Rie Tsutsumi

Diversity of Calcium Signaling by Metabotropic Glutamate Receptors

During prolonged application of glutamate (20 min), patterns of increase in intracellular Ca2+concentration ([Ca2+] i ) were studied in HEK-293 cells expressing metabotropic glutamate receptor, mGluR1α or mGluR5a. Stimulation of mGluR1α induced an increase in [Ca2+] i that consisted of an initial transient peak with a subsequent steady plateau or an oscillatory increase in [Ca2+] i . The transient phase was largely attributed to Ca2+mobilization from the intracellular Ca2+ stores, but the sustained phase was solely due to Ca2+ influx through the mGluR1α receptor-operated Ca2+ channel. Prolonged stimulation of mGluR5a continuously induced [Ca2+] i oscillations through mobilization of Ca2+ from the intracellular Ca2+ stores. Studies on mutant receptors of mGluR1α and mGluR5a revealed that the coupling mechanism in the sustained phase of Ca2+ response is determined by oscillatory/non-oscillatory patterns of the initial Ca2+response but not by the receptor identity. In mGluR1α-expressing cells, activation of protein kinase C selectively desensitized the pathway for intracellular Ca2+ mobilization, but the mGluR1α-operated Ca2+ channel remained active. In mGluR5a-expressing cells, phosphorylation of mGluR5a by protein kinase C, which accounts for the mechanism of mGluR5a-controlled [Ca2+] i oscillations, might prevent desensitization and result in constant oscillatory mobilization of Ca2+ from intracellular Ca2+ stores. Our results provide a novel concept in which oscillatory/non-oscillatory mobilizations of Ca2+ induce different coupling mechanisms during prolonged stimulation of mGluRs.

Signaling Responses to Pulsatile Gonadotropin-releasing Hormone in LβT2 Gonadotrope Cells

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile fashion by hypothalamic neurons, and alterations in pulse frequency and amplitude differentially regulate gonadotropin synthesis and release. In this study, we investigated the kinetics of Gs and Gq signaling in response to continuous or pulsatile GnRH using fluorescence resonance energy transfer reporters in live mouse LβT2 gonadotrope cells. cAMP and protein kinase A-dependent reporters showed a rapid but transient increase in fluorescence resonance energy transfer signal with increasing doses of constant GnRH, and in contrast diacylglycerol (DAG) and calcium reporters showed a rapid and sustained signal. Multiple pulses of GnRH caused multiple pulses of cAMP and protein kinase A activation without desensitization, but the DAG and calcium reporters were rapidly desensitized resulting in inhibition of calcium and DAG responses. At the transcriptional level, both a cAMP-dependent cAMP-response element reporter and a DAG/calcium-dependent AP-1 reporter showed a pulse frequency-dependent increase in luciferase activity. However, constant GnRH stimulation gave very little cAMP-response element activation but very strong AP-1 activation. Based on these data, we propose that both the GnRH-R-Gs and Gq pathways are responsive to pulses of GnRH, but only the Gq pathway is responsive to constant GnRH. Furthermore, the Gq pathway is subject to desensitization with multiple GnRH pulses, but the Gs pathway is not.

In-vitro Characterization of YM872, a Selective, Potent and Highly Water-soluble α-Amino-3-hydroxy-5-methylisoxazole-4-propionate Receptor Antagonist

The in‐vitro pharmacological properties of (2,3‐dioxo‐7‐(1H‐imidazol‐***1‐yl)‐6‐nitro‐1,2,3,4‐tetrahydro‐1‐quinoxalinyl)‐acetic acid monohydrate, YM872, a novel and highly water‐soluble α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)‐receptor antagonist were investigated.

Compound K, a metabolite of ginsenosides, induces cardiac protection mediated nitric oxide via Akt/PI3K pathway.

AIMS Compound K (C-K; 20-O-D-glucopyranosyl-20(S)-protopanaxadiol) is a novel ginsenoside metabolite formed by intestinal bacteria and does not occur naturally in ginseng. In this study, we investigated whether administration of C-K has protective effects on myocardial ischemia-reperfusion injury and its potential mechanisms. MAIN METHODS We used in vivo mouse models of ischemia-reperfusion injury and performed biochemical assays in excised hearts. KEY FINDINGS C-K reduced infarct size compared with the control group after ischemia-reperfusion. Immunoblot analysis showed that C-K significantly enhanced protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) activity. Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, blocked cardiac protection in vivo and attenuated phosphorylation of Akt and eNOS. Additionally, the hearts of C-K pretreated mice showed inhibition of mitochondrial swelling induced by Ca(2+). SIGNIFICANCE This study showed that Compound K pretreatment has protective effects on myocardial ischemia-reperfusion injury, partly by mediating the activation of PI3K pathway and phosphorylation of Akt and eNOS.

tRFs: miRNAs in disguise

tRFs and tiRNAs are two new classes of regulatory non-coding small RNAs that are derived from the cleavage of pre-existing tRNAs. tRFs are 18-22 nt long and are classified into the tRF-5, tRF-3, and tRF-1 series. Here, we discuss in detail the regulatory roles of tRFs in translation, viral infections, and carcinogenesis. Moreover, we have reviewed the association of tRFs with Argonaute proteins, including their potential to function as miRNAs. Interestingly, few miRNAs are generated from pre-existing tRNAs. Hence, tRNAs generate similar-sized tRFs and miRNAs, leading to misannotations due to cross mapping of tRFs and tRNA-derived miRNAs during deep sequencing data analysis. Therefore, it is important to catalogue the overlapping sequences between tRNA-derived miRNAs and tRFs. We have catalogued the miRNAs that overlap with tRFs sequences in humans using miRBase. We identified 20 tRNA-derived miRNAs that share sequences with tRFs. Of the 20 miRNAs, 5 miRNAs (miR-3182, miR-4521, miR-1260a, miR-1260b, and miR-7977) showed significant prediction scores. Furthermore, we have identified a lysine degradation pathway as a common regulatory pathway for miR-1260a, miR-1260b, and miR-3182 by using DIANA-mirPath.

Role of the O-linked β-N -acetylglucosamine in the Cardioprotection Induced by Isoflurane

Background: Cardiac protection by volatile anesthetic-induced preconditioning and ischemic preconditioning have similar signaling pathways. Recently, it was reported that augmentation of protein modified with O-linked &bgr;-N-acetylglucosamine (O-GlcNAc) contributes to cardiac protection. This study investigated the role of O-GlcNAc in cardiac protection induced by anesthetic-induced preconditioning. Methods: O-GlcNAc-modified proteins were visualized by immunoblotting. Tolerance against ischemia or reperfusion was tested in vivo (n = 8) and in vitro (n = 6). The opening of the mitochondrial permeability transition pore (mPTP) upon oxidative stress was examined in myocytes treated with calcein AM (n = 5). Coimmunoprecipitation and enzymatic labeling were performed to detect the mitochondrial protein responsible for the mPTP opening. Results: Isoflurane treatment and the consequent augmentation of O-GlcNAc concentrations reduced the infarct size (26 ± 5% [mean ± SD], P < 0.001) compared with the control. The protective effect of O-GlcNAc was eliminated in the group pretreated with the O-GlcNAc transferase inhibitor alloxan (39 ± 5%, P < 0.001). Myocyte survival also showed the same result in vitro. Formation of the mPTP was abrogated in the isoflurane-treated cells (86 ± 4%, P < 0.001) compared with the control and alloxan-plus-isoflurane–treated cells (57 ± 7%, P < 0.001). Coimmunoprecipitation and enzymatic labeling studies revealed that the O-GlcNAc–modified, voltage-dependent anion channel restained the mPTP opening. Conclusions: Isoflurane induced O-GlcNAc modification of mitochondrial voltage-dependent anion channel. This modification inhibited the opening of the mPTP and conferred resistance to ischemia-reperfusion stress.

Non-coding RNAs: Functions and applications in endocrine-related cancer

A significant fraction of the human genome is transcribed as non-coding RNAs (ncRNAs). This non-coding transcriptome has challenged the notion of the central dogma and its involvement in transcriptional and post-transcriptional regulation of gene expression is well established. Interestingly, several ncRNAs are dysregulated in cancer and current non-coding transcriptome research aims to use our increasing knowledge of these ncRNAs for the development of cancer biomarkers and anti-cancer drugs. In endocrine-related cancers, for which survival rates can be relatively low, there is a need for such advancements. In this review, we aimed to summarize the roles and clinical implications of recently discovered ncRNAs, including long ncRNAs, PIWI-interacting RNAs, tRNA- and Y RNA-derived ncRNAs, and small nucleolar RNAs, in endocrine-related cancers affecting both sexes. We focus on recent studies highlighting discoveries in ncRNA biology and expression in cancer, and conclude with a discussion on the challenges and future directions, including clinical application. ncRNAs show great promise as diagnostic tools and therapeutic targets, but further work is necessary to realize the potential of these unconventional transcripts.

New insights into the genetic basis of infertility

Infertility is a disease of the reproductive system characterized by inability to achieve pregnancy after 12 or more months of regular unprotected sexual intercourse. A variety of factors, including ovulation defects, spermatogenic failure, parental age, obesity, and infections have been linked with infertility, in addition to specific karyotypes and genotypes. The study of genes associated with infertility in rodent models has expanded the field of translational genetics in identifying the underlying cause of human infertility problems. Many intriguing aspects of the molecular basis of infertility in humans remain poorly understood; however, application of genetic knowledge in this field looks promising. The growing literature on the genetics of human infertility disorders deserves attention and a critical concise summary is required. This paper provides information obtained from a systematic analysis of the literature related to current research into the genetics of infertility affecting both sexes.

Exendin-4 ameliorates cardiac ischemia/reperfusion injury via caveolae and caveolins-3

BackgroundExendin-4, an exogenous glucagon-like peptide-1 receptor (GLP-1R) agonist, protects the heart from ischemia/reperfusion injury. However, the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for exendin-4 induced cardiac protection using in vitro and in vivo studies in control and caveolin-3 (Cav-3) knockout mice (Cav-3 KO).MethodsMyocytes were treated with exendin-4 and then incubated with methyl-β-cyclodextrin (MβCD) to disrupt caveolae formation. This was then followed by simulated ischemia/reperfusion (SI/R). In addition, cardiac protection in vivo was assessed by measuring infarct size and cardiac troponin levels.ResultsExendin-4 protected cardiac myocytes (CM) from SI/R [35.6 ± 12.6% vs. 64.4 ± 18.0% cell death, P = 0.034] and apoptosis but this protection was abolished by MβCD (71.8 ± 10.8% cell death, P = 0.004). Furthermore, Cav-3/GLP-1R co-localization was observed and membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD  + exendin-4 revealed that buoyant (caveolae enriched) fractions decreased Cav-3 compared to CM treated with exendin-4 exclusively. Furthermore, exendin-4 induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 25.1 ± 8.2% vs. 41.4 ± 4.1%, P < 0.001; troponin: 36.9 ± 14.2 vs. 101.1 ± 22.3 ng/ml, P < 0.001). However, exendin-4 induced cardiac protection was abolished in Cav-3 KO mice (infarct size: 43.0 ± 6.4%, P < 0.001; troponin: 96.8 ± 26.6 ng/ml, P = 0.001).ConclusionsWe conclude that caveolae and caveolin-3 are critical for exendin-4 induced protection of the heart from ischemia/reperfusion injury.

Effects of chemotherapy on gene expression of lingual taste receptors in patients with head and neck cancer.

We aimed to test the hypothesis that chemotherapy changes the gene expression of taste receptors in the tongue to induce dysgeusia in patients with head and neck cancer.