Male Stressed Mice Having Behavioral Control Exhibit Escalations in Dorsal Dentate Adult-Born Neurons and Spatial Memory.

An escapable (ES)/inescapable stress (IS) paradigm was used to study whether behavioral control and repeated footshock stressors may affect adult neurogenesis and related cognitive function. Male stressed mice having behavioral control (ES) had a short-term escalation in dorsal dentate gyrus (DG) neurogenesis, while similarly stressed mice having no such control had unaltered neurogenesis as compared to control mice receiving no stressors. Paradoxically, ES and IS mice had comparable stress-induced corticosterone elevations throughout the stress regimen. Appetitive operant conditioning and forced running procedures were used to model learning and exercise effects in this escapable/inescapable paradigm. Further, conditioning and running procedures did not seem to affect the mice's corticosterone or short-term neurogenesis. ES and IS mice did not show noticeable long-term changes in their dorsal DG neurogenesis, gliogenesis, local neuronal density, apoptosis, autophagic flux, or heterotypic stress responses. ES mice were found to have a greater number of previously labeled and functionally integrated DG neurons as compared to IS and control mice 6 weeks after the conclusion of the stressor regimen. Likewise, ES mice outperformed IS and non-stressed control mice for the first two, but not the remaining two, trials in the object location task. Compared to non-stressed controls, temozolomide-treated ES and IS mice having a lower number of dorsal DG 6-week-old neurons display poor performance in their object location working memory. These results, taken together, prompt us to conclude that repeated stressors, albeit their corticosterone secretion-stimulating effect, do not necessary affect adult dorsal DG neurogenesis. Moreover, stressed animals having behavioral control may display adult neurogenesis escalation in the dorsal DG. Furthermore, the number of 6-week-old and functionally-integrated neurons in the dorsal DG seems to confer the quality of spatial location working memory. Finally, these 6-week-old, adult-born neurons seem to contribute spatial location memory in a use-dependent manner.

mGluR5 upregulation and the effects of repeated methamphetamine administration and withdrawal on the rewarding efficacy of ketamine and social interaction.

Repeated, recreational ketamine (KE) or methamphetamine (MA) administration seldom produce neurotoxicity, while combining MA and KE administration have been thought to render changes in neural plasticity and motivational behavior. In this study, we sought to assess whether pre-exposure to multiple MA injections and withdrawal may affect low-dose KE-produced rewarding effects, social interaction behavior and its neurochemical underpinnings. A 10-day MA injections (2 mg/kg/day) and 10-day withdrawal regimen was found to cause reliable behavioral sensitization. While KE (1 mg/kg) induced weak conditioned place preference (CPP), pre-exposure to this MA-withdrawal regimen enhanced such KE CPP magnitude. This MA-withdrawal regimen also caused impairments in the social interaction behavior in the sociability, social novelty test. Compared with the mice undergoing the 10-day saline-withdrawal or MA regimen, mice receiving the 10-day MA-withdrawal regimen exhibited lower dopamine-releasing probability in the nucleus accumbens, inferring the MA-withdrawal regimen-primed preference for KE rewarding effects. Likewise, mice receiving the MA-withdrawal regimen had high expression in mGluR5 protein but unaltered EAAT3, Homer2 expression in hippocampal tissues. Pretreatment with MPEP, an mGluR5 antagonist, prevented the MA-withdrawal regimen-induced increment in the KE CPP magnitude and impairments in social interaction behavior. We, thus, conclude that repeated MA administration and abstinence may enhance KE rewarding effects and produce eminent deficits in social recognition and interest. And these effects correlate with the mGluR5 over-expression and modulation of the KE-stimulating effect on dopamine release.

A sensitive LC-MS/MS method for simultaneous determination of cabozantinib and its metabolite cabozantinib N-oxide in rat plasma and its application in a pharmacokinetic study.

Cabozantinib (CBZ) is used for the treatment of progressive, metastatic medullary thyroid cancer. Its major oxidative metabolite is cabozantinib N-oxide (CBN), which contains a structural alert associated with mutagenicity, yet the pharmacokinetics studies lack the simultaneous investigation of CBN and dose proportionality. In the current study a simple LC-MS/MS method was developed and validated for the simultaneous estimation and pharmacokinetic investigation of CBZ and CBN in rat plasma. The analytes were separated on a Waters Atlantics C18 column (2.1 × 150 mm, 3 µm). The mass spectrometry analysis was conducted in positive ionization mode with multiple reaction monitoring. Good linearity was observed over the concentration ranges of 0.500-5000 ng/mL for CBZ and 0.525-2100 ng/mL for CBN. The extraction recoveries were constant and the intra- and inter-batch precision and accuracy were acceptable for the analysis of biological samples. The method was successfully applied for the simultaneous estimation of CBZ and CBN in a pharmacokinetic study in Sprague-Dawley rats. After oral administration of CBZ (1, 5 and 12.6 mg/kg), although CBZ showed dose proportionality, the metabolite CBN showed obvious nonlinear elimination pharmacokinetics with greater than dose-proportional increases in exposure.

Synergistic Effects of Psychosocial Stress and Mild Peripheral Infection on Inducing Microglial Activation in the Hippocampal Dentate Gyrus and Long-Lasting Deficits in Hippocampus-Related Memory.

Lipopolysaccharide (LPS) treatment and stress may cause immune activation in the brain, an event which has been thought to play a role in mediating stress-induced cognitive dysfunction. However, the enduring impact of psychosocial stress on brain immune activation or cognitive deficits has not been well investigated. Likewise, it remains unexplored whether there exist synergistic effects of psychosocial stress and a weak systemic LPS treatment on brain immune activation and/or cognitive function. In this work, a 10-day social defeat regimen was used to model psychosocial stress and the number and density of ionized calcium-binding adaptor molecule 1 (Iba1)-stained microglia was used to reveal brain immune activation in male Balb/C mice. The social defeat regimen did not cause observable microglial activation in dentate gyrus (DG) 24 h after the conclusion of the regimen. Microglial activation peaked in DG 24 h following a single 1 mg/kg intra-peritoneal LPS injection. At this time point, DG microglial activation was not evident providing 0.125 mg/kg or lower of LPS was used, this dose of LPS was, thus, regarded as the "sub-threshold" in this study. Twenty-four h after the conclusion of the defeat regimen, mice received a social interaction test to determine their defeat stress susceptibility and a "sub-threshold" LPS injection. DG microglial activation was observed in the defeat-stress susceptible, but not in the resilient, mice. Furthermore, the stress-susceptible mice showed impairment in object location and Y maze tasks 24 and 72 h after the "sub-threshold" LPS injection. These results suggest that psychosocial stress, when combined with a negligible peripheral infection, may induce long-lasting hippocampus-related memory deficits exclusively in subjects susceptible to psychosocial stresses.

Combining brief recall and ketamine treatment prevents stress-primed methamphetamine memory reinstatement via heightening mPFC GABA activity.

This study aimed to assess whether brief recall of methamphetamine (MA) memory, when combined with ketamine (KE) treatment, may prevent stress-primed MA memory reinstatement. Combining 3-min recall and KE facilitated MA memory extinction and resistance to subsequent stress-primed reinstatement. Such combination also produced glutamate metabotropic receptor 5 (mGluR5) upregulation in animals' medial prefrontal cortex (mPFC) γ-amino-butyric acid (GABA) neuron. Accordingly, chemogenetic methods were employed to bi-directionally modulate mPFC GABA activity. Following brief recall and KE-produced MA memory extinction, intra-mPFC mDlx-Gi-coupled-human-muscarinic-receptor 4 (hM4Di)-infused mice receiving compound 21 (C21) treatment showed eminent stress-primed reinstatement, while their GABA mGluR5 expression seemed to be unaltered. Intra-mPFC mDlx-Gq-coupled-human-muscarinic-receptor 3 (hM3Dq)-infused mice undergoing C21 treatment displayed MA memory extinction and resistance to stress-provoked reinstatement. These results suggest that combining a brief recall and KE treatment and exciting mPFC GABA neuron may facilitate MA memory extinction and resistance to stress-primed recall. mPFC GABA neuronal activity plays a role in mediating brief recall/KE-produced effects on curbing the stress-provoked MA seeking.

Medial and dorsal lateral septum involving social disruption stress-primed escalation in acid-induced writhes.

Introduction: Stress may cause prospective escalations in abdominal pain magnitude and accumbal TRPV1 expression, while central neural circuits mediating these stress effects remain unclear. Methods: Using retrograde tracing methods, we first demonstrated the existence of a medial septal-dorsal lateral septal -accumbal circuit very likely involving social disruption stress-primed escalations in acid-induced writhes and accumbal TRPV1 level. An intersectional viral strategy and virus-carrying hM3Dq and hM4Di DREADDs were, then, employed to selectively modulate GABAergic and cholinergic neuronal activity in medial and dorsal lateral septum. Results: Exciting medial septal GABAergic neuron was found to prevent social disruption stress-primed escalations in acid-induced writhes and accumbal TRPV1 and PKCε expressions. Likewise, inactivating dorsal lateral septal cholinergic neurons was also effective in abolishing these stress-primed escalations. Inactivating GABAergic neuron in non-stressed animals' medial septum was found to reproduce the stress-primed effects in causing heightened acid-induced writhes and accumbal TRPV1 and PKCε levels. Discussion: These results, taken together, prompt us to conclude that social disruption stress may produce plastic changes in a newly-identified medial septal-dorsal lateral septal-accumbal circuit. Moreover, medial septal GABAergic hypoactivity and dorsal lateral septal cholinergic hyperactivity are, at least, two likely causes reflecting such stress-produced escalations in abdominal pain magnitude and pain transduction-related protein over-expression in nucleus accumbens.

Stress while lacking of control induces ventral hippocampal autophagic flux hyperactivity and a depression-like behavior.

BACKGROUND: Stressed animals may perform depression-like behavior insomuch as stress-provoking blood-brain barrier (BBB) disruption, central immune activation, and autophagic flux changes. This study was undertaken to assess whether adult mice having (executive) vs. lacking (yoke) of behavioral control in otherwise equivalent stress magnitude condition, may display differences in their BBB integrity, ventral hippocampal (VH) interleukin-6 (IL-6) and autophagic flux level and VH-related depression-like behavior. To further understand the causative relation of enhanced autophagic flux and stress-primed depression-like behavior, we assessed the effects of bilateral intra-VH 3-methyladenine (3-MA), an autophagic flux inhibitor, infusion in stressed mice. METHODS: Adult mice used had comparable genetic background and housing condition. Executive/yoke pairs of mice received a 10-day (1 h/day) footshock stressor regimen. Throughout the regimen, the ongoing footshock was terminated immediately contingent on the executive mouse', while irrelevant to the respective yoke mouse' voluntary behavior, or lasting for 7 s. Each dyad's cage-mate receiving no such regimen served as no stressor controls. RESULTS: Yoke mice displayed disrupted BBB integrity (escalated Evans blue extravasation and decreased VH ZO-1, claudin-5 expression), increases in VH autophagic flux (increased LC3II/LC3I and decreased p62) and immobility duration in forced swimming test. Most of these indices remained unaltered in executive mice. Administration of 3-MA did not affect immobility duration in control mice, while prevented the increases in immobility duration in yoke mice. CONCLUSIONS: (1) stress susceptibility may be determined by their differences in stress-coping results; (2) VH autophagic flux increase plays a permissive role in priming the stressed animals susceptible to exhibit depression-like behavior.

Effects of Septin-14 Gene Deletion on Adult Cognitive/Emotional Behavior.

While various septin GTPases have been reported for their physiological functions, their roles in orchestrating complex cognitive/emotional functions in adult mammals remained scarcely explored. A comprehensive behavioral test battery was administered to two sexes of 12-week-old Septin-14 (SEPT14) knockout (KO) and wild-type (WT) mice. The sexually dimorphic effects of brain SEPT14 KO on inhibitory avoidance (IA) and hippocampal mGluR5 expression were noticed with greater IA latency and elevated mGluR5 level exclusively in male KO mice. Moreover, SEPT14 KO appeared to be associated with stress-provoked anxiety increase in a stress-related navigation task regardless of animals' sexes. While male and female WT mice demonstrated comparable cell proliferation in the dorsal and ventral hippocampal dentate gyrus (DG), both sexes of SEPT14 KO mice had increased cell proliferation in the ventral DG. Finally, male and female SEPT14 KO mice displayed dampened observational fear conditioning magnitude and learning-provoked corticosterone secretion as compared to their same-sex WT mice. These results, taken together, prompt us to conclude that male, but not female, mice lacking the Septin-14 gene may exhibit increased aversive emotion-related learning and dorsal/ventral hippocampal mGluR5 expressions. Moreover, deletion of SEPT14 may be associated with elevated ventral hippocampal DG cell proliferation and stress-provoked anxiety-like behavior, while dampening vicarious fear conditioning magnitudes.

Observer's adrenal corticosterone secretion involvement in vicarious fear conditioning.

Vicarious learning represents a far-reaching value for the survival of social animals. Adrenal hormones are known to affect many forms of learning, yet the roles of adrenal hormones in vicarious learning remain unexplored. This study was undertaken to assess whether observation-stimulated corticosterone (CORT) secretion may affect the magnitude of a vicarious fear conditioning. Mouse observers were individually subjected to an observational compartment next to the training compartment wherein three their cage-mate demonstrators received (1) 5 days of 15 randomly-scheduled footshocks (0.5 mA, 2 s in duration over a 30 min session) (G1); (2) a 30-min presentation of vanilla odors (G2); or (3) footshock delivery and vanilla odors in combination (G3). Demonstrator mice receiving G3 training session and their respective observer mice were found to exhibit greater training-induced and slightly greater observation-stimulated CORT secretion, greater vanilla odors-induced fear responses (FR) and conditioned place aversion (CPA), as compared with the observers vicariously learning from demonstrators receiving G1 or G2 sessions. Observers held in their home cages during demonstrators' trainings and those receiving null demonstrator (No Demonstrator) failed to exhibit vanilla odors-induced FR. Moreover, observers undergoing adrenalectomy (ADX) and G3 sessions exhibited lower vanilla odors-induced FR and CPA as compared to sham surgical (Sham) observers observing G3 sessions. Furthermore, systemic metyrapone injections (50 and 100 mg/kg) prior to daily vicarious G3 training session resulted in decreases in vanilla odors-induced FR and CPA magnitudes in observers. Finally, CORT (1 mg/kg)-pretreated G2 observers failed to display odors-induced FR escalation. These results, taken together, suggest that observation-stimulated CORT secretion is necessary for reliable establishment of vicarious fear conditioning in observer mice.

Social disruption-induced stress pre-exposure aggravates, while the presence of conspecifics diminishes, acetic acid-induced writhing.

RATIONALE AND OBJECTIVE: This study was undertaken to assess the modulating effects of (1) pre-exposure to repeated social disruption and (2) group testing on writhing associated with visceral pain induced by intraperitoneal administration of acetic acid. MATERIALS AND METHODS: Six consecutive days of social disruption were used to prime for stress, while group testing referred to 3 mouse cage-mates receiving the acetic acid-induced writhing test as a group. RESULTS: Social disruption-induced stress-pre-exposed mice displayed a greater number acid-induced writhes compared to mice not receiving the pre-exposure. However, mice displayed fewer acid-induced writhes in a triad group vs. individually, suggesting group-mediated writhing-reducing effects. Likewise, group testing prevented the stress pre-exposure escalation in acid-induced writhes. Additional studies revealed that the stress-pre-exposed mice had increased expression in accumbal TRPV1 receptors. Systemic (0.25 mg/kg) and bilateral intra-accumbal (0.2 ng/0.2 µl/side) administration of SB366791, a TRPV1 receptor antagonist, reliably prevented the stress pre-exposure escalation in acid-induced writhing; SB366791 treatment alone did not affect acid-induced writhing, stress pre-exposure anxiety-like behavior, or the group testing effects. Furthermore, lower neuronal activation was found in the medial septal nucleus in group vs. individual tested mice. Intra-medial septum (0.2 µg/0.5 µl) infusion with bicuculline, a GABAA receptor antagonist, effectively prevented group-mediated writhing-reducing effects, but not individual acid-induced writhing effects. CONCLUSIONS: These findings suggest that social disruption-induced stress pre-exposure may upregulate accumbal TRPV1 receptor expression and consequently aggravate acid-induced writhing. Group testing prevents such stress pre-exposure escalation of acid-induced writhing most likely by strengthening the GABAergic inhibition on local neural activity in the medial septum.

Carbon material-immobilized ionic liquids were applied on absorption of Hg2+ from water phase.

In this study, several immobilized ionic liquid adsorbents on carbon materials were synthesized with impregnation method. The carrier materials were activated carbon and three kinds of multi-walled carbon nanotubes. And the synthetic adsorbents immobilized different kinds of ionic liquids were characterized by Boehm titration, FT-IR, XPS, TG, and BET analysis, respectively. Finally, carbon materials after [C4mim]HSO4 immobilization were selected as adsorbent to remove Hg2+ from water phase. The optimum conditions of adsorption test of ionic liquid immobilized by multi-walled carbon nanotubes were as follows: the initial concentration of Hg2+ was 400 mg/L, the adsorbent addition amount was 40 mg, the temperature was 20 °C, the reaction time was 200 min, the removal rate of Hg2+ peaked at 62.95%, the adsorption capacity was reached 79.00 mg/g. The optimum conditions of adsorption test of ionic liquid immobilized by activated carbon were as follows: the initial concentration of Hg2+ was 300 mg/L, the adsorbent addition amount was 0.2 g, the temperature was 20 °C, pH was 2.0, the reaction time was 100 min, the removal rate of Hg2+ was more than 99%, the adsorption capacity was 118.65 mg/g. The adsorption isotherm fitting study found that the adsorption of adsorbent on Hg2+ was more in line with the Langmuir model, and the adsorption kinetics study shows that the adsorption process is consistent with the pseudo-second-order kinetic equation. The results of kinetic analysis are further verified by thermodynamic analysis.

Adsorption Separation of Cr(VI) from a Water Phase Using Multiwalled Carbon Nanotube-Immobilized Ionic Liquids.

Three types of multiwalled carbon nanotubes (MWCNTs, MWCNTs-OH, and MWCNTs-COOH) were used as carriers and five types of ionic liquids (ILs) were immobilized on each carrier by an impregnation method. Boehm titration, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, specific surface area analysis by the Brunauer-Emmett-Teller method, and thermogravimetric analysis were performed to investigate [C4mim]HSO4 adsorption by the MWCNTs. The MWCNT-immobilized IL was used for Cr(VI) removal from a water phase. The adsorption properties of MWCNTs-COOH-immobilized [C4mim]HSO4 were investigated by single-factor analysis. The results showed that the Cr(VI) removal rate was 52.14% and the adsorption capacity was 31.29 mg/g. The optimum adsorption conditions were as follows: initial Cr(VI) concentration, 60 mg/L; adsorbent dosage, 50 mg; pH 2.0; adsorption temperature 40 °C; and adsorption time, 200 min. Adsorption isotherm data fitted the Freundlich model, which indicates that the adsorption process was in line with the multimolecular layer adsorption theory. The Cr(VI) adsorption behaviors of the three adsorbents were consistent with a pseudo-second-order dynamic model. Thermodynamic analysis of the reaction systems was also performed. The Cr(VI) removal rates of MWCNTs-3, MWCNTs-OH-3, and MWCNTs-COOH-3 were 27.97, 9.39, and 7.34% lower than the initial removal rates after five cycles.

Relevance of number and physiological status of conspecifics in preventing stress-induced decreases in newly proliferated cells and neuroblasts.

RATIONALE AND OBJECTIVE: The presence of three conspecifics prevents stress-induced decreases in newly proliferated cells and neuroblasts in mouse dentate gyrus (DG). In this study, we sought to determine how many conspecifics are required to exert these protective effects against stress. In addition, we manipulated the physiological status of those conspecifics in the context of their stress-buffering effects and used airborne oxytocin exposure as a substitute for the presence of conspecifics. MATERIALS AND METHODS: Bromodeoxyuridine staining was used to indicate the newly proliferated cells and co-staining with doublecortin to reveal the proliferative neuroblasts. RESULTS: Presentation of three intact and lipopolysaccharide-treated conspecifics prevented the stress-induced decreases in the number of newly proliferated cells and neuroblasts in DG. Presentation of one saline- or oxytocin (OT)-treated conspecific did not exert observable stress-buffering effects. In contrast, airborne oxytocin prevented the stress-induced decreases in DG cell proliferation and early neurogenesis, while pretreatment with L-371,257, a selective OT receptor antagonist, abolished the buffering effects of OT. CONCLUSIONS: Physical interaction with the conspecifics and conspecifics' sickness, at best, play a minor role in mediating the buffering effects against stress-induced decreases in DG cell proliferation or early neurogenesis. Moreover, stress-buffering effects are negligible with the presence of only one conspecific. Finally, airborne OT produced stress-buffering effects possibly via its stimulation of OT receptors. Oxytocin merits further study as a substitute for the stress-buffering effects of companions.

Development and validation of a highly sensitive LC-MS/MS method for determination of brain active agent dianhydrogalactitol in mouse plasma and tissues: Application to a pharmacokinetic study.

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitative analysis of 1,2:5,6-dianhydrogalactitol (DAG) in mouse plasma and tissues. Sodium diethyldithiocarbamate (DDTC) was used as the derivatization reagent to improve its LC-MS/MS behavior. Analytes were separated on a Welch Ultimate XB-CN column with a mobile phase consisting of acetonitrile and 0.1% formic acid solution (65:35). The MS analysis was conducted by positive electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity (r2 > 0.9958) was observed over the concentration range of 1-1000 ng/mL in plasma and tissue homogenates (brain, liver, heart, spleen, lung and kidney). The intra- and inter-batch precision and accuracy of DAG in plasma and brain samples were all within the acceptable limits. The extraction recovery was stable and no significant matrix effects were observed. The method was successfully applied to study the pharmacokinetic and tissue distribution of DAG in mice after intravenous administration. DAG could cross the blood-brain barrier and had limited liver distribution. Rat primary hepatocytes in vitro experiments demonstrated that DAG had a safe profile in liver.

Bioactives in Chinese Proprietary Medicine Modulates 5α-Reductase Activity and Gene Expression Associated with Androgenetic Alopecia.

Androgenetic alopecia (AGA) is characterized by a progressive and patterned transformation of thick, pigmented terminal scalp hairs into short, hypo-pigmented vellus-like hairs. The use of Minoxidil and Finasteride to treat AGA are often associated with complications in safety and efficacy. However, herbal remedies are deemed to have lesser side effects in many societies. This study aims to identify potential hair growth properties of individual compounds from a Chinese proprietary medicine known as Yangxue Shengfa capsule (YSC), used in China for many years for improving AGA. Six marker compounds, including 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), Chlorogenic acid, Emodin, Ferulic acid, Isoimperatorin, and Paeoniflorin were used for simultaneous HPLC quantification and anti-AGA in-vitro screening. Simultaneous quantification of these components was performed on 75% (v/v) methanol extracts of YSC, using a Welch Ultimate XB-C18 column and gradient elution. Five compounds significantly promoted cell proliferation in cultured immortalized human Dermal Papilla Cells (DPC). Multiple genes associated with the progression of AGA, including IGF-1, DKK-1, and TGF-ß1, were found to be regulated by some of these compounds. Interestingly, Ferulic acid and Emodin demonstrated good pharmacological properties against AGA, thereby concluding the potential of these bioactives to be used in the treatment against AGA.

Critical Role of Hepatic Cyp450s in the Testis-Specific Toxicity of (5R)-5-Hydroxytriptolide in C57BL/6 Mice.

Low solubility, tissue accumulation, and toxicity are chief obstacles to developing triptolide derivatives, so a better understanding of the pharmacokinetics and toxicity of triptolide derivatives will help with these limitations. To address this, we studied pharmacokinetics and toxicity of (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative immunosuppressant in a conditional knockout (KO) mouse model with liver-specific deletion of CYP450 reductase. Compared to wild type (WT) mice, after LLDT-8 treatment, KO mice suffered severe testicular toxicity (decreased testicular weight, spermatocytes apoptosis) unlike WT mice. Moreover, KO mice had greater LLDT-8 exposure as confirmed with elevated AUC and Cmax, increased drug half-life, and greater tissue distribution. γ-H2AX, a marker of meiosis process, its localization and protein level in testis showed a distinct meiosis block induced by LLDT-8. RNA polymerase II (Pol II), an essential factor for RNA storage and synapsis in spermatogenesis, decreased in testes of KO mice after LLDT-8 treatment. Germ-cell line based assays confirmed that LLDT-8 selectively inhibited Pol II in spermatocyte-like cells. Importantly, the analysis of androgen receptor (AR) related genes showed that LLDT-8 did not change AR-related signaling in testes. Thus, hepatic CYP450s were responsible for in vivo metabolism and clearance of LLDT-8 and aggravated testicular injury may be due to increased LLDT-8 exposure in testis and subsequent Pol II reduction.

ALDH2 activator inhibits increased myocardial infarction injury by nitroglycerin tolerance.

Nitroglycerin, which treats impaired cardiac function through vasodilation as it is converted to nitric oxide, is used worldwide for patients with various ischemic and congestive cardiac diseases, including angina pectoris. Nevertheless, after continuous treatment, the benefits of nitroglycerin are limited by the development of tolerance to the drug. Nitroglycerin tolerance is a result of inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme essential for cardioprotection in animals subjected to myocardial infarction. Here, we tested the hypothesis that the tolerance that develops as a result of sustained nitroglycerin treatment increases cardiac injury by subsequent myocardial infarction. In a rat model of myocardial infarction, 16 hours of prior, sustained nitroglycerin treatment resulted in infarcts that were twice as large as those in untreated control animals and in diminished cardiac function at 3 days and 2 weeks after the myocardial infarction. We also sought to identify a potential treatment to protect against this increased cardiac damage. Nitroglycerin inhibited ALDH2 activity in vitro, an effect that was blocked by Alda-1, an activator of ALDH2. Co-administration of Alda-1 with the nitroglycerin prevented the nitroglycerin-induced increase in cardiac dysfunction after myocardial infarction in rats, at least in part by enhancing metabolism of reactive aldehyde adducts that impair normal protein functions. If our animal studies showing that nitroglycerin tolerance increases cardiac injury upon ischemic insult are corroborated in humans, activators of ALDH2 such as Alda-1 may help to protect patients with myocardial infarction from this nitroglycerin-induced increase in cardiac injury while maintaining the cardiac benefits of the increased nitric oxide concentrations produced by nitroglycerin.

Mitochondrial aldehyde dehydrogenase and cardiac diseases.

Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular 'aldehydic load'. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases.

Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.

AIMS: Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCepsilon. METHODS AND RESULTS: Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction. CONCLUSION: Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.

Protein kinase C in heart failure: a therapeutic target?

Heart failure (HF) afflicts about 5 million people and causes 300,000 deaths a year in the United States alone. An integral part of the pathogenesis of HF is cardiac remodelling, and the signalling events that regulate it are a subject of intense research. Cardiac remodelling is the sum of responses of the heart to causes of HF, such as ischaemia, myocardial infarction, volume and pressure overload, infection, inflammation, and mechanical injury. These responses, including cardiomyocyte hypertrophy, myocardial fibrosis, and inflammation, involve numerous cellular and structural changes and ultimately result in a progressive decline in cardiac performance. Pharmacological and genetic manipulation of cultured heart cells and animal models of HF and the analysis of cardiac samples from patients with HF are all used to identify the molecular and cellular mechanisms leading to the disease. Protein kinase C (PKC) isozymes, a family of serine-threonine protein kinase enzymes, were found to regulate a number of cardiac responses, including those associated with HF. In this review, we describe the PKC isozymes that play critical roles in specific aspects of cardiac remodelling and dysfunction in HF.