A Store-operated Calcium Channel in Drosophila S2 Cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells

Cryin\u27 for the Moon

Woman sitting down looking at moon through windowhttps://scholarsjunction.msstate.edu/cht-sheet-music/9116/thumbnail.jp

The mtDNA diversity of captive ruffed lemurs (Varecia spp.): Implications for conservation

Ruffed lemurs (Varecia variegata and V. rubra) are considered Critically Endangered, and genetic studies are therefore needed for assessing the conservation value of captive populations. Using 280 mitochondrial DNA (mtDNA) D-loop sequences, we studied the genetic diversity and structure of captive ruffed lemurs in Madagascar, Europe and North America. We found 10 new haplotypes, one from the European captive V. rubra population, three from captive V. variegata subcincta (one from Europe and two from Madagascar), and six from other captive V. variegata in Madagascar. There was low mtDNA genetic diversity in the European and North American captive populations of V. variegata. Several founder individuals shared the same mtDNA haplotype, and therefore should perhaps not be considered as unrelated founders for making breeding recommendations. The captive population in Madagascar has high genetic diversity, including haplotypes not yet identified in wild populations. The likely geographical provenance of founders of captive populations was determined by comparison with previous studies; all reported haplotypes from captive ruffed lemurs were identical to, or clustered with, haplotypes from wild populations located north of the Mangoro River in Madagascar. Effective conservation strategies for wild populations, with potentially unidentified genetic diversity, should still be considered the priority for conserving ruffed lemurs. However, our results illustrate that the captive population in Madagascar has conservation value as a source of potential release stock for reintroduction or reinforcement projects, and that cross-regional transfers within the global captive population could increase the genetic diversity and therefore the conservation value of each regional population

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth human (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa Gh was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26576/1/0000115.pd

Estimating genetic diversity across the neutral genome with the use of dense marker maps

<p>Abstract</p> <p>Background</p> <p>With the advent of high throughput DNA typing, dense marker maps have become available to investigate genetic diversity on specific regions of the genome. The aim of this paper was to compare two marker based estimates of the genetic diversity in specific genomic regions lying in between markers: IBD-based genetic diversity and heterozygosity.</p> <p>Methods</p> <p>A computer simulated population was set up with individuals containing a single 1-Morgan chromosome and 1665 SNP markers and from this one, an additional population was produced with a lower marker density i.e. 166 SNP markers. For each marker interval based on adjacent markers, the genetic diversity was estimated either by IBD probabilities or heterozygosity. Estimates were compared to each other and to the true genetic diversity. The latter was calculated for a marker in the middle of each marker interval that was not used to estimate genetic diversity.</p> <p>Results</p> <p>The simulated population had an average minor allele frequency of 0.28 and an LD (r²) of 0.26, comparable to those of real livestock populations. Genetic diversities estimated by IBD probabilities and by heterozygosity were positively correlated, and correlations with the true genetic diversity were quite similar for the simulated population with a high marker density, both for specific regions (r = 0.19-0.20) and large regions (r = 0.61-0.64) over the genome. For the population with a lower marker density, the correlation with the true genetic diversity turned out to be higher for the IBD-based genetic diversity.</p> <p>Conclusions</p> <p>Genetic diversities of ungenotyped regions of the genome (i.e. between markers) estimated by IBD-based methods and heterozygosity give similar results for the simulated population with a high marker density. However, for a population with a lower marker density, the IBD-based method gives a better prediction, since variation and recombination between markers are missed with heterozygosity.</p

Identifying priorities in methodological research using ICD-9-CM and ICD-10 administrative data: report from an international consortium

BACKGROUND: Health administrative data are frequently used for health services and population health research. Comparative research using these data has been facilitated by the use of a standard system for coding diagnoses, the International Classification of Diseases (ICD). Research using the data must deal with data quality and validity limitations which arise because the data are not created for research purposes. This paper presents a list of high-priority methodological areas for researchers using health administrative data. METHODS: A group of researchers and users of health administrative data from Canada, the United States, Switzerland, Australia, China and the United Kingdom came together in June 2005 in Banff, Canada to discuss and identify high-priority methodological research areas. The generation of ideas for research focussed not only on matters relating to the use of administrative data in health services and population health research, but also on the challenges created in transitioning from ICD-9 to ICD-10. After the brain-storming session, voting took place to rank-order the suggested projects. Participants were asked to rate the importance of each project from 1 (low priority) to 10 (high priority). Average ranks were computed to prioritise the projects. RESULTS: Thirteen potential areas of research were identified, some of which represented preparatory work rather than research per se. The three most highly ranked priorities were the documentation of data fields in each country's hospital administrative data (average score 8.4), the translation of patient safety indicators from ICD-9 to ICD-10 (average score 8.0), and the development and validation of algorithms to verify the logic and internal consistency of coding in hospital abstract data (average score 7.0). CONCLUSION: The group discussions resulted in a list of expert views on critical international priorities for future methodological research relating to health administrative data. The consortium's members welcome contacts from investigators involved in research using health administrative data, especially in cross-jurisdictional collaborative studies or in studies that illustrate the application of ICD-10