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We examined diet composition of Wilson's Snipe (Gallinago delicata) (n = 372) collected along the central Gulf Coast of Texas based solely on upper digestive tract contents. Food items included 11 invertebrate orders, one invertebrate class, and eight plant genera. Oligochaetes were the predominant food throughout the non-breeding period, but snipe consumed fewer (P = 0.021) earthworms in spring than in fall. Aquatic insects were frequently consumed by snipe and during spring represented approximately the same proportion of the diet as earthworms. Plant foods consisted almost entirely of seeds and comprised 9.7–26.8% of the diet throughout the non-breeding period. Wilson's Snipe consumed dipteran larvae more often during spring than fall (P = 0.056). Female snipe consumed crustaceans during spring (14.8%), while only trace amounts were found in the diet of male snipe. Differences in the diet of Wilson's Snipe between males and females were probably related to differences in habitat use as well as...

SUMMARY 1 ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2 Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3 Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4 The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I− > Cl− > Asp−) and inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5 Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6 In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.

We analyzed carcass lipid, ash and protein dynamics of male and female Wilson's Snipe (Gallinago gallinago delicata) from arrival on wintering areas in coastal Texas until departure for spring migration. In addition, we analyzed seasonal variation in mass and length of digestive organs to document changes that may increase gut capacity and digestive efficiency of migrating and wintering Wilson's Snipe. Lipid reserves of female snipe remained relatively stable from fall to winter, whereas body protein increased by 3% during this time. Females catabolized lipids and protein prior to spring migration. Conversely, lipid reserves of male snipe increased by 44% from fall to winter and were at least maintained from winter to spring. Furthermore, we observed a trend (P = 0.021) for increasing lipid content in males during spring. Because male snipe accumulate fat reserves prior to spring departure, it appears they employ a “time-selected” strategy for spring migration. In contrast, female snipe migrate ...

4-AP Facilitates L-Type Ca Current. Background: Transient outward current (I to ) and L-type calcium current (I Ca ) are important repolarization currents in cardiac myocytes. These two currents often undergo disease-related remodeling while other currents are spared, suggesting a functional coupling between them. Here, we investigated the effects of I to channel blockers, 4-aminopyridine (4-AP) and heteropodatoxin-2 (HpTx2), on I Ca in cardiac ventricular myocytes. Methods and Results: I Ca was recorded in enzymatically dissociated mouse and guinea pig ventricular myocytes using the whole-cell voltage clamp method. In mouse ventricular myocytes, 4-AP (2 mM) significantly facilitated I Ca by increasing current amplitude and slowing inactivation. These effects were not voltage-dependent. Similar facilitating effects were seen when equimolar Ba 2+ was substituted for external Ca 2+ , indicating that Ca 2+ influx is not required. Measurements of Ca 2+ /calmodulin-dependent protein kinase (CaMKII) activity revealed significant increases in cells treated with 4-AP. Pretreatment of cells with 10μM KN93, a specific inhibitor of CaMKII, abolished the effects of 4-AP on I Ca . To test the requirement of I to , we studied guinea pig ventricular myocytes, which do not express I to channels. In these cells, 2 mM 4-AP had no effect on I Ca amplitude or kinetics. In both cell types, Ca 2+ -induced I Ca facilitation, a CaMKII-dependent process, was observed. However, 4-AP abolished Ca 2+ -induced I Ca facilitation exclusively in mouse ventricular myocytes. Conclusion: 4-AP, an I to blocker, facilitates L-type Ca 2+ current through a mechanism involving the I to channel and CaMKII activation. These data indicate a functional association of I Ca and I to in cardiac myocytes.

We used double-knockout mice (ABKO) lacking both predominant myocardial alpha(1)-adrenergic receptor (AR) subtypes (alpha(1A) and alpha(1B)) to determine if alpha(1)-ARs are required for normal myocardial contraction. Langendorff-perfused ABKO hearts had higher developed pressure than wild type (WT) hearts (123 +/- 3 mmHg n = 22 vs. 103 +/- 3 mmHg, n = 38, P0.001). Acutely inhibiting alpha(1)-ARs in WT hearts with prazosin did not increase pressure, suggesting that the increased pressure of ABKO hearts was mediated by long-term trophic effects on contraction rather than direct regulatory effects of alpha(1)-AR removal. Similar to perfused hearts, ABKO ventricular trabeculae had higher submaximal force at 2 mM extracellular [Ca(2+)] than WT (11.4 +/- 1.7 vs. 6.9 +/- 0.6 mN/mm(2), n = 8, P0.05); however, the peaks of fura-2 Ca(2+) transients were not different (0.79 +/- 0.11 vs. 0.75 +/- 0.16 microM, n = 10-12, P0.05), suggesting ABKO myocardium had increased myofilament Ca(2+)-sensitivity. This conclusion was supported by measuring the Ca(2+)-force relationship using tetanization. Increased myofilament Ca(2+)-sensitivity was not explained by intracellular pH, which did not differ between ABKO and WT (7.41 +/- 0.01 vs. 7.39 +/- 0.02, n = 4-6, P0.05; from BCECF fluorescence). However, ABKO displayed impaired troponin I phosphorylation, which may have played a role. In contrast to increased submaximal force, ABKO trabeculae had lower maximal force than WT at high extracellular [Ca(2+)] (29.6 +/- 1.9 vs. 37.6 +/- 1.4 mN/mm(2), n = 7, P0.01). However, peak cytosolic [Ca(2+)] was not different (1.13 +/- 0.15 vs. 1.19 +/- 0.04 microM, n = 6-7, P0.05), suggesting ABKO myocardium had impaired myofilament function. Finally, ABKO myocardium had decreased responsiveness to beta-AR stimulation. We conclude: alpha(1)-ARs are required for normal myocardial contraction; alpha(1)-ARs mediate long-term trophic effects on contraction; loss of alpha(1)-AR function causes some of the functional abnormalities that are also found in heart failure.

Two functional α1-adrenergic receptor (AR) subtypes (α1Aand α1B) have been identified in the mouse heart. However, it is unclear whether the third known subtype, α1D-AR, is also present. To investigate this, we determined whether there were α1-AR responses in hearts from a novel mouse model lacking α1A- and α1B-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, α1-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 ± 2% and 86 ± 4% of initial, respectively, n = 11, P < 0.01). These effects were blocked by prazosin and 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione} dihydrochloride, suggesting that α1D-AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that α1-AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking α1A- and α1B-ARs retain functional α1-AR responses involving decreases of coronary flow and ventricular pressure that reflect α1D-AR-mediated vasoconstriction. Furthermore, α1-AR inotropic responses depend critically on the experimental conditions.

D. T. McCloskey, D. G. Rokosh, T. D. O'Connell, E. C. Keung, P. C. Simpson and A. J. Baker. α1-Adrenoceptor Subtypes Mediate Negative Inotropy in Myocardium from α1A/C-Knockout and Wild Type Mice. Journal of Molecular and Cellular Cardiology (2002) 34, 1007–1017. Cardiac α1-adrenoceptors (AR) have two predominant subtypes (α1A-AR and α1B-AR) however, their roles in regulating contraction are unclear. We determined the effects of stimulating α1A-AR (using the subtype-selective agonist A61603) and α1B-AR (using a gene knockout mouse lacking α1A-AR) separately, and together (using phenylephrine) on Ca2+ transients, intracellular pH, and contraction of mouse cardiac trabeculae. Stimulation of α1-AR subtypes separately or together caused a triphasic contractile response. After a transient (≈3%) force rise (phase 1), force declined markedly (phase 2), then partially recovered (phase 3). In phase 2, the force decline (% of initial) with combined α1A-AR plus α1B-AR stimulation (50±3%) was more than with separate subtype stimulation (P

Abstract —Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during β-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TnI. The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca 2+ ] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from ≈150 to ≈90 ms at 22°C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f min (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of β-adrenoceptors with isoproterenol increased f min from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f min in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.

There is little information concerning differences in migration chronology between male and female Common Snipe (Gallinago gallinago) and virtually no accounts of sex-related differences in winter habitat use. We collected 372 Common Snipe in five different habitat types during the non-breeding period along the central Gulf Coast of Texas. Proportions of male and female snipe collected on wintering areas during the beginning of the fall period (i.e., between 6 and 21 October) indicated a tendency for females to arrive ahead of males. Sex ratios during the latter part of spring (16 March–10 April 1998) suggested male snipe leave wintering areas before females. During the winter period (14 November 1997–4 February 1998), female snipe were more common than males along the Texas Gulf Coast. Differences in sex ratios during winter are likely due to sex-related differences in habitat use. During winter, females were more common than males in heavily vegetated habitats (e.g., coastal marshes and cultiva...

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174–179, 2000. © 2000 Wiley-Liss, Inc.

Ecological risk assessment can be enhanced with predictive models for metal toxicity. Modelings of published data were done under the simplifying assumption that intermetal trends in toxicity reflect relative metal-ligand complex stabilities. This idea has been invoked successfully since 1904 but has yet to be applied widely in quantitative ecotoxicology. Intermetal trends in toxicity were successfully modeled with ion characteristics reflecting metal binding to ligands for a wide range of effects. Most models were useful for predictive purposes based on an F-ratio criterion and cross-validation, but anomalous predictions did occur if speciation was ignored. In general, models for metals with the same valence (i.e., divalent metals) were better than those combining mono-, di-, and trivalent metals. The softness parameter (sigma p) and the absolute value of the log of the first hydrolysis constant ([symbol: see text] log KOH [symbol: see text]) were especially useful in model construction. Also, delta E0 contributed substantially to several of the two-variable models. In contrast, quantitative attempts to predict metal interactions in binary mixtures based on metal-ligand complex stabilities were not successful.

Predictive models for relative toxicity of divalent metal ions using ion characteristics have been produced with both Microtox®, a 15 min microbial bioassay, and the 24 h Caenorhabditis elegans bioassay. Relative toxicity of mono-, di- and trivalent metal ions has also been successfully modeled using ion characteristics with the Microtox® bioassay. This study extends this approach to include longer exposure durations (24 h) and a more complex organism (metazoan). Twenty-four-hour LC50s (expressed as total and free ion concentrations) for the free-living soil nematode, C. elegans , were determined for Li, Na, Mg, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Sr, Cd, Cs, Ba, La, and Pb in an aqueous medium. Relative metal toxicity was predicted with least squares linear regression and several ion characteristics. Toxicity was most effectively predicted ( r 2 =0.85) with a two-variable model containing |log K OH | (where K OH is the first hydrolysis constant) and χ 2 m r (the covalent index). The first hydrolysis constant reflects a metal ion's tendency to bind to intermediate ligands such as biochemical groups with O donor atoms, while χ 2 m r reflects binding to soft ligands such as those with S donor atoms. The use of LC50s based on free ion concentrations did not significantly improve model fit. The results of this study are consistent with earlier models generated with Microtox® data, with the exception of barium, which was much more toxic to C. elegans than would be predicted from the model. We conclude that, with thoughtful application, ion characteristics can be used to predict the relative toxicity of metal ions that vary widely in both valence and binding tendency.

Quantitative Structure Activity Relationships (QSAR) predict relative toxicity of a family of chemicals from fundamental and surrogate molecular qualities. Most QSARs are developed for organic toxicants, with inorganic toxicants (metals) being under-represented. Successful predictive models for relative toxicity of divalent metal ions using ion characteristics have been produced using Microtox®, a 15 min microbial bioassay. The present study extends this approach to longer exposure durations (24 h), and a more complex organism (metazoan). Twenty-four hour LC50s (expressed as total metal concentration) for the free-living soil nematode, C. elegans were determined for Ca, Cd, Cu, Hg, Mg, Mn, Ni, Pb, and Zn in an aqueous medium. Relative metal toxicity was predicted with least squares linear regression and several ion characteristics. Toxicity was most effectively predicted (r2 = 0.89) with ¦ log K OH ∣ (where KOH is the first hydrolysis constant), which reflects a metal ion's tendency to bind to intermediate ligands such as biochemical functional groups with O donor atoms. The best fitting model was obtained using LC50 metameters based on total metal concentration, indicating that the identification of the bioactive species of metals can be ambiguous, and does not necessarily aid in the prediction of relative metal toxicity with ion characteristics. The modelling of relative metal toxicity using ion characteristics was successful for 24 h exposure durations using this more complex organism.

Quantitative structure-activity relationships have been used to predict the relative toxicity of organic compounds. Although not as common, ion characteristics have also proven useful for predicting the relative toxicity of metal ions. The purpose of this study was to determine if the relative toxicity of metal ions using the Microtox® bioassay was predictable using ion characteristics. Median effect concentrations (EC50s) were determined for 20 metals in a NaNO 3 medium, which reflected freshwater speciation conditions, using the Microtox bacterial assay. The log of EC50 values was modeled using several ion characteristics, and Akaike's Information Criterion was calculated to determine which ion characteristics provided the best fit. Whether modeling total ion (unspeciated) or free ion (speciated) EC50 values, the one variable which best modeled EC50s was the softness index (σ p , i.e., [coordinate bond energy of the metal fluoride - coordinate bond energy of the metal iodide]/[coordinate bond energy of the metal fluoride]), while a combination of χ m 2 r (χ m = electronegativity, r = Pauling ionic radius) and |log K OK | (absolute value of the log of the first hydrolysis constant, K OH for M n+ + H 2 O → MOH n-1 + H + ) was the best two-variable model. Other variables, including ΔE o and χ m 2 r (one-variable models) and (AN/ΔIP, ΔE o ) and (χ m 2 r, Z 2 /r) (two-variable models), also gave adequate fits. Modeling with speciated (free ion) versus unspeciated (total ion) EC50 values did not improve fits. Modeling mono-, di-, and trivalent metal ions separately improved the models. We conclude that ion characteristics can be used to predict the relative toxicity of metal ions whether in freshwater (NaNO 3 medium) or saltwater (NaCl medium) speciation conditions and that this approach can be applied to metal ions varying widely in both valence and binding tendencies.

Both relative toxicity and interactions between paired metal ions were predicted with least-squares linear regression and various ion characteristics. Microtox{reg_sign} 15 min EC50s (expressed as free ion) for Ca(II), Cd(II), Cu(II), Hg(II), Mg(II), Mn(II), Ni(II), Pb(II), and Zn(II) were most effectively modeled with the constant for the first hydrolysis (K{sub H} for M{sup n+} + H{sub 2}O {yields} MOH{sup a{minus}1} + H{sup +}) although other ion characteristics were also significant in regression models. The {vert_bar}log K{sub H}{vert_bar} is correlated with metal ion affinity to intermediate ligands such as many biochemical functional groups with O donor atoms. Further, ordination of metals according to ion characteristics, e.g., {vert_bar}log K{sub H}{vert_bar} facilitated prediction of paired metal interactions. Pairing metals with strong tendencies to complex with intermediate or soft ligands such as those with O or S donor atoms resulted in strong interactions.

We develop an adaptive and flexible framework for engaging experts and stakeholders at the household- and community-level that have different livelihoods and land use interests within Kenya’s Athi-Kaputiei Plains (AKP). We use Bayesian Belief Networks linked to GIS data layers to integrate empirical data and elicited stakeholder knowledge. The process is designed to address problems with past conservation-development strategies by allowing participants to build relationships among people with different land use interests in order to clarify opportunities and constraints, examine assumptions at the design phase of a project, and determine future actions and potential development scenarios. We use an example of four different livelihood groups in the AKP to demonstrate how the process might work to identify suitable areas for alternative land uses (e.g., wildebeest and livestock grazing, crop cultivation, and urban development), and to identify future compatibilities and conflicts between these different land use interests. The modeling process provides a maximal coverage strategy that allows decision makers to target and prioritize areas for protection or development, and to set specific strategies in the face of changing ecological, social, or economic processes. The process is iterative so that revised models can be developed as new data and knowledge arise, thereby

Anthracene, a polycyclic aromatic hydrocarbon, is acutely toxic to fish when they are exposed simultaneously to solar ultraviolet radiation (SUVR). However, the physiological mechanism of acute anthracene photo-induced toxicity is not known. The purpose of this study was to investigate possible modes of action associated with simultaneous anthracene and SUVR exposure using blood and gill biochemistry in bluegill sunfish (Lepomis macrochirus). Fish were exposed to anthracene (< 0.01 and 7.04 μg/l) and SUVR in a flow-through system. Following 96 h of anthracene and SUVR exposure, fish exhibited significant increases in hematocrit and significant decreases in whole blood hemoglobin content when compared to no-anthracene controls. Evidence of hemolysis was also observed in anthracene and SUVR exposed fish. Using in vitro enzyme analysis, both Na,K-ATPase and Mg-ATPase were signficantly inhibited in gill tissue homogenates exposed to anthracene and SUVR when compared to homogenates exposed to SUVR alone. These blood and gill measurements provide evidence of osmotic stress in exposed fish. Given the results presented in this and in previous studies, we conclude that there are numerous sites of acute toxic action with anthracene and SUVR exposure in fish and that this toxicity appears to be associated with a general disruption of cell membrane function.

Low-angle inelastic neutron scattering was used to study the temperature and wave vector dependence of the spin waves in La0.67Ca0.33MnO3 perovskite-based colossal magnetoresistance material. At low q the spin waves show Heisenberg ferromagnetic dispersion (E=Dq2+Δ) where D is the spin stiffness, q is the wave vector, and Δ is the energy gap. However, the temperature renormalization of the spin stiffness D is anomalous, and as T increases toward Tc, D does not show the expected power law collapse, but rather exhibits a sudden sharp drop suggestive of a first-order phase transition. Detailed neutron measurements of the order parameter in zero applied field showed a similar first-order-like transition. However, no temperature hysteresis was observed in either D or in the magnetization.

Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants in the aquatic environment and demonstrate a wide variety of toxic effects in freshwater and marine organisms. We have previously documented photo-induced toxic effects of a model PAH (i.e. anthracene) in fish. The goal of our ongoing studies is to examine environmental factors which may enhance or mitigate this toxicity in order to better estimate the risks of phototoxic PAHs to aquatic organisms. The purpose of the present study was to determine the effect of water temperature and oxygen concentration on the photo-induced toxicity of anthracene to juvenile bluegill sunfish (Lepomis macrochirus). Bluegill sunfish were exposed to anthracene ( μg l ) and solar ultraviolet radiation (SUVR) at different water temperatures (20 and 30°C) and oxygen concentrations (5.0, 6.9 and 8.1 mg O2/1 for 120 h in a laboratory flow-through system. A trend toward an inverse relationship was found between water temperature and LC50 values, although no significant temperature effect was observed. A nonlinear relationship was found between oxygen concentration and LC50 values, with significantly increased toxicity at the middle oxygen concentration. Opercular ventilation rates were inversely related to oxygen concentration and directly related to anthracene concentration. A statistical relationship was developed on the basis of oxygen concentration to predict LC50 values. We conclude that dissolved oxygen concentration is an important environmental factor in the assessment of photo-induced toxicity of anthracene to fish.

Increased signaling by Gi-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted Gi-coupled receptor (Ro1). Activation of Gisignaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist nor-binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum Ca2+-ATPase ( P < 0.05); thereafter, there was progressive impairment of both Ca2+handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic Ca2+concentration was reduced to 0.61 ± 0.08 vs. 0.91 ± 0.07 μM for control ( n = 6–8; P < 0.05), diastolic Ca2+concentration was elevated to 0.41 ± 0.07 vs. 0.23 ± 0.06 μM for control ( n = 6–8; P < 0.01), and the decline phase of the Ca2+transient (time from peak to 50% decline) was slowed to 0.25 ± 0.02 s vs. 0.13 ± 0.02 s for control ( n = 6–8; P < 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 ( P < 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, Ca2+-saturated myofilament force in skinned trabeculae was reduced to 21 ± 2 vs. 38 ± 0.1 mN/mm2for controls ( n = 3; P < 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of Gi-coupled receptors leads to impaired Ca2+handling and myofilament injury, contributing to impaired ventricular pump function and heart failure.

Introduction: EUROFORS was a 2-yr prospective, randomized trial of postmenopausal women with established osteoporosis, designed to investigate various sequential treatments after teriparatide 20 μg/d for 1 yr. The present secondary analysis examined the effects of 2 yr of open-label teriparatide in women previously treated with antiresorptive drugs for at least 1 yr. Methods: A subgroup of 245 women with osteoporosis who had 2 yr of teriparatide treatment were stratified by previous predominant antiresorptive treatment into four groups: alendronate (n = 107), risedronate (n = 59), etidronate (n = 30), and non-bisphosphonate (n = 49). Bone mineral density (BMD) at the lumbar spine and hip was determined after 6, 12, 18, and 24 months, and bone formation markers were measured after 1 and 6 months. Results: Significant increases in bone formation markers occurred in all groups after 1 month of teriparatide treatment. Lumbar spine BMD increased at all visits, whereas a transient decrease in hip BMD, which was subsequently reversed, was observed in all groups. BMD responses were similar in all previous antiresorptive groups. Previous etidronate users showed a higher increase at the spine but not at the hip BMD. Duration of previous antiresorptive therapy and lag time between stopping previous therapy and starting teriparatide did not affect the BMD response at any skeletal site. Treatment-emergent adverse events were similar to those reported in treatment-naive postmenopausal women with osteoporosis treated with teriparatide. Conclusions: Teriparatide induces positive effects on BMD and markers of bone formation in postmenopausal women with established osteoporosis, regardless of previous long-term exposure to antiresorptive therapies. Copyright © 2008 by The Endocrine Society.

Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

The left ventricle (LV) and right ventricle (RV) have differing hemodynamics and embryological origins, but it is unclear whether they are regulated differently. In particular, no previous studies have directly compared the LV versus RV myocardial inotropic responses to α1-adrenergic receptor (α1-AR) stimulation. We compared α1-AR inotropy of cardiac trabeculae from the LV versus RV of adult mouse hearts. As previously reported, for mouse RV trabeculae, α1-AR stimulation with phenylephrine (PE) caused a triphasic contractile response with overall negative inotropy. In marked contrast, LV trabeculae had an overall positive inotropic response to PE. Stimulation of a single subtype (α1A-AR) with A-61603 also mediated contrasting LV/RV inotropy, suggesting differential activation of multiple α1-AR-subtypes was not involved. Contrasting LV/RV α1-AR inotropy was not abolished by inhibiting protein kinase C, suggesting differential activation of PKC isoforms was not involved. However, contrasting LV/RV α1-AR inotropic responses did involve different effects on myofilament Ca2+ sensitivity: submaximal force of skinned trabeculae was increased by PE pretreatment for LV but was decreased by PE for RV. For LV myocardium, α1-AR-induced net positive inotropy was abolished by the myosin light chain kinase inhibitor ML-9. This study suggests that LV and RV myocardium have fundamentally different inotropic responses to α1-AR stimulation, involving different effects on myofilament function and myosin light chain phosphorylation.

The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac alpha-myosin heavy chain promoter (alphaMHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing alphaMHC-tTA mice with mice containing a tTA-responsive promoter linked to a target gene yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Using the tet-system, some studies use nontransgenic mice for the control group, whereas others use single-transgenic alphaMHC-tTA mice. However, previous studies found that high-level expression of a modified activator protein caused cardiomyopathy. Therefore, we tested whether cardiac expression of tTA was associated with altered function of alphaMHC-tTA mice compared with wild-type (WT) littermates. We monitored in vivo and in vitro function and gene expression profiles for myocardium from WT and alphaMHC-tTA mice. Compared with WT littermates, alphaMHC-tTA mice had a greater heart-to-body weight ratio (approximately 10%), ventricular dilation, and decreased ejection fraction, suggesting mild cardiomyopathy. In vitro, submaximal contractions were greater compared with WT and were associated with greater myofilament Ca2+ sensitivity. Gene expression profiling revealed that the expression of 153 genes was significantly changed by20% when comparing alphaMHC-tTA with WT myocardium. These findings demonstrate that introduction of the alphaMHC-tTA construct causes significant effects on myocardial gene expression and major functional abnormalities in vivo and in vitro. For studies using the tet-system, these results suggest caution in the use of controls, since alphaMHC-tTA myocardium differs appreciably from WT. Furthermore, the results raise the possibility that the phenotype conferred by a target gene may be influenced by the modified genetic background of alphaMHC-tTA myocardium.

Two functional alpha(1)-adrenergic receptor (AR) subtypes (alpha(1A) and alpha(1B)) have been identified in the mouse heart. However, it is unclear whether the third known subtype, alpha(1D)-AR, is also present. To investigate this, we determined whether there were alpha(1)-AR responses in hearts from a novel mouse model lacking alpha(1A)- and alpha(1B)-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, alpha(1)-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 +/- 2% and 86 +/- 4% of initial, respectively, n = 11, P0.01). These effects were blocked by prazosin and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione] dihydrochloride, suggesting that alpha(1D)-AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that alpha(1)-AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking alpha(1A)- and alpha(1B)-ARs retain functional alpha(1)-AR responses involving decreases of coronary flow and ventricular pressure that reflect alpha(1D)-AR-mediated vasoconstriction. Furthermore, alpha(1)-AR inotropic responses depend critically on the experimental conditions.

The purpose of this study was to determine the effect of metal and metalloid contaminated sediment on the movement and spatial distribution of asiatic clam (Corbicula fluminea) populations in the laboratory. One hundred clams were randomly placed in one meter diameter pools containing sediment collected from three sites of varying levels of metal contamination. The location of each clam was determined on days 0, 7, 14, 21 and 28. Ripley's L(t) function was used to measure the randomness or clustering of clams in experimental pools. There was no significant evidence of clustering of clam locations throughout the experiment. Further, sediment type had no significant effect on the distribution of clams in the pools, which suggests that clams were moving independently of one another. This information allowed us to estimate whether individual clams move sufficient distances to escape from patches of contaminated sediment of different sizes. Simulation results showed that after 150 weeks, clams can effectively escape from smaller contaminated patches of sediment (25-, 50-, and 100-cm radius), while less than 50% of clams could escape from a contaminated patch of 200-cm radius. These simulations suggest that an avoidance of contaminated sediment will not result in effective escape from moderately sized patches of contaminated sediment (>100-cm radius).

Sediment preference experiments were performed with the asiatic clam (Corbicula fluminea) and viviparid snail (Campeloma decisum) to determine the potential use of clam and snail behavior as a response to low-level metal and metalloid contamination. Three sediment types with varying levels of metal contamination were paired in various combinations. Clams and snails were placed in aquaria along the interface between the sediment types. Daily location and burial status were noted for two weeks. Clams spent significantly more days in the uncontaminated sediment when paired with one of the contaminated sediments. Snails spent more days in contaminated sediments when paired with the uncontaminated sediment, but none of these differences was statistically significant. Clams moved fewer days in tanks with the two most contaminated sediment types. Burrowing of snails was relatively unaffected by sediment treatments. The behavior of clams was more sensitive than the behavior of snails to sediment metal contamination. Consequently, clam behavior appears to be a better behavioral indicator of metal contamination.

PURPOSE To develop an active high-dose chemotherapy regimen for the treatment of ovarian carcinoma. Due to the rapid development a drug resistance, conventional chemotherapy cures only 20% of patients with advanced disease. However, in vitro data demonstrate a steep dose-response curve to a variety of agents, most notably mitoxantrone. PATIENTS AND METHODS A phase I study of escalated bolus mitoxantrone (10 to 25 mg/m2 x 3) and cyclophosphamide (30 to 50 mg/kg x 3) with a 5-day infusion of carboplatin (1,500 mg/m2) and an autologous bone marrow transplant (ABMT) was performed. Mitoxantrone pharmacokinetics were performed to document levels required to kill platinum-resistant ovarian carcinoma in vitro. RESULTS We treated 25 patients; the maximum-tolerated total doses (MTD) were 75 mg/m2 for mitoxantrone, 120 mg/kg for cyclophosphamide, and 1,500 mg/m2 for carboplatin. The dose-limiting toxicity was gastrointestinal, with severe diarrhea, ileus, and resulting sepsis. Transient partial deafness was seen in four patients, and acute renal failure (ARF) occurred in one patient at the first dose level, but was eliminated in subsequent patients with aggressive hydration. There were four early deaths due to ARF (n = 1), Legionella pneumonia (n = 1), and sepsis (n = 2). Peak mitoxantrone levels at the MTD were 623 to 2,810 ng/mL, and the area under the curve (AUC) values of the concentration versus time measurements were 560 to 1,700 ng/mL/h. Of 20 assessable patients, 65% responded, with a 45% complete remission (CR) rate. All six of the assessable patients with ovarian cancer responded: CR in five (83%) and partial remission (PR) in one (17%); the CRs have lasted 7 to 30+ months. Responses were also seen in testicular and breast carcinoma. CONCLUSION This regimen was well tolerated at the MTD and appears promising for relapsed/refractory ovarian carcinoma, with mitoxantrone levels achieved that are active in vitro against platinum-resistant ovarian carcinoma cells.

Long-wavelength spin waves in La 1− x Ca x MnO 3 CMR materials exhibit conventional Heisenberg dispersion ( E = Dq 2 + Δ ), but with an anomalous temperature dependence for the spin dispersion parameter D . For x ≈0.33, near the optimum CMR composition, D remains approximately 50% of its T =0 K value before exhibiting a near-first-order transition at T c , instead of following the expected power law decrease as T approaches T c . No temperature hysteresis was observed in either D or in the magnetization. Compositions with x =0.40 and 0.45 show more conventional temperature renormalization.

Common snipe (Gallinago gallinago delicata) harvest surveys and banding programs have been hampered by the inability to externally distinguish between age and sex cohorts. We measured 11 skeletal and 34 plumage characteristics on 152 male and 220 female snipe that had been internally sexed. We also compared coloration of plumage and soft parts between 131 hatch-year (HY) and 93 after-hatch-year (AHY) snipe that were aged internally using presence or absence of the bursa of Fabricius. Using discriminant analysis of morphological characteristics, we developed 5 discriminant models to sex common snipe. Discriminant equations for sexing common snipe contained 1-4 variables and were tested using cross-validation (i.e., jacknifing) and an independent sample of 99 birds. Common snipe could be correctly sexed 73-91% of the time, depending on the model. To facilitate a parts-collection survey to determine sex ratio in the harvest, we derived 3 discriminant models using strictly feather measurements. After-hatch-year and HY common snipe showed no consistent differences in plumage and soft part coloration. However, once snipe were sexed, they could be aged using 1 of 4 discriminant functions that aged snipe with 77.5-84.1% accuracy. These aging and sexing techniques will be useful in monitoring snipe populations, establishing harvest surveys, and conducting future research on common snipe.

Predominant methods for analyzing dose- or concentration-effect data (i.e., probit analysis) are based on the concept of individual tolerance or individual effective dose (IED, the smallest characteristic dose needed to kill an individual). An alternative explanation (stochasticity hypothesis) is that individuals do not have unique tolerances: death results from stochastic processes occurring similarly in all individuals. These opposing hypotheses were tested with two types of experiments. First, time to stupefaction (TTS) was measured for zebra fish (Brachydanio rerio) exposed to benzocaine. The same 40 fish were exposed during five trials to test if the same order for TTS was maintained among trials. The IED hypothesis was supported with a minor stochastic component being present. Second, eastern mosquitofish (Gambusia holbrooki) were exposed to sublethal or lethal NaCl concentrations until a large portion of the lethally exposed fish died. After sufficient time for recovery, fish sublethally exposed and fish surviving lethal exposure were exposed simultaneously to lethal NaCl concentrations. No statistically significant effect was found of previous exposure on survival time but a large stochastic component to the survival dynamics was obvious. Repetition of this second type of test with pentachlorophenol also provided no support for the IED hypothesis. We conclude that neither hypothesis alone was the sole or dominant explanation for the lognormal (probit) model. Determination of the correct explanation (IED or stochastic) or the relative contributions of each is crucial to predicting consequences to populations after repeated or chronic exposures to any particular toxicant.

In classical pharmacology, oral bioavailability of a toxicant is defined as that fraction of an orally administered dose reaching the systemic circulation of the animal. The present study estimates the bioavailability of methylmercury in channel catfish (Ictalurus punctatus) by comparing concentrations in the blood through time after oral and intra-arterial (IA) administration. Catfish were cannulated in the dorsal aorta and gavaged a pelleted feed that had been spiked with methylmercury. Each catfish was gavaged an increasing amount of spiked feed. Following oral dosing, serial blood samples were removed for more than 1,500 h. One month after removal of the last blood sample, the same fish were injected IA with methylmercury and serial blood samples were removed for more than 3,000 h. The area under the curve of the blood concentration-time curve extrapolated to infinity (AUC0→∞) was calculated from fish dosed orally and IA using both noncompartmental (trapezoidal) and compartmental methods. Bioavailability was estimated as the ratio of the dose-corrected oral AUC0→∞ to the IA AUC0→∞. Average bioavailability estimates from this approach were 33% using noncompartmental (range 14–55%) and 29% using compartmental (range 12–42%) methods and were correlated with the amount of food gavaged to the fish (r2 = 0.95, p = 0.026). Bioavailability estimates using the present methods were much lower than estimates using more conventional methods (i.e., assimilation efficiency estimates using mass balance), suggesting that conventional methods may overestimate the true bioavailability of toxicants in fish.