Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Effect of digitonin on rat myometrium subcellular membrane fractions

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry

Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Electronic Cigarette and Traditional Cigarette Use among Middle and High School Students in Florida, 2011–2014

Recent youth trends in the prevalence of e-cigarette and traditional cigarette use in Florida were examined in a cross-sectional, representative state sample from 2011 to 2014. Traditional cigarette use among youth declined during the study period. Experimentation with and past 30-day use of e-cigarettes among Florida youth tripled over 4 years. Past 30-day e-cigarette use exceeded traditional cigarette use in 2014; 10.8% of high school and 4.0% of middle school students reported recent e-cigarette use, compared with 8.7% of high school and 2.9% of middle school students for traditional cigarettes (P<0.001). By 2014, 20.5% of high school and 8.5% of middle school students reported ever use of e-cigarettes. Among ever e-cigarette users in 2014, 30.3% of high school and 42.2% of middle school students had never smoked traditional cigarettes. Given the concern that significant rates of e-cigarette use by U.S. adolescents may have a negative effect on public health, further review of e-cigarette advertising, marketing, sales, and use among U.S. youth is warranted.