Chini, Eduardo N.

The molecular mechanism that initiates Ca2+ signaling in sea urchin egg fertilization has not yet been clarified. To determine whether sea urchin sperm may generate and possibly supply cyclic ADP-ribose (cADPR) as a Ca2+-releasing factor in the course of sea urchin egg fertilization, we determined cADPR content and the capacity for cADPR synthesis in sea urchin sperm. cADPR content was determined using the sea urchin egg homogenate Ca2+-release bioassay combined with high-performance liquid chromatography (HPLC). We found that sperm homogenates synthesized cADPR from beta-NAD but did not synthesize cADPR when alpha-NAD was the substrate. The identity of cADPR generated by sperm homogenates was verified by HPLC analysis, use of specific Ca2+-release antagonists, and homologous desensitization of the sea urchin egg homogenate Ca2+-release bioassay. The ambient content of cADPR was approximately 0.3 nmol cADPR/g wet wt sea urchin sperm. Our results show that sperm can synthesize cADPR and that they contain cADPR levels comparable to other tissues.

In our previous study of plasma membrane Ca2+ pump (PMCA) isozymes in the rat kidney, we found that the mRNA coding isoform PMCA3 was detected primarily in the outer medullary zone of rat kidney tissue. We now investigated the location of the mRNAs coding for isoforms PMCA3 and PMCA4 of Ca2+ pump in the nephron segments that are present in outer medullary parenchyma using the method of reverse transcription and polymerase chain reaction. In mRNA extracted from whole dissected outer medulla we detected mRNA encoding three splice variants (a, b, and c) of isoform PMCA3; isoform PMCA4 was found in the outer medulla almost exclusively as variant b. Analysis of mRNA from microdissected tubule segments show that proximal straight tubules (PST), medullary thick ascending limbs, outer medullary collecting ducts, and descending thin limb of Henle's loop (DTL) all contained mRNA for isoform 4b. In contrast, the mRNA encoding isoform 3 was detected exclusively in DTL and not in other nephron segments. The unique presence of isoform 3 in DTL is rather surprising, since the specific role of this nephron segment in vectorial Ca2+ transport or in intracellular Ca2+ signaling is not known. The data suggest that isoform 3 in cells of the DTL may have a hitherto unrecognized specific role in Ca2+ signaling or transport of Ca2+, which is distinct from the role of the isoforms of the PMCA in all other nephron segments.

Volatile anesthetics have multiple actions on intracellular Ca2+homeostasis, including activation of the ryanodine channel (RyR) and sensitization of this channel to agonists such as caffeine and ryanodine. Recently it has been described that the nucleotide cADP-ribose (cADPR) is the endogenous regulator of the RyR in many mammalian cells, and cADPR has been proposed to be a second messenger in many signaling pathways. I investigated the effect of volatile anesthetics on the cADPR signaling system, using sea urchin egg homogenates as a model of intracellular Ca2+stores. Ca2+uptake and release were monitored in sea urchin egg homogenates by using the fluo-3 fluorescence technique. Activity of the ADP-ribosyl cyclase was monitored by using a fluorometric method using nicotinamide guanine dinucleotide as a substrate. Halothane in concentrations up to 800 μM did not induce Ca2+release by itself in sea urchin egg homogenates. However, halothane potentiates the Ca2+release mediated by agonists of the ryanodine channel, such as ryanodine. Furthermore, other volatile anesthetics such as isoflurane and sevoflurane had no effect. Halothane also potentiated the activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca2+release was decreased about three times by addition of 800 μM halothane. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to halothane. In contrast, all the volatile anesthetics used had no effect on the activity of the enzyme that synthesizes cADPR. I propose that the complex effect of volatile anesthetics on intracellular Ca2+homeostasis may involve modulation of the cADPR signaling system.

Abstract Purpose: Recent studies suggest that SIRT1-activating compounds (STAC) are a promising class of anticancer drugs, although their mechanism of action remains elusive. The main goal of this study is to determine the role of STACs as a potential therapy for pancreatic cancer. In addition, we also explored the mechanism by which these compounds affect pancreatic cancer. Experimental design: Using in vitro (cell culture experiments) and in vivo (xenograft experiments) approaches, we studied the role of SIRT1 agonists (STAC) in human pancreatic cancer cell viability and growth. Results: We show that SIRT1 is highly expressed in pancreatic cancer cells and that the STACs SRT1720, SRT1460, and SRT3025 inhibited cell growth and survival of pancreatic cancer cells. STACs enhanced the sensitivity of pancreatic cells to gemcitabine and paclitaxel, indicating that these drugs could be used in combination with other chemotherapy drugs. We also show that STACs were very effective in inhibiting tumor xenograft growth. In mechanistic studies, we observed that STACs activated a SIRT1 lysosomal–dependent cell death. Furthermore, the effect of STACs on cell viability was also dependent on the expression of the endogenous SIRT1 inhibitor DBC1. Conclusions: Taken together, our results reveal an essential role for SIRT1 and lysosomes in the death pathway regulated by STACs in pancreatic cancer cells. Clin Cancer Res; 22(10); 2496–507. ©2015 AACR.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the accumulation of kidney cysts that ultimately leads to loss of renal function and kidney failure. At present, the treatment for ADPKD is largely supportive. Multiple studies have focused on pharmacologic approaches to slow the development of the cystic disease; however, little is known about the role of nutrition and dietary manipulation in PKD. Here, we show that food restriction (FR) effectively slows the course of the disease in mouse models of ADPKD. Mild to moderate (10%–40%) FR reduced cyst area, renal fibrosis, inflammation, and injury in a dose-dependent manner. Molecular and biochemical studies in these mice indicate that FR ameliorates ADPKD through a mechanism involving suppression of the mammalian target of the rapamycin pathway and activation of the liver kinase B1/AMP-activated protein kinase pathway. Our data suggest that dietary interventions such as FR, or treatment that mimics the effects of such interventions, may be potential and novel preventive and therapeutic options for patients with ADPKD.

Abstract Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKα facilitates its interaction with RelB and IKKα, indicating that DBC1-mediated suppression of alternative NF-κB is regulated by IKKα. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation.