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Burrowing organisms augment the availability of important resources for other species. The gopher tortoise (Gopherus polyphemus) is a keystone excavator in open canopy pine-forest ecosystems in the southeastern United States because its burrows are utilized by over 360 species. Across its range, the gopher tortoise is declining, which is thought to negatively affect burrow-associated species and ecosystem functionality. The nine-banded armadillo (Dasypus novemcinctus) is another burrower of similar size that has become syntopically distributed with the gopher tortoise as a result of range expansion. Recent studies have documented vertebrates utilizing armadillo burrows, linking armadillo burrowing to support of local biodiversity similar to the gopher tortoise. We sought to determine the potential for ecological redundancy between gopher tortoises and armadillos and test quantitatively for differences in associate events at their burrows in a mixed-pine–hardwood forest where they co-occur. Using motion activated game cameras to monitor burrows, we compared metrics of vertebrate occurrence between armadillo and tortoise burrows and examined the effects of environmental variables using a series of regression models. A total of 40 vertebrate taxa were observed visiting burrows between October 2019 and December 2020. Richness, diversity, and community composition were not significantly different between the two burrow types. However, associate event counts were significantly greater at tortoise burrows. Burrow and microhabitat variables had varying effects on associate event counts, with consistently positive effects for tortoise burrows, active burrows, and increased richness of tree species, while negative effects were detected for increased canopy cover as well as increased proportions of hardwood trees. Our study provides a framework for testing redundancy of function between syntopic ecosystem engineers, adds to the growing body of work on the ecological significance of armadillo range expansion, and identifies aspects of the habitat that cause fluctuations in the importance of burrows for associate species. [ABSTRACT FROM AUTHOR]

The electrocardiogram (ECG) empowered clinician scientists to measure the electrical activity of the heart noninvasively to identify arrhythmias and heart disease. Shortly after the standardization of the 12-lead ECG for the diagnosis of heart disease, several families with autosomal recessive (Jervell and Lange-Nielsen Syndrome) and dominant (Romano–Ward Syndrome) forms of long QT syndrome (LQTS) were identified. An abnormally long heart rate-corrected QT-interval was established as a biomarker for the risk of sudden cardiac death. Since then, the International LQTS Registry was established; a phenotypic scoring system to identify LQTS patients was developed; the major genes that associate with typical forms of LQTS were identified; and guidelines for the successful management of patients advanced. In this review, we discuss the molecular and cellular mechanisms for LQTS associated with missense variants in KCNQ1 (LQT1) and KCNH2 (LQT2). We move beyond the "benign" to a "pathogenic" binary classification scheme for different KCNQ1 and KCNH2 missense variants and discuss gene- and mutation-specific differences in K+ channel dysfunction, which can predispose people to distinct clinical phenotypes (e.g., concealed, pleiotropic, severe, etc.). We conclude by discussing the emerging computational structural modeling strategies that will distinguish between dysfunctional subtypes of KCNQ1 and KCNH2 variants, with the goal of realizing a layered precision medicine approach focused on individuals. [ABSTRACT FROM AUTHOR]

This study was undertaken to determine whether combined elastic and free weight resistance (CR) provides different strength and power adaptations than free weight resistance (FWR) training alone. Forty-four young (age 20 [+ or -] 1 years), resistance-trained (4 [+ or -] 2 years' experience) subjects were recruited from men's basketball and wrestling teams and women's basketball and hockey teams at Cornell University. Subjects were stratified according to team, then randomly assigned to the control (C; n = 21) or experimental group (E; n = 23). Before and after 7 weeks of resistance training, subjects were tested for lean body mass, 1 repetition maximum back squat and bench press, and peak and average power. Both C and E groups performed identical workouts except that E used CR (i.e., elastic resistance) for the back squat and bench press, whereas the C group used FWR alone. CR was performed using an elastic bungee cord attached to a standard barbell loaded with plates. Elastic tension was accounted for in an attempt to equalize the total work done by each group. Statistical analyses revealed significant (P < 0.05) between-group differences after training. Compared with C, improvement for E was nearly three times greater for back squat (16.47 [+ or -] 5.67 vs. 6.84 [+ or -] 4.42 kg increase), two times greater for bench press (6.68 [+ or -] 3.41 vs. 3.34 [+ or -] 2.67 kg increase), and nearly three times greater for average power (68.55 [+ or -] 84.35 vs. 23.66 [+ or -] 40.56 watt increase). Training with CR may be better than FWR alone for developing lower and upper body strength, and lower body power in resistance-trained individuals. Long-term effects are unclear, but CR training makes a meaningful contribution in the short term to performance adaptations of experienced athletes. KEY WORDS variable resistance, physical training, bungee cords, rubber bands

KCNH2 (hERG1) encodes the [alpha]-subunit proteins for the rapidly activating delayed rectifier [K.sup.+] current ([I.sub.Kr]), a major [K.sup.+] current for cardiac myocyte repolarization. In isolated myocytes [I.sub.Kr] frequently is small in amplitude or absent, yet KCNH2 channels and [I.sub.Kr] are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and [I.sub.Kr] are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed [K.sup.+], [Na.sup.+], and L-type [Ca.sup.2+] channels, and in ventricular myoctyes [I.sub.Kr], slowly activating delayed rectifier [K.sup.+] current ([I.sub.Ks]), and inward rectifier [K.sup.+] current ([I.sub.1]), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current ([I.sub.KCNH2]) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished [I.sub.Kr]; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of [I.sub.KCNH2] and its mature channel protein over several hours. Thus the channel protein for [I.sub.KCNH2] and [I.sub.Kr] is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low [I.sub.Kr] density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells. rapidly activating delayed rectifier potassium current; enzymes; myocyte isolation

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1) to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2) to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies. ion channels; arrhythmia; protein trafficking; patch clamp

Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels. Am J Physiol Heart Circ Physiol 286: H2434-H2441. 2004. First published February 19, 2004; 10.1152/ajpheart.00891.2003.--Long QT syndrome (LQTS) is a cardiac repolarization disorder that can lead to arrhythmias and sudden death. Chromosome 7-linked inherited LQTS (LQT2) is caused by mutations in human ether-a-go-go-related gene (HERG; KCNH2), whereas drug-induced LQTS is caused primarily by HERG channel block. Many common polymorphisms are functionally silent and have been traditionally regarded as benign and without physiological consequence. However, the identification of common nonsynonymous single nucleotide polymorphisms (nSNPs; i.e., amino-acid coding variants) with functional phenotypes in the SCN5A N[a.sup.+] channel and MiRP1 [K.sup.+] channel [beta]-subunit have challenged this viewpoint. In this report, we test the hypothesis that common missense HERG polymorphisms alter channel physiology. Comprehensive mutational analysis of HERG was performed on genomic DNA derived from a population-based cohort of sudden infant death syndrome and two reference allele cohorts derived from 100 African American and 100 Caucasian individuals. Amino acid-encoding variants were considered common polymorphisms if they were present in at least two of the three study cohorts with an allelic frequency >0.5%. Four nSNPs were identified: K897T, P967L, R1047L, and Q1068R. Wild-type (WT) and polymorphic channels were heterologously expressed in human embryonic kidney cells, and biochemical and voltage-clamp techniques were used to characterize their functional properties. All channel types were processed similarly, but several electrophysiological differences were identified: 1) K897T current density was lower than the other polymorphic channels; 2) K897T channels activated at more negative potentials than WT and R1047L; 3) K897T and Q1068R channels inactivated and recovered from inactivation faster than WT, P967L, and R1047L channels; and 4) K897T channels showed subtle differences compared with WT channels when stimulated with an action potential waveform. In contrast to K897T and Q1068R channels, P967L and R 1047L channels were electrophysiologically indistinguishable from WT channels. All HERG channels had similar sensitivity to block by cisapride. Therefore, some HERG polymorphic channels are electrophysiologically different from WT channels. ion channels; genetics; long QT syndrome; arrhythmia; sudden death

Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol 283: H77-H84, 2002. First published March 7, 2002; 10.1152/ajpheart.00008. 2002.--The human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability. heart; arrhythmia; ion channels; mutations; patch clamp

Some large-seeded plants lack effective seed dispersal agents when they are introduced as ornamental plants to new areas, but can rapidly colonize a landscape if seed dispersal functions are restored. We examined whether Gopherus polyphemus (Gopher Tortoise) facilitated the spread of Chrysobalanus icaco (Cocoplum; Chrysobalanaceae) over a 14-year period in a suburban nature preserve (in Jupiter, FL, USA) by: (i) comparing germination patterns among gut-passed, hand-depulped and whole fruit treatments, and (ii) testing hypotheses about environmental predictors of the spatial distribution of C. icaco , including information about G. polyphemus movement pathways and burrow locations. While we did not find a significant difference in the total proportion of C. icaco seeds that germinated in each treatment, time to event analysis revealed that seeds that were found in faeces germinated significantly earlier than seeds that were hand-depulped or that were planted as whole fruits, supporting a lone scarification effect. Point process modeling revealed that the density of C. icaco bushes was higher near G. polyphemus movement pathways and was lower inside Serenoa repens (Saw Palmetto) patches, supporting a positive effect of tortoise movement patterns on plant distributions. The density of C. icaco increased from west to east, consistent with westward dispersal from the four founder bushes on the east side of the study area. After removal of outliers, we also detected a negative association between C. icaco spatial density and G. polyphemus burrow density that was presumably explained by the fact that seeds defecated deep within burrows were unlikely to germinate and establish without secondary movement. The results suggest that G. polyphemus contributed to the rapid dispersal of C. icaco by scatter dispersal of seeds (via faeces) in areas where tortoises were active and that movement pathways provided suitable conditions for colonization. The spread of C. icaco by G. polyphemus over a relatively short period of time provides a valuable window into the earliest stages of the colonization process and further supports the role of Chelonians as effective seed dispersal agents for large-seeded plants. [ABSTRACT FROM AUTHOR]

Prohibitin (PHB) is a critical protein involved in many cellular activities. In brain, PHB resides in mitochondria, where it forms a large protein complex with PHB2 in the inner TFmembrane, which serves as a scaffolding platform for proteins involved in mitochondrial structural and functional integrity. PHB overexpression at moderate levels provides neuroprotection in experimental brain injury models. In addition, PHB expression is involved in ischemic preconditioning, as its expression is enhanced in preconditioning paradigms. However, the mechanisms of PHB functional regulation are still unknown. Observations that nitric oxide (NO) plays a key role in ischemia preconditioning compelled us to postulate that the neuroprotective effect of PHB could be regulated by NO. Here, we test this hypothesis in a neuronal model of ischemia-reperfusion injury and show that NO and PHB are mutually required for neuronal resilience against oxygen and glucose deprivation stress. Further, we demonstrate that NO post-translationally modifies PHB through protein S-nitrosylation and regulates PHB neuroprotective function, in a nitric oxide synthase-dependent manner. These results uncover the mechanisms of a previously unrecognized form of molecular regulation of PHB that underlies its neuroprotective function. [ABSTRACT FROM AUTHOR]

The nine-banded armadillo (Dasypus novemcinctus) is unique among mammals because females produce litters of genetically identical quadruplets via monozygotic polyembryony. This unusual form of reproduction could have profound impacts on the spatial genetic structure of populations of armadillos, but at present it is unclear whether littermates remain together as adults, or if sex-biased dispersal occurs. The goal of our study was to determine whether fine-scale spatial genetic structure (FSGS) within a population of armadillos diminishes with age (i.e., from juvenile to adult due to the dispersal of littermates away from one another), and if the degree of FSGS for a given age class differs between the sexes. We obtained genotype data at seven microsatellite DNA loci for 421 individuals in a wild population of armadillos inhabiting the Yazoo National Wildlife Refuge in western Mississippi. Correlogram analyses based on measures of spatial genetic autocorrelation showed weak but significant FSGS that was driven by positive spatial genetic autocorrelation among both male and female juveniles and adult males, but not adult females. Positive spatial genetic autocorrelation among adult males could be due to either female-biased dispersal or high variance in male reproductive success. Further work is required to discriminate between these two possibilities. [ABSTRACT FROM AUTHOR]

In southern Florida, Gopher Tortoises (Gopherus polyphemus) occupy mesic flatwoods and Florida scrub communities where habitat and climatic conditions differ from other portions of the species' range. Both of these habitats appear suboptimal for tortoises due to saturated soils in mesic flatwoods and low forage abundance in scrub. Nonetheless, these habitats support large numbers of tortoises in southern Florida, albeit at low intensities. We assessed influences of elevation and microtopography on the spatial distributions of tortoise burrows and examined burrow use patterns within six sites at Avon Park Air Force Range in south-central Florida. The six sites differed in dominant soil types and vegetation communities, allowing comparisons of burrow distributions among mesic flatwoods, Florida scrub, and mixed flatwoods-scrub habitats (two replicate sites each). Point-process modeling identified significant influences of microtopography on burrow intensities that superseded the effects of site-wide elevation trends in five of the six sites. The effects of microtopography were most pronounced in flatwoods, suggesting greater reliance on areas of slightly higher elevation in mesic habitat, presumably in response to saturated soils and frequent flooding. Burrow use patterns during an exceedingly wet year also suggested that tortoises respond behaviorally to unsuitable hydrology by moving frequently among burrows that were flooded with groundwater. Microtopographic variation may be an important predictor of small-scale habitat use for fossorial reptiles, especially in mesic soils, which could be readily explored using increasingly available LiDAR-derived elevation data combined with the modeling approach demonstrated here. [ABSTRACT FROM AUTHOR]

Abstract: Prohibitin (PHB) is a ubiquitously expressed and evolutionarily conserved mitochondrial protein with multiple functions. We have recently shown that PHB up‐regulation offers robust protection against neuronal injury in models of cerebral ischemia in vitro and in vivo, but the mechanism by which PHB affords neuroprotection remains to be elucidated. Here, we manipulated PHB expression in PC12 neural cells to investigate its impact on mitochondrial function and the mechanisms whereby it protects cells exposed to oxidative stress. PHB over‐expression promoted cell survival, whereas PHB down‐regulation diminished cell viability. Functionally, manipulation of PHB levels did not affect basal mitochondrial respiration, but it increased spare respiratory capacity. Moreover, PHB over‐expression preserved mitochondrial respiratory function of cells exposed to oxidative stress. Preserved respiratory capacity in differentiated PHB over‐expressing cells exposed to oxidative stress was associated with an elongated mitochondrial morphology, whereas PHB down‐regulation enhanced fragmentation. Mitochondrial complex I oxidative degradation was attenuated by PHB over‐expression and increased in PHB knockdown cells. Changes in complex I degradation were associated with alterations of respiratory chain supercomplexes. Furthermore, we showed that PHB directly interacts with cardiolipin and that down‐regulation of PHB results in loss of cardiolipin in mitochondria, which may contribute to destabilizing respiratory chain supercomplexes. Taken together, these data demonstrate that PHB modulates mitochondrial integrity and bioenergetics under oxidative stress, and suggest that the protective effect of PHB is mediated by stabilization of the mitochondrial respiratory machinery and its functional capacity, by the regulation of cardiolipin content. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/ [ABSTRACT FROM AUTHOR]

KCNH2 encodes the Kv11.1 a-subunit that underlies the rapidly activating delayedrectifier K+ current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805CKv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805CKv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular retention. [ABSTRACT FROM AUTHOR]

Background: Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome.Methods: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential.Results: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration.Conclusions: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying congenital long QT syndrome (LQTS) are now beginning to be understood. New insights into the etiology and therapeutic strategies are emerging from heterologous expression studies of LQTS-linked mutant proteins, as well as inducible pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from LQTS patients. This review focuses on the major molecular mechanism that underlies LQTS type 2 (LQT2). LQT2 is caused by loss of function (LOF) mutations in KCNH2 (also known as the human Ether-à-go-go-Related Gene or hERG ). Most LQT2-linked mutations are missense mutations and functional studies suggest that ~90% of them disrupt the intracellular transport (trafficking) of KCNH2 -encoded Kv11.1 proteins to the cell membrane. Trafficking deficient LQT2 mutations disrupt Kv11.1 protein folding and misfolded Kv11.1 proteins are retained in the endoplasmic reticulum (ER) until they are degraded in the ER associated degradation pathway (ERAD). This review focuses on the quality control mechanisms in the ER that contribute to the folding and ERAD of Kv11.1 proteins; the mechanism for ER export of Kv11.1 proteins in the secretory pathway; different subclasses of trafficking deficient LQT2 mutations; and strategies being developed to mitigate or correct trafficking deficient LQT2-related phenotypes. [ABSTRACT FROM AUTHOR]

The nine-banded armadillo ( Dasypus novemcinctus) is the only known nonhuman reservoir of Mycobacterium leprae, the causative agent of Hansen's disease or leprosy. We conducted a 6-yr study on a wild population of armadillos in western Mississippi that was exposed to M. leprae to evaluate the importance of demographic and spatial risk factors on individual antibody status. We found that spatially derived covariates were not predictive of antibody status. Furthermore, analyses revealed no evidence of clustering by antibody-positive individuals. Lactating females and adult males had higher odds of being antibody positive than did nonlactating females. No juveniles or yearlings were antibody positive. Results of these analyses support the hypothesis that M. leprae infection patterns are spatially homogeneous within this armadillo population. Further research related to movement patterns, contact among individuals, antibody status, and environmental factors could help address hypotheses related to the role of environmental transmission on M. leprae infection and the mechanisms underlying the differential infection patterns among demographic groups. [ABSTRACT FROM AUTHOR]

Ten microsatellite loci were characterized for 34 locations from roundtail chub (Gila robusta complex) to better resolve patterns of genetic variation among local populations in the lower Colorado River basin. This group has had a complex taxonomic history and previous molecular analyses failed to identify species diagnostic molecular markers. Our results supported previous molecular studies based on allozymes and DNA sequences, which found that most genetic variance was explained by differences among local populations. Samples from most localities were so divergent species-level diagnostic markers were not found. Some geographic samples were discordant with current taxonomy due to admixture or misidentification; therefore, additional morphological studies are necessary. Differences in spatial genetic structure were consistent with differences in connectivity of stream habitats, with the typically mainstem species, G. robusta, exhibiting greater genetic connectedness within the Gila River drainage. No species exhibited strong isolation by distance over the entire stream network, but the two species typically found in headwaters, G. nigra and G. intermedia, exhibited greater than expected genetic similarity between geographically proximate populations, and usually clustered with individuals from the same geographic location and/or sub-basin. These results highlight the significance of microevolutionary processes and importance of maintaining local populations to maximize evolutionary potential for this complex. Augmentation stocking as a conservation management strategy should only occur under extreme circumstances, and potential source populations should be geographically proximate stocks of the same species, especially for the headwater forms. [ABSTRACT FROM AUTHOR]

Several mutations in the human ether-a-go-go-related gene (HERG of KCNH2)-encoded [K.sup.+] channel disrupt intracellular ion channel transport (trafficking) and cause long QT syndrome (LQT2). Compared with WT HERG, trafficking-defective LQT2 channels have a phenotype of minimal of absent complex glycosylation and poor surface membrane expression. We report a novel HERG mutation in the sixth transmembrane segment (F640V) in a family with a history of sudden death, syncope, and long QT syndrome. Heterologous expression of F640V showed a trafficking-defective phenotype, and this phenotype was corrected by a 24-h incubation in the HERG channel blocker E4031(10 mM). We then tested the hypothesis that mutating the putative drug-binding domain at position Y652 would alter the effect of E4031. In a WT background, the Y652C mutation did not alter the WT glycosylation pattern but reduced [I.sub.HERG] (WT = 108 [+ or -] 20pA/pF, n = 9; Y652C 31 [+ or -] 8pA/pF, n = 6), presumably by altering channel function. Y652C [I.sub.HERG] also was less sensitive than WT to E4031(100 nM) block; [I.sub.HERG] WT = 17 + 1% (n = 5) and Y652C = 78 [+ or -] 3% (n = 3). We next tested the double mutation F640V-Y652C. In contrast to F640V, F640V-Y652C was complexly glycosylated; confocal microscopy confirmed cell surface expression; and [I.sub.HERG] was increased (F640V = 8 [+ or -] 1 pA/pF, n = 13; F640V-Y652C = 22 [+ or -] 2 pA/pF, n = 10) to a level similar to Y652C alone. We also tested the trafficking-defective G601S mutation and obtained similar findings; G601S-Y652C was complexly glycosylated and [I.sub.HERG] density was comparable to that of Y652C alone (G601S = 39 [+ or -] 8 pA/pF, n = 6; G601S-Y652C = 25 [+ or -] 7 pA/pF, n = 5). E4031 (10 [micro]M) incubation (24 h) did not alter the glycosylation pattern or [I.sub.HERG] in F640V-Y652C of G601S-Y652C. We conclude that mutagenic modification of the inner pore can restore the processing of several trafficking-defective LQT2 channel proteins. (Supported in part by RO1 HL60723 to Craig T. January and F32 HL071476-01 to Brian Delisle.)

The objective of this study was to optimize the methodology for spectrophotometric determination of sperm concentration in Atlantic croaker Micropogonias undulatus L. milt and to estimate its potential for short-term cold-storage. The spectrophotometric determination of sperm concentration was evaluated using milt samples from six males serially diluted in Hank's balanced salt solution at 200 mOsm kg−1 (HBSS). The predictive power of regression models between sperm concentration and absorbance was determined from 200 to 500 nm and found to be highest within the visible spectrum despite a peak of milt absorbance at 288 nm. Absorbance reading at 400 nm was selected for further analysis to maximize the absorbance of the sample hence the sensitivity of the method while minimizing the impact of potential sample contamination with blood. The standard-curve of correlation between sperm absorbance at 400 nm and concentration was validated and held an accuracy ranging between −7.40% and +4.56% across males. Total sperm motility duration and the proportion of motile spermatozoa were significantly higher in milt samples diluted 1:3 in HBSS than in the undiluted control during up to 30 h of cold-storage.The methodologies investigated in this study can be applied to optimize sperm usage and achieve predictable artificial fertilization protocols in Atlantic croaker. [ABSTRACT FROM AUTHOR]

The addition of α-deprotonated nitriles to azaheterocycles followed by rearomatization is described. A simple two-step, one-pot procedure for the sequence is also presented. [ABSTRACT FROM AUTHOR]

Abstract: Calcitonin gene-related peptide (CGRP) has been implicated in acute migraine pathogenesis. In an effort to identify novel CGRP receptor antagonists for the treatment of migraine, we have discovered thiazolidinone 49, a potent (K i =30pM, IC50 =1nM), orally bioavailable, CNS-penetrant CGRP antagonist with good pharmacokinetic properties. [Copyright &y& Elsevier]

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubuledependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant- negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential. [ABSTRACT FROM AUTHOR]