Synergic Effects of β-Estradiol and Erythromycin on hERG Currents

The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties, the underlying mechanisms of the gender-based differences are not fully understood. In particular I _Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents were inhibited by β-estradiol maximally to 62% of control with an IC₅₀ of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin, which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced. Namely, hERG currents were inhibited maximally to 45.8% of control with an IC₅₀ of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher risk of drug-induced TDP in women than men.     

Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.     

Pharmacokinetics and Efficacy of Topically Applied Nonsteroidal Anti-Inflammatory Drugs in Retinochoroidal Tissues in Rabbits

Purpose

To evaluate the pharmacokinetics and efficacy of topically applied nonsteroidal anti-inflammatory drugs (NSAIDs) in the retinochoroidal tissues of rabbits.

Methods

The cyclooxygenase (COX) inhibitory activity of diclofenac, bromfenac, and amfenac, an active metabolite of nepafenac, were determined using human-derived COX-1 and COX-2. Each of the three NSAIDs was applied topically to rabbits, and after 0.5 to 8 hrs, the concentration of each drug in the aqueous humor and the retinochoroidal tissues was measured by liquid chromatography-tandem mass spectrometry. The pharmacokinetics of the drugs in the tissues after repeated doses as is done on patients was calculated by a simulation software. The inhibitory effect of each NSAID on the breakdown of the blood-retinal barrier was assessed by the vitreous protein concentration on concanavalin A-induced retinochoroidal inflammation in rabbits.

Results

The half-maximal inhibitory concentration (IC₅₀) of diclofenac, bromfenac, and amfenac was 55.5, 5.56, and 15.3 nM for human COX-1, and 30.7, 7.45, and 20.4 nM for human COX-2, respectively. The three NSAIDs were detected in the aqueous humor and the retinochoroidal tissue at all-time points. Simulated pharmacokinetics showed that the levels of the three NSAIDs were continuously higher than the IC₅₀ of COX-2, as an index of efficacy, in the aqueous humor, whereas only the bromfenac concentration was continuously higher than the IC₅₀ at its trough level in the retinochoroidal tissues. The intravitreous concentration of proteins was significantly reduced in rabbits that received topical bromfenac (P = 0.026) but not the other two NSAIDs.

Conclusions

Topical bromfenac can penetrate into the retinochoroidal tissues in high enough concentrations to inhibit COX-2 and exerts its inhibitory effect on the blood-retinal barrier breakdown in an experimental retinochoroidal inflammation in rabbits. Topical bromfenac may have a better therapeutic benefit than diclofenac and nepafenac for retinochoroidal inflammatory diseases.     

Developmental changes in chemotactic response and choice of two attractants, sodium acetate and diacetyl, in the nematode Caenorhabditis elegans

The chemotactic behavior of the nematode Caenorhabditis elegans to chemical attractants, water-soluble sodium acetate and odorant diacetyl, was investigated using nematodes at various developmental stages to examine the effects of postembryonic development on chemotactic response and spontaneous locomotion. The chemotactic responses to attractants increased as development progressed, and the largest responses to either 1.0 M sodium acetate or 0.1% diacetyl were seen at the young adult (YA) or day adult (A1) stage, respectively. Responses to the chemicals declined thereafter in-line with increasing age. The chemotaxis indices for attractants correlated with activity of spontaneous locomotion (p<0.01), suggesting that a change in spontaneous locomotion is one of the factors involved with the change in chemotactic responses during development. We also investigated the effect of aging on attractant choice by the simultaneous presentation of 0.6 M sodium acetate and 0.1% diacetyl. In the presence of both attractants, the fraction of larval animals at the sodium acetate location was greater than that at the diacetyl location (p<0.05). The fractions of YA animals that gathered at either location were almost identical, whereas the fraction of adult animals at the diacetyl location was greater than that at the sodium acetate location (p<0.05). The patterns of attractant choice of the long-lived daf-2 mutants and short lifespan mev-1 mutants showed the same tendency as those of wild type nematodes in the presence of both attractants. These results suggest that a change in the neuronal mechanisms controlling attractant choice and preference occurs during developmental progression.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.     

Effects of Prenatal Leydig Cell Function on the Ratio of the Second to Fourth Digit Lengths in School-Aged Children

Prenatal sex hormones can induce abnormalities in the reproductive system and adversely impact on genital development. We investigated whether sex hormones in cord blood influenced the ratio of the second to fourth digit lengths (2D/4D) in school-aged children. Of the 514 children who participated in a prospective cohort study on birth in Sapporo between 2002 and 2005, the following sex hormone levels were measured in 294 stored cord blood samples (135 boys and 159 girls); testosterone (T), estradiol (E), progesterone, LH, FSH, inhibin B, and insulin-like factor 3 (INSL3). A total of 350 children, who were of school age and could be contacted for this survey, were then requested via mail to send black-and-white photocopies of the palms of both the left and right hands. 2D/4D was calculated in 190 children (88 boys and 102 girls) using photocopies and derived from participants with the characteristics of older mothers, a higher annual household income, higher educational level, and fewer smokers among family members. 2D/4D was significantly lower in males than in females (p<0.01). In the 294 stored cord blood samples, T, T/E, LH, FSH, Inhibin B, and INSL3 levels were significantly higher in samples collected from males than those from females. A multivariate regression model revealed that 2D/4D negatively correlated with INSL3 in males and was significantly higher in males with <0.32 ng/mL of INSL3 (p<0.01). No correlations were observed between other hormones and 2D/4D. In conclusion, 2D/4D in school-aged children, which was significantly lower in males than in females, was affected by prenatal Leydig cell function.     

Accumulation of Squalene in a Microalga Chlamydomonas reinhardtii by Genetic Modification of Squalene Synthase and Squalene Epoxidase Genes

Several microalgae accumulate high levels of squalene, and as such provide a potentially valuable source of this useful compound. However, the molecular mechanism of squalene biosynthesis in microalgae is still largely unknown. We obtained the sequences of two enzymes involved in squalene synthesis and metabolism, squalene synthase (CrSQS) and squalene epoxidase (CrSQE), from the model green alga Chlamydomonas reinhardtii. CrSQS was functionally characterized by expression in Escherichia coli and CrSQE by complementation of a budding yeast erg1 mutant. Transient expression of CrSQS and CrSQE fused with fluorescent proteins in onion epidermal tissue suggested that both proteins were co-localized in the endoplasmic reticulum. CrSQS-overexpression increased the rate of conversion of ¹⁴C-labeled farnesylpyrophosphate into squalene but did not lead to over-accumulation of squalene. Addition of terbinafine caused the accumulation of squalene and suppression of cell survival. On the other hand, in CrSQE-knockdown lines, the expression level of CrSQE was reduced by 59–76% of that in wild-type cells, and significant levels of squalene (0.9–1.1 μg mg^–1 cell dry weight) accumulated without any growth inhibition. In co-transformation lines with CrSQS-overexpression and CrSQE-knockdown, the level of squalene was not increased significantly compared with that in solitary CrSQE-knockdown lines. These results indicated that partial knockdown of CrSQE is an effective strategy to increase squalene production in C. reinhardtii cells.     

Cage evaluation of augmentative biological control of Thrips palmi with Wollastoniella rotunda in winter greenhouses

Cage trials of an anthocorid predator, Wollastoniella rotunda Yasunaga et Miyamoto, as a biological control agent of Thrips palmi Karny were conducted in Fukuoka, Japan, under winter greenhouse production conditions. Females of W. rotunda were released on caged eggplants, and placed in two greenhouses on 27 October. The development, population growth, and effectiveness of W. rotunda were observed until early March. Results from the cage trials showed that W. rotunda successfully developed, reproduced, and suppressed T. palmi populations under the conditions found in winter greenhouses. During the experiment, one full generation and a second generation of adult predators occurred. The T. palmi population which was exposed to predators remained at a low density throughout the trial period, but it increased dramatically on eggplants without W. rotunda. The maximum difference between predator treatments and controls was approximately 10‐fold by the end of January. Wollastoniella rotunda has the potential to be an effective control agent for T. palmi on eggplant, even during the winter in temperate regions.     

Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library

Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.