebra mussel (Dreissena polymorpha Pallas, 1771), are invaders capable of adversely affecting the functioning and biodiversity of freshwater ecosystems. Despite efforts to implement substantial population-control measures, both species continue to spread and persist within freshwater environments. As bivalve beds often become exposed during low-water conditions, this study examined the efficacy of steam-spray (≥100 °C, 350 kPa) and open-flame burn treatments (~1000 °C) to kill exposed individuals. Direct steam exposure lasting for 5 min caused 100% mortality of C. fluminea buried at a depth of 3 cm. Further, combined rake and thermal shock treatments, whereby the substrate is disturbed between each application of either a steam or open flame, caused 100% mortality of C. fluminea specimens residing within a 4-cm deep substrate patch, following three consecutive treatment applications. However, deeper 8-cm patches and water-saturated substrate reduced maximum bivalve species mortality rates to 77% and 70%, respectively. Finally, 100% of D. polymorpha specimens were killed following exposure to steam and open-flame treatments lasting for 30 s and 5 s, respectively. Overall, our results confirm the efficacy of thermal shock treatments as a potential tool for substantial control of low-water-exposed bivalves. Although promising, our results require validation through upscaling to field application, with consideration of other substrate types, increased substrate depth, greater bivalve densities, non-target and long-term treatment effects.

Invasive species lose parasites in the process of invasion and tend to be less parasitized than conspecifics in the native range and sympatric native species in the invasive range (enemy release). We evaluated enemy release in an invasive freshwater fish in Ireland, common dace Leuciscus leuciscus, using helminth parasite community surveys at the core and front of the invasive range of common dace. Furthermore, we undertook a systematic literature review of helminth infection in common dace across its native range in Great Britain and Europe and invasive range in Ireland. The helminth parasite community survey revealed that invasive common dace were infected with fewer helminth species at the invasion front than at the core. Four helminth taxa - Acanthocephala, Monogenea, Digenea and Nematoda - were present in dace at the invasion core compared to only a single helminth species (Pomphorhynchus tereticollis) at the front. The systematic review revealed that invasive common dace in Ireland hosted fewer species of helminths than common dace in the native range. We report a total of three helminth species in common dace in Ireland compared to 24 in Great Britain and 84 in Continental Europe. Our results support the hypotheses that invasive populations are less parasitized than native populations and that more recently established populations host fewer parasites. However, we demonstrate that invasive species may continue to experience release from parasites long after initial invasion.

The most commonly used method for measuring vegetation cover is visual estimation, which is highly subjective, potentially leading to measurement errors. This poses serious implications to the assessment and continued management of plant species cover, for example in the control of invasive plant species. Morphological analysis of digital imagery has, to date, been primarily applied in the classification of landscape features. Our novel application of morphological image analysis provides an objective method for detection and accurate cover assessment of an invasive alien plant species (IAS), giving reduced measurement errors when compared to visual estimation. Importantly, this method is entirely based on free software. Guidos Toolbox is a collection of generic raster image processing routines, including Morphological Spatial Pattern Analysis (MSPA), which classifies and quantifies features according to shape. MSPA was employed in this study to detect and quantify cover of invasive Petasites pyrenaicus (Winter heliotrope) in digital images of 1 m × 1 m plots. Its efficacy was compared to that of two other methods- GIS Digitisation (used as an accurate baseline) and Visual Estimation (standard method). We tested the limit of MSPA usability on images of varying complexity, i.e. "simple", intermediate" or "complex", depending on presence/absence of other vascular plant species and the species richness of plot. Our results show good agreement between all three methods. MSPA measurement of P. pyrenaicus cover was most closely aligned with the GIS Digitisation (concordance correlation coefficients of 0.966). Visual Estimation was less closely aligned with GIS Digitisation (concordance correlation coefficients of 0.888). However, image complexity resulted in differing levels of agreement; with the closest agreement being achieved between MSPA and GIS Digitisation when used on images of lower and higher complexity. MSPA consistently provides higher accuracy and precision for P. pyrenaicus cover measurement than the standard Visual Estimation method. Our methodology is applicable to a range of focal vegetation species, both herbaceous and graminoid. Future application of MSPA for larger-scale surveying and monitoring via remote sensing is discussed, potentially reducing resource demands and increasing cover measurement consistency and accuracy. We recommend this method forms part of vegetation management toolkits for not only environmental managers, but for anyone concerned with plant cover assessment, from agricultural systems to sustainable resource use., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Biosecurity protocols designed to prevent the spread of invasive alien species (IAS) are now an essential aspect of IAS management. However, the effectiveness of various biosecurity treatments requires further exploration. Killer shrimp, Dikerogammarus villosus, a notoriously high impact and ecosystem destabilising invader, has rapidly spread across Europe, and is of concern to invade Northern America. In this study, we examine the effectiveness of three commonly used, broad-spectrum disinfectants to cause mortality of D. villosus: Virasure Aquatic, Virkon Aquatic, and Virkon S. Immersion and spray treatments of 1%, 2% and 4% disinfectant solutions were examined for applications of up to 300 secs immersion and for up to ten consecutive sprays. Furthermore, we assessed the effectiveness of steam (≥100 °C) treatments for up to 120 secs. For all disinfectants, immersion in 1% solutions caused 100% mortality at ≥120 secs. At higher concentrations, shorter immersion times caused complete mortality: 60 and 15 secs for 2% and 4% solutions, respectively. Five sprays of 2% and 4% solutions resulted in 100% mortality, for all disinfectants. Direct steam exposure was highly effective, with complete D. villosus mortality occurring at ≥10 secs. Overall, brief exposure to broad-spectrum disinfectants and direct steam could be used to limit D. villosus spread.

Increased organic loading to sediments from eutrophication often results in hypoxia, reduced nitrification and increased production of hydrogen sulfide, altering the balance between nitrogen removal and retention. We examined the effect of short-term exposure to various oxygen and sulfide concentrations on sediment nitrification, denitrification and DNRA from a chronically hypoxic basin in Roskilde Fjord, Denmark. Surprisingly, nitrification rates were highest in the hypoxic and anoxic treatments (about 5 μmol cm-3 d-1) and the high sulfide treatment was not significantly different than the oxic treatment. Denitrification in the hypoxic treatment was highest at 1.4 μmol cm-3 d-1 and significantly higher than the high sulfide treatment. For DNRA, the rate in high sulfide treatment was 2 μmol cm-3 d-1. This was significantly higher than all oxygen treatments that were near zero. In this system, nitrifiers rapidly recovered from conditions typically considered inhibiting, while denitrifiers had a more muted response. DNRA bacteria appear to depend on sulfide for nitrate reduction. Anammox was insignificant. Thus, in estuaries and coastal systems that experience short-term variations in oxygen and sulfide, capabilities of microbial communities are more diverse and tolerant of suboptimal conditions than some paradigms suggest.

Seasonal responses in estuarine metabolism (primary production, respiration, and net metabolism) were examined using two complementary approaches. Total ecosystem metabolism rates were calculated from dissolved oxygen time series using Odum's open water method. Water column rates were calculated from oxygen-based bottle experiments. The study was conducted over a spring-summer season in the Pensacola Bay estuary at a shallow seagrass-dominated site and a deeper bare-bottomed site. Water column integrated gross production rates more than doubled (58.7 to 130.9 mmol O 2 m -2 d -1 ) from spring to summer, coinciding with a sharp increase in water column chlorophyll-a, and a decrease in surface salinity. As expected, ecosystem gross production rates were consistently higher than water column rates, but showed a different spring-summer pattern, decreasing at the shoal site from 197 to 168 mmol O 2 m -2 d -1 and sharply increasing at the channel site from 93.4 to 197.4 mmol O 2 m -2 d -1 . The consistency among approaches was evaluated by calculating residual metabolism rates (ecosystem - water column). At the shoal site, residual gross production rates decreased from spring to summer from 176.8 to 99.1 mmol O 2 m -2 d -1 , but were generally consistent with expectations for seagrass environments, indicating that the open water method captured both water column and benthic processes. However, at the channel site, where benthic production was strongly light-limited, residual gross production varied from 15.7 mmol O 2 m -2 d -1 in spring to 86.7 mmol O 2 m -2 d -1 in summer. The summer rates were much higher than could be realistically attributed to benthic processes, and likely reflected a violation of the open water method due to water column stratification. While the use of sensors for estimating complex ecosystem processes holds promise for coastal monitoring programs, careful attention to the sampling design, and to the underlying assumptions of the methods, is critical for correctly interpreting the results. This study demonstrated how using a combination of approaches yielded a fuller understanding of the ecosystem response to hydrologic and seasonal variability.

Oysters provide a critical habitat, are a food resource for higher trophic levels and support important commercial fisheries throughout the world. Oyster reefs can improve water quality by removing phytoplankton. While sediment denitrification may be enhanced adjacent to oyster reefs, little is known about nitrification and denitrification associated with living oysters and their shells. We measured nitrification and denitrification in living oysters (Crassostrea virginica and Crassostrea gigas) and empty oyster shells. Nitrification was similar between live oysters and empty oyster shells, however, denitrification was enhanced significantly on living oysters compared to shells. This is the first demonstration of nitrification and denitrification associated with living oysters and their shells. Our data suggest that loss of historic oyster reefs has likely affected the resilience of estuaries to eutrophication. The additional benefit of oyster mediated denitrification should be considered in restoration of oyster reefs as a tool for managing eutrophication., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mercury (Hg) wet deposition, transfer from the atmosphere to Earth's surface by precipitation, in the United States is highest in locations and seasons with frequent deep convective thunderstorms, but it has never been demonstrated whether the connection is causal or simple coincidence. We use rainwater samples from over 800 individual precipitation events to show that thunderstorms increase Hg concentrations by 50% relative to weak convective or stratiform events of equal precipitation depth. Radar and satellite observations reveal that strong convection reaching the upper troposphere (where high atmospheric concentrations of soluble, oxidized mercury species (Hg(II)) are known to reside) produces the highest Hg concentrations in rain. As a result, precipitation meteorology, especially thunderstorm frequency and total rainfall, explains differences in Hg deposition between study sites located in the eastern United States. Assessing the fate of atmospheric mercury thus requires bridging the scales of global transport and convective precipitation.

Ecosystem respiration is the biotic conversion of organic carbon to carbon dioxide by all of the organisms in an ecosystem, including both consumers and primary producers. Respiration exhibits an exponential temperature dependence at the subcellular and individual levels, but at the ecosystem level respiration can be modified by many variables including community abundance and biomass, which vary substantially among ecosystems. Despite its importance for predicting the responses of the biosphere to climate change, it is as yet unknown whether the temperature dependence of ecosystem respiration varies systematically between aquatic and terrestrial environments. Here we use the largest database of respiratory measurements yet compiled to show that the sensitivity of ecosystem respiration to seasonal changes in temperature is remarkably similar for diverse environments encompassing lakes, rivers, estuaries, the open ocean and forested and non-forested terrestrial ecosystems, with an average activation energy similar to that of the respiratory complex (approximately 0.65 electronvolts (eV)). By contrast, annual ecosystem respiration shows a substantially greater temperature dependence across aquatic (approximately 0.65 eV) versus terrestrial ecosystems (approximately 0.32 eV) that span broad geographic gradients in temperature. Using a model derived from metabolic theory, these findings can be reconciled by similarities in the biochemical kinetics of metabolism at the subcellular level, and fundamental differences in the importance of other variables besides temperature—such as primary productivity and allochthonous carbon inputs—on the structure of aquatic and terrestrial biota at the community level.

Native Eurasian otter (Lutra lutra) and introduced American mink (Mustela vison) carcasses collected throughout Ireland were screened for biliary parasites. Secondary intermediate hosts, Cyprinid fish, were also examined for Opistorchiid metacercariae. Twenty-nine mink and 24 otter gall bladders were screened for biliary parasites. A single mink and three otters were found to be infected with the digenetic trematode Pseudamphistomum truncatum. Eighty-nine percent of roach (Rutilus rutilus) from the River Shannon were infected with P. truncatum metacercariae, confirming the persistence of the parasite. This is the first record of the species in Ireland, and its recent introduction is probably related to the movement and release of Cyprinid fishes by anglers.

Nitrification, the oxidation of NH(4)(+) to NO(2)(-) and subsequently to NO(3)(-), plays a central role in the nitrogen cycle and is often a critical first step in nitrogen removal from estuarine and coastal environments. The first and rate-limiting step in nitrification is catalyzed by the enzyme ammonia monooxygenase (AmoA). We evaluate the relationships between the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) amoA genes; potential nitrification rates and environmental variables to identify factors influencing AOA abundance and nitrifier activity in estuarine sediments. Our results showed that potential nitrification rates increased as abundance of AOA amoA increased. In contrast, there was no relationship between potential nitrification rates and AOB amoA abundance. This suggests that AOA are significant in estuarine nitrogen cycling. Surprisingly, more of the variability in potential nitrification rates was predicted by salinity and pore water sulfide than by dissolved oxygen history.

Diamond-Blackfan anemia (DBA) typically presents with red blood cell aplasia that usually manifests in the first year of life. The only gene currently known to be mutated in DBA encodes ribosomal protein S19 (RPS19). Previous studies have shown that the yeast RPS19 protein is required for a specific step in the maturation of 40S ribosomal subunits. Our objective here was to determine whether the human RPS19 protein functions at a similar step in 40S subunit maturation. Studies where RPS19 expression is reduced by siRNA in the hematopoietic cell line, TF-1, show that human RPS19 is also required for a specific step in the maturation of 40S ribosomal subunits. This maturation defect can be monitored by studying rRNA-processing intermediates along the ribosome synthesis pathway. Analysis of these intermediates in CD34- cells from the bone marrow of patients with DBA harboring mutations in RPS19 revealed a pre-rRNA-processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34+ cells from patients with DBA who have mutations in RPS19.

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3'-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis.

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3' end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3' end of 18S rRNA and formation of active 40S ribosomal subunits.

Concentrations of Cu and Pb were determined in the roots and shoots of six salt marsh plant species, and in sediment taken from between the roots of the plants, sampled from the lower salt marsh zone at four sites along the Suir Estuary in autumn 1997. Cu was mainly accumulated in the roots of monocotyledonous and dicotyledonous species. Pb was mainly accumulated in the roots of monocotyledons, while dicotyledons tended to accumulate Pb in the shoots. In the case of Aster tripolium there was a clear differentiation in the partitioning of Pb within the plant, between low and high salinity sites. At the low salinity sites, Pb accumulated only in the roots while at the high salinity sites there was a marked translocation to the shoots. The increase in Pb concentrations in roots and shoots of A. tripolium was accompanied by a concomitant decrease in sediment concentrations of Pb. This inverse correlation between sediment and plant concentrations of Pb was also recorded for Spartina spp. and Schoenoplectus tabernaemontani but in the case of these species the roots contained higher concentrations of Pb regardless of salinity levels. These differences in accumulation of Cu and Pb in various salt marsh species, and the influence of salinity on the translocation of Pb in A. tripolium in particular, should be taken into account when using these plants for biomonitoring purposes.

Primary production, respiration, and net ecosystem metabolism (NEM) are useful indicators of ecosystem level trophic conditions within estuaries. In this study, dissolved oxygen data collected every half hour between January 1996 to December 1998 by the National Estuarine Research Reserve System Wide Monitoring Program were used to calculate primary production, respiration, and net ecosystem metabolism. Data from two sites at each of 14 Reserves were analyzed. On average, three quarters of the data available could be used to calculate metabolic rates. Data from two of the Reserves were used to evaluate the assumption of homogeneity of water masses moving past the oxygen sensor. Temperature was the single most important factor controlling metabolic rates at individual sites, although salinity was also important at about half the sites. On an annual basis, respiration exceeded gross primary production demonstrating that all but 4 of the 28 sites were heterotrophic.

Macroscopic kinetics and single-channel properties of skeletal-muscle-type calcium currents were studied in the non-fusing, clonal muscle cell line, BC3H1. Slowly activating, dihydropyridine(DHP)-sensitive currents, associated with T-tubular DHP receptors and ion channels, could be isolated from rapidly activating, DHP-resistant currents. Description of macroscopic current activation kinetics required only a brief delay term (tau o <1 ms), two ascending exponential terms with voltage-dependent time constants (2 < tau 1 > 20 ms and 10 < tau 2 < 200 ms), and a single exponential decay term (0.5 < tau 3 < or = 5 s). Steady-state activation voltage dependence required description by two Boltzmann distribution terms with V 1/2 and slope factors differing by 20 mV and 3.5- to 4-fold respectively. These two distributions were correlated with the steady-state voltage dependence of the two ascending kinetic terms described by tau 1 and tau 2 respectively. Rundown of the DHP-sensitive slow current was correlated with a negative shift in the voltage dependence of current decay (tau 3). Three conductance levels (4.5 pS, 8 pS and 12 pS) were detected in single-channel records, two of which (the 8-pS and 12-pS events) were prolonged by BayK8644 and thus associated with DHP-sensitive single-channel events. Description of single-channel open time distributions required a minimum of two exponential terms (2.5 +/- 0.9 ms and 10.3 +/- 5.4 ms at -10 mV). Slow transitions among closed states results in biexponential latency-to-first-event distributions (47 +/- 12 ms and 470 +/- 123 ms at -10 mV).

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.

The mechanism of how chloroethylamine-treated agarose greatly simplifies the purification of ceruloplasmin (Cp) by preferentially binding this protein from blood plasma has been investigated. Chloroethylamine readily cyclizes to ethylenimine under alkaline conditions which then polymerizes to polyethylenimine (PEI). Ethylenimine and PEI were detected in the reaction mixture used to generate the resin. PEI polymers are grown from the matrix and form "tentacle" ligands, while PEI-silica and PEI-cellulose which are not tentacle gels did not bind Cp as readily. Spermine and spermidine, naturally occurring polyamine compounds, were attached to CNBr activated-agarose and showed weak affinity for Cp. [It should be noted that ethylenimine is a potential human carcinogen and presents an inhalation hazard.]

Neurons and glia exhibit complex homeostatic interactions via shared extracellular space which can involve metabolites, inorganic ions, and neurotransmitters. Focal application of glutamate to both human and rat central nervous system astrocytes in primary culture produced a rapid, transient increase in both cytoplasmic and nuclear Ca2+. These Ca2+ waves can propagate at up to 15-20 micron/s for long distances (millimetres) through the astrocyte syncitium. Oscillatory Ca2+ signals were frequently observed under control conditions and were enhanced by glutamate application. These Ca2+ signals were paralleled by rapid extensions of filopodia from the astrocyte cell margin and apical surface near the point of glutamate application. Focal application of glutamate to rat hippocampal neurons also elicited rapid, transient increases in intracellular Ca2+. Levels of Ca2+ signals were consistently two- to three-fold greater in pyramidal neurons cultured from CA1 than in those from CA3. Filopodial extension was extensive in CA1 neurons, but rare in CA3 neurons, and in either case observable only during the first few days of primary culture. Diversity of glial and neuronal responses to binding the glutamate receptors may reflect their roles in homeostatic interactions.

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.

To evaluate developmental and physiological signals that may influence expression of the dihydropyridine-sensitive "slow" Ca2+ channel, we analyzed dihydropyridine receptor (DHPR) mRNA abundance in mouse skeletal muscle. Using synthetic oligonucleotide probes corresponding to the rabbit skeletal muscle DHPR, a 6.5 kb DHPR transcript was identified in postnatal skeletal muscle and differentiated C2 or BC3H1 myocytes, but not cardiac muscle or brain. DHPR gene expression was reversibly suppressed by 0.4 nM transforming growth factor beta-1 or by transfection with a mutant c-H-ras allele, nominal inhibitors of myogenesis that block the appearance of slow channels and DHPR. In contrast, both BC3H1 and C2 myocytes containing the activated ras vector expressed the gene encoding the nicotinic acetylcholine receptor delta subunit, demonstrating that not all muscle-specific genes are extinguished by ras. Denervation stimulated DHPR gene expression less than 0.6-fold, despite 8-fold upregulation of delta-subunit mRNA and reciprocal effects on the skeletal and cardiac alpha-actin genes. Thus, DHPR gene induction is prevented by inhibitors of other muscle-specific genes, whereas, at most, relatively small changes in DHPR mRNA abundance occur during adaptation to denervation.

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.

A monoclonal antibody, mAb 1A, previously shown to recognize the dihydropyridine (DHP)-binding complex in rabbit muscle transverse tubules, inhibits voltage-sensitive calcium channel activity in a mouse muscle cell line. mAb 1A applied externally to BC3H1 muscle cells produced a concentration-dependent attenuation of the slowly-activating, DHP-sensitive, high-threshold calcium current. mAb 1A had no direct effect on either the rapidly-activating, DHP-insensitive, low-threshold calcium current or the delayed outward potassium current. However, sodium current kinetics were altered. On Western blot analysis of immunoaffinity-purified protein solubilized from BC3H1 membranes, mAb 1A recognized a Mr 210,000 polypeptide that is an analogous to the alpha 1 subunit of the DHP-binding complex from skeletal muscle. These results suggest that an extracellular segment of the alpha 1 subunit of the DHP-binding complex plays a functionally important role in voltage-gated calcium currents.

A slowly inactivating inward calcium current was identified in the rat osteosarcoma cell line ROS 17/2.8 using a combination of ion flux and electrophysiological techniques. Voltage dependence, dihydropyridine sensitivity, divalent cation selectivity, and single channel properties identified this current as a high threshold, "L-type" calcium current. Ion flux experiments using 45Ca2+ confirmed that calcium uptake through these channel represents a major pathway for calcium entry into osteosarcoma cells. In resting cells, i.e. at negative membrane potentials, stimulation of both calcium current and rapid 45Ca2+ influx could be elicited by concentrations of 1,25-(OH)2-vitamin D3 between 0.1 and 3 nM. At these concentrations, 1,25-(OH)2-vitamin D3 shifted the threshold for activation of inward calcium current to more negative potentials. At higher concentrations (5-10 nM), inhibitory effects became predominant. These opposing effects are functionally similar to those of the dihydropyridine BAY K 8644. Other vitamin D3 metabolites (25-(OH)-D3 and 24,25-(OH)2-D3) exhibited less potent stimulatory effects and greater inhibition of calcium current than 1,25-(OH)2-D3. These results suggest that (i) vitamin D3 acts as a potent modulator of calcium channel function in osteosarcoma cells, and (ii) intracellular Ca2+-dependent signaling processes may be affected acutely by physiological concentrations of vitamin D3 metabolites.

The rate of hemolysis and the decline in glutathione (GSH) in rabbit erythrocytes caused by copper (Cu) ions were determined. Prior investigations have proposed that the oxidative stress induced by Cu ion depleted the normal cell protective mechanisms. The decline in GSH has been proposed as a necessary prerequisite for hemolysis. We have observed that both GSH decline and hemolysis are Cu dependent, but are two concurrent and independent processes. We have confirmed that oxygen is a necessary reactant for hemolysis and responsible for a major portion of GSH decline. However, in the presence of Cu ion, a slow decline in GSH occurs even in a deaerated system.

To determine whether expression of the 1,4-dihydropyridine receptor of skeletal muscle Ca2+ channels is regulated by signals that impinge on muscle-specific gene expression, BC3H1 muscle cells were analyzed using (+)[3H]PN200-110 as a probe for the receptor. No dihydropyridine binding sites were detected in proliferating cells. Binding site density increased following serum withdrawal, peaking at day six, with little or no change in Kd (approximately equal to 250 pM, similar to that seen in skeletal muscle). No DHP binding sites were detected in BC3H1 cells bearing an activated c-H-ras oncogene. Induction of the dihydropyridine receptor was reversibly blocked by 200 pM transforming growth factor beta. The results indicate that formation of dihydropyridine-sensitive Ca2+ channels may require up-regulation of the dihydropyridine receptor itself, and that transforming growth factor beta is a potent, reversible inhibitor of this receptor in BC3H1 muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.

Reduced cytochrome-c, reduced myoglobin and oxyhemoglobin respectively have been oxidized to oxidized cytochrome-c, metmyoglobin and methemoglobin by ceruloplasmin. Metmyoglobin and methemoglobin formation was stoichiometric while oxidized cytochrome-c reacted catalytically. Only 50% methemoglobin was formed which suggested that two hemes out of four could transfer electrons. Hydrogen peroxide was formed in the reaction of reduced cytochrome-c with ceruloplasmin.

The nonfusing muscle cell line BC3H1 expresses functional Na and Ca channels similar to those found in skeletal muscle (1). We have utilized a monoclonal antibody that cross-reacts with a 45-50-kDa glycoprotein in differentiated BC3H1 myocytes but not in cardiac or neuronal cells. This antibody, MAb1223, specifically modulates 45Ca influx and slowly activating, dihydropyridine-sensitive Ca currents when added to BC3H1 myocytes. Low-threshold, dihydropyridine-insensitive Ca currents and Na and K currents are unaffected. MAb1223 action is voltage dependent. At very negative holding potentials, similar to those of the resting cell, MAb1223 increases slow Ca current without significant changes in the voltage dependence of activation. At more positive potentials, at which channel opening probability is reduced by inactivation, exposure to MAb1223 reduces current. Agonist and antagonist dihydropyridines modulate the action of MAb1223 on the slow Ca channel in a manner that suggests that their respective binding sites may interact directly with the channel.

1. Erythrocytes from different vertebrate classes were tested for susceptibility towards copper ion-induced lysis under identical copper ion concentration and per cent cell volumes. 2. The susceptibility towards lysis was found to be correlated with the rate of copper ion entry into the erythrocytes. 3. GSH levels decline in red blood cells at a rate proportional to the rate of copper ion entry. 4. Hemolysis does not seem to be causally related to the level of GSH in the erythrocytes.

Oocytes of Xenopus laevis, after microinjection with mRNA from rat heart, display typical high-threshold calcium (Ca) whole cell currents. To prepare to study structure-function relationships of the cardiac Ca channel molecule, we examined the fidelity of expression of biophysical and pharmacological properties at the molecular level. Cell-attached gigaseal recordings in five K-depolarized oocytes injected with adult rat heart mRNA showed single channel Ba currents with mean amplitude 1.3-1.5 pA at 0 mV, slope conductance 18-25 pS, and extrapolated reversal potential 57-68 mV. Openings were predominantly brief (mean 1.2 ms) but longer openings (mean 9 ms) were greatly enhanced in 10(-6) M BAY-K 8644, increasing the ensemble average current at 0 mV by more than fivefold. These features are typical of high-threshold cardiac Ca channels. In two patches from one injected oocyte, we saw multiple Ca channel conductances, as recently observed in other preparations. We conclude that X. laevis oocytes injected with adult rat heart mRNA produce high-threshold cardiac Ca channels with molecular properties identical to native cells.