[Glutamate transporter dysfunction and major mental illnesses].

Glutamate is the main excitatory neurotransmitter in the central nervous system and plays an important role in most aspects of normal brain function. In spite of its importance as a neurotransmitter, excess glutamate is toxic to neurons. Clearance of extracellular glutamate is critical for maintenance of low extracellular glutamate concentration, and occurs in large part through the activity of GLT1 (EAAT2) and GLAST (EAAT1), which are primarily expressed by astrocytes. Rare variants and down-regulation of GLT1 and GLAST, in psychiatric disorders have been reported. In this review, we demonstrate that various kinds of GLT1 and/or GLAST knockout mice replicate many aspects of the behavioral abnormalities seen in major mental illnesses including schizophrenia, depression, obsessive -compulsive disorders, autism, epilepsy and addiction.

Regiospecificity and stereospecificity of human UDP-glucuronosyltransferases in the glucuronidation of estriol, 16-epiestriol, 17-epiestriol, and 13-epiestradiol.

The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B was examined. UGT1A10 is highly active in the conjugation of the 3-OH in all these estriols, whereas UGT2B7 is the most active UGT toward one of the ring D hydroxyls, the 16-OH in estriol and 16-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses indicated that the 17-OH configuration plays a major role in the affinity of UGT2B7 for estrogens. The glucuronidation of the different estriols by the human liver and intestine microsomes reflects the activity of UGT1A10 and UGT2B7 in combination with the tissues' difference in UGT1A10 expression. The UGT1A10 mutant 1A10-F93G exhibited much higher V(max) values than UGT1A10 in estriol and 17-epiestriol glucuronidation, but a significantly lower value in 16-epiestriol glucuronidation. To this study on estriol glucuronidation we have added experiments with 13-epiestradiol, a synthetic estradiol in which the spatial arrangement of the methyl on C18 and the hydroxyl on C17 is significantly different than in other estrogens. In comparison with estradiol glucuronidation, the C13 configuration change decreases the turnover of UGTs that conjugate the 3-OH, but increases it in UGTs that primarily conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol. The combined results reveal the strong preference of UGT1A10 for the 3-OH of physiologic estrogens and the equivalently strong preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of such steroid hormones.

Ca2+-dependent release of Munc18-1 from presynaptic mGluRs in short-term facilitation.

Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca(2+) in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca(2+)-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC(50) for Ca(2+) of 168 nM, close to the resting Ca(2+) concentration, and that the interaction was disrupted by Ca(2+)-activated calmodulin (CaM) at higher concentrations of Ca(2+). Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca(2+) level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.

The bone-protective effect of genistein in the animal model of bilateral ovariectomy: roles of phytoestrogens and PTH/PTHR1 against post-menopausal osteoporosis.

Genistein, a major phytoestrogen of soy, is considered a potential drug for the prevention and treatment of post-menopausal osteoporosis. Mounting evidence suggested a positive correlation between genistein consumption and bone health both in vivo and in vitro. Earlier studies have revealed that genistein acted as a natural estrogen analogue which activated estrogen receptor and exerted anti-osteoporotic effect. However, it remains unclear whether PTH, the most crucial hormone that regulates mineral homeostasis, participates in the process of genistein-mediated bone protection. In the present study, we compared the therapeutic effects between genistein and nilestriol and investigated whether PTH and its specific receptor PTHR1 altered in response to genistein-containing diet in the animal model of ovariectomy. Our results showed that genistein administration significantly improved femoral mechanical properties and alleviates femoral turnover. Genistein at all doses (4.5 mg/kg, 9.0 mg/kg and 18.0 mg/kg per day, respectively) exerted improved bending strength and b-ALP limiting effects than nilestriol in the present study. However, genistein administration did not exert superior effects on bone protection than nilestriol. We also observed circulating PTH restoration in ovariectomized rats receiving genistein at the dose of 18 mg/kg per day. Meanwhile, PTHR1 abnormalities were attenuated in the presence of genistein as confirmed by RT-PCR, Western blot and immunohistochemistry. These findings strongly support the idea that besides serving as an estrogen, genistein could interact with PTH/PTHR1, causing a superior mineral restoring effect than nilestriol on certain circumstance. In conclusion, our study reported for the first time that the anti-osteoporotic effect of genistein is partly PTH/PTHR1-dependent. Genistein might be a potential option in the prevention and treatment of post-menopausal osteoporosis with good tolerance, more clinical benefits and few undesirable side effects.

2-Methoxyestradiol-bis-sulfamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line.

In anticancer research where the focus is on finding agents that induces cell death while leaving non-tumorigenic cells less affected, a novel 2-methoxyestradiol derivative has come forth. 2-Methoxyestradiol-bis-sulfamate (2-MeOE2bisMATE) is a 2-methoxyestradiol derivative produced by bis-sulphamoylation, which possesses increased antiproliferative activity and biological availability. Several questions remain regarding the type of cell death mechanisms and possible induction of autophagy by 2-MeOE2bisMATE. The aim of this in vitro study was to investigate the cell death mechanisms exerted by 2-MeOE2bisMATE in an adenocarcinoma cell line (MCF-7) by analyzing its influence on cell growth, morphology, and possible induction of cell death. Spectrophotometry (crystal violet staining), transmission electron microscopy (TEM), light microscopy (hematoxylin and eosin staining), and fluorescent microscopy (Hoechst 33342, propidium iodide and acridine orange) were employed. Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 75% in MCF-7 cells after 24 h and to 47% after 48 h of exposure. TEM demonstrated membrane blebbing, nuclear fragmentation, and chromatin condensation indicating the hallmarks of apoptosis. Light microscopy revealed the presence of several cells blocked in metaphase, and apoptotic cells were also observed. Fluorescent microscopy demonstrated increased lysosomal staining; suggesting the induction of autophagy. 2-MeOE2bisMATE shows therapeutic potential, as an, anticancer agent, and the investigation of the cell death mechanisms used by 2-MeOE2bisMATE, thus, warrants further investigation.

Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse.

Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.

Endocytosis and recycling of AMPA receptors lacking GluR2/3.

Excitatory synapses in the mammalian brain contain two types of ligand-gated ion channels: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). AMPARs are responsible for generating excitatory synaptic responses, whereas NMDAR activation triggers long-lasting changes in these responses by modulating the trafficking of AMPARs toward and away from synapses. AMPARs are tetramers composed of four subunits (GluR1-GluR4), which current models suggest govern distinct AMPAR trafficking behavior during synaptic plasticity. Here, we address the roles of GluR2 and GluR3 in controlling the recycling- and activity-dependent endocytosis of AMPARs by using cultured hippocampal neurons prepared from knockout (KO) mice lacking these subunits. We find that synapses and dendritic spines form normally in cells lacking GluR2/3 and that upon NMDAR activation, GluR2/3-lacking AMPARs are endocytosed in a manner indistinguishable from GluR2-containing AMPARs in wild-type (WT) neurons. AMPARs lacking GluR2/3 also recycle to the plasma membrane identically to WT AMPARs. However, because of their permeability to calcium, GluR2-lacking but not WT AMPARs exhibited robust internalization throughout the dendritic tree in response to AMPA application. Dendritic endocytosis of AMPARs also was observed in GABAergic neurons, which express a high proportion of GluR2-lacking AMPARs. These results demonstrate that GluR2 and GluR3 are not required for activity-dependent endocytosis of AMPARs and suggest that the most important property of GluR2 in the context of AMPAR trafficking may be its influence on calcium permeability.

Production and radioprotective effects of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ) was produced by fermentation of the Methylovorus sp. MP688 strain and purified by ion-exchange chromatography, crystallization and recrystallization. The yield of PQQ reached approximately 125 mg/L and highly pure PQQ was obtained. To determine the optimum dose of PQQ for radioprotection, three doses (2 mg/kg, 4 mg/kg, 8 mg/kg) of PQQ were orally administrated to the experimental animals subjected to a lethal dose of 8.0 Gy in survival test. Survival of mice in the irradiation + PQQ (4 mg/kg) group was found to be significantly higher in comparison with the irradiation and irradiation + nilestriol (10 mg/kg) groups. The numbers of hematocytes and bone marrow cells were measured for 21 days after sublethal 4 Gy gamma-ray irradiation with per os of 4 mg/kg of PQQ. The recovery of white blood cells, reticulocytes and bone marrow cells in the irradiation + PQQ group was faster than that in the irradiation group. Furthermore, the recovery of bone marrow cell in the irradiation + PQQ group was superior to that in irradiation + nilestriol group. Our results clearly indicate favourable effects on survival under higher lethal radiation doses and the ability of pyrroloquinoline quinine to enhance haemopoietic recovery after sublethal radiation exposure.

[Effects of exercise and nilestriol on bone size and bone mass in ovariectomized rats].

OBJECTIVE: To investigate the influence of nilestriol (CCE3) and exercise on bone size and bone mass in ovariectomized (OVX) rats. METHODS: Forty eight (48) female Sprague-Dawley (SD) rats were divided randomly into six groups: (1) Normal control group, (2) Sham OVX group, (3) OVX group, (4) OVX + CCE3 group, (5)) OVX + exercise group, (6) OVX + CCE3 + exercise group. CCE (0.5 mg/kg per week) was given to the rats in OVX + CCE3 group and OVX + CCE3 + exercise group from the 2nd day after the operation for 11 weeks. Exercise training was loaded to the rats in exercise groups from the 7th day after operation, each rat was subjected to running on 0 dig angle runway with the speed of 16 m per minute, 45 minutes per day, 5 days per week for 10 weeks. RESULTS: The bone diameter, bone volume, bone wet weight, bone dry weight, bone ashes weight in the OVX group were lower than those in the sham OVX group (P < 0.01). All of these measurements were increased in OVX + CCE3 group and OVX + exercise group when compared with the OVX group, but were still lower than those in sham OVX group (P < 0.01). Exercise enhances the effect of CCE3 (P < 0.01). CONCLUSION: Exercise enhances the effects of CCE3 on the improvement of both bone size and quantity in OVX rats.

Stability of chimpanzee (Pan troglodytes) urinary reproductive hormones during long-term preservation on filter paper.

Urine contains multiple water-soluble hormones, which are valuable non-invasive biomarkers for the monitoring of reproductive status and health. An effective method for drying urine on filter paper was previously developed to preserve wildlife urine samples where electrical equipment was not available for this; however, the stability of samples preserved in this way remains to be verified. Here, we developed and validated a method to elute multiple water-soluble reproductive hormones from filter paper that had been stored for an extended period of time. Aliquots of urine from chimpanzees were adsorbed on filter papers, air dried and stored for 1 year at room temperature. Estrone-3-conjugate (E1C), pregnanediol-3-glucuronide (PdG), estriol-3-glucuronide (E3G), and chorionic gonadotropin (CG) were eluted into deionized water from the filter papers and measured using enzyme immunoassays (EIAs). The mean recoveries of E1C, PdG, and creatinine from filter papers stored for 1 year were 69.5%, 128.7%, and 83.8%, respectively. The profiles of E1C and PdG from preserved filter papers significantly correlated with those derived from a direct analysis of the frozen urine of menstruating chimpanzees. We detected E3G and CG from 1-year-old filter papers for urine collected during early pregnancy, but the recovery of E3G was low and CG profiles did not correlate with those of the original frozen urine samples. The method proposed here for the elution and measurement of reproductive hormones in urine preserved for a long period of time on filter paper provides a practical and simple way to monitor the reproductive status of chimpanzees. We propose that this method can also be utilized in field studies of other wild nonhuman primates.

Influence of estradiol analogue on cell growth, morphology and death in esophageal carcinoma cells.

2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.

In vitro effects of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and cell cycle dynamics in the MCF-7 breast adenocarcinoma cell line.

In the search for new and improved anticancer therapies, researchers have identified several potentially useful compounds. One of these agents is 2-methoxyestradiol-bis-sulphamate (2ME-BM), a sulphamoylated derivative of 2-methoxyestradiol. The objective of this study was to evaluate 2ME-BM's in vitro efficacy as antiproliferative agent in the MCF-7 breast adenocarcinoma cell line. Light- and fluorescent microscopy showed decreased cell density, increased apoptotic characteristics and significant ultrastructural aberrations indicative of autophagic cell death after 24 hours of exposure at a concentration of 0.4 microM. In addition, mitotic indices revealed that 2ME-BM induces a G2M block. The latter was confirmed by flow cytometric analyses where increased sub-G1 and G2/M fractions, as well as an increase in cyclin B1 levels were observed. Further in vitro research into the mechanism of this potentially useful anticancer compound is thus warranted.

[The estrogenic effect of formononetin and its effect on the expression of rats' atrium estrogen receptors].

OBJECTIVE: To observe the estrogenic effect of formononetin and its effect on the expressions of atrial estrogen receptor subtypes alpha and beta (ERalpha and ERbeta). METHODS: 50 femal rats were randomly divided into five groups: sham group, model group, nilestriol group, formononetin groups of low and high dose. Rats in sham group were cut a piece of fat before closing the abdomen, the others were ovariectomized. Vaginal exfoliated cell were observed from the fifth day to the tenth after operation to test if the model is successful. The sham and model group were given nomal saline in 10 mL/kg by gavage, the remaining three groups were given nilestriol 2.5 mg/(kg x w), low [20 mg/(kg x d) land high dose [100 mg/(kg x d)) of formononetin by gavage respectively. In the 8th week, vaginal exfoliated cell were observed, then decapitated the rats, removed the uterus, weighed and take wright staining microscopy. The relative expressions of ERalpha and ERbeta of right atrium were detected by RT-PCR. RESULTS: The vaginal cells exhibit a change of estrus after had been fed with high dose of formononetin after 8 weeks. Formononetin increase the uterus coefficient and the expression of atrial ERbeta (P < 0.01), but it dose not have any effect on the expression of ERalpha (P > 0.05). CONCLUSION: Formononetin have estrogenic effect in ovariectomized rats, and it can markedly upregulate the expression of rats' atrial ERbeta.

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC-MS/MS study.

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ±â€¯SD, 8.6 ±â€¯3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ±â€¯2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ±â€¯3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ±â€¯10.7 ng/mL), androsterone sulfate (AnS: 9.2 ±â€¯7.4 ng/mL), estrone sulfate (E1S: 3.0 ±â€¯3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ±â€¯4.0 ng/mL) and estriol (E3: 1.2 ±â€¯0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine.

We report a hydrophilic interaction liquid chromatography (HILIC) separation with tandem mass spectrometry (MS) detection method for analysis of seven urinary estrogen conjugates. HILIC separation employing a mobile phase with high organic solvent content resulted in enhanced electrospray ionization efficiency and MS sensitivity compared with reversed-phase (RP) LC-MS methods. Solid-phase extraction (SPE) was used to further improve the limit of detection and to eliminate interferences for the analysis of urine samples. No hydrolysis or derivatization was required in the sample pretreatment. This SPE/HILIC-MS/MS method provided limits of quantification (LOQs at S/N = 10) for the seven conjugates ranging from 2 to 1000 pg/mL with only 1 mL of urine sample, representing an improvement of 1 order of magnitude over the RPLC tandem MS methods previously reported. This method provided a linear dynamic range of 3 orders of magnitude, recovery of 92-109%, intraday accuracy of 84-109%, intraday precision of 1-14%, interday accuracy of 80-111%, and interday precision of 1-22%. We have successfully applied this technique to determine the seven estrogen conjugates in urine samples of a pregnant woman and found unique concentration changes of six estrogen conjugates at different stages of pregnancy while the concentration of estriol-3-glucuronide (E3-3G) remained constant. We further studied the profiles of individual estrogen conjugates in breast cancer patients before and after treatment and found patient-dependent effects of aromatase inhibitor treatment on estrogen phase-II metabolism, which have not been reported previously. This study demonstrates the potential clinical application of the HILIC-MS/MS technique for sensitive monitoring of the changes of urinary estrogen conjugates in a clinical setting.

Establishment of an HPLC identification system for detection of counterfeit steroidal drugs.

A set of simple HPLC methods employing UV detection were developed for detection of counterfeit drugs by the qualitative and quantitative analysis of nine steroidal drugs, ethinylestradiol, diethylstilbestrol, norethisterone, norgestrel, methyltestosterone, medroxyprogesterone acetate, progesterone, testosterone propionate and nilestriol. The methods were based on studies of the relationships between the retention factors (k) of the nine compounds and the percentages of water to methanol in the mobile phases on a reverse phase Alltima C(18) column giving reliable separation of the compounds under three sets of chromatographic conditions. The methods were validated using statistical tests and were used on nine commercial samples for detection of possible counterfeit drugs.

GSK3ß-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting synaptic and mitochondrial functions.

BACKGROUND: Although diabetic retinopathy (DR) has long been considered as a microvascular disorder, mounting evidence suggests that diabetic retinal neurodegeneration, in particular synaptic loss and dysfunction of retinal ganglion cells (RGCs) may precede retinal microvascular changes. Key molecules involved in this process remain poorly defined. The microtubule-associated protein tau is a critical mediator of neurotoxicity in Alzheimer's disease (AD) and other neurodegenerative diseases. However, the effect of tau, if any, in the context of diabetes-induced retinal neurodegeneration has yet to be ascertained. Here, we investigate the changes and putative roles of endogeneous tau in diabetic retinal neurodegeneration. METHODS: To this aim, we combine clinically used electrophysiological techniques, i.e. pattern electroretinogram and visual evoked potential, and molecular analyses in a well characterized high-fat diet (HFD)-induced mouse diabetes model in vivo and primary retinal ganglion cells (RGCs) in vitro. RESULTS: We demonstrate for the first time that tau hyperphosphorylation via GSK3ß activation causes vision deficits and synapse loss of RGCs in HFD-induced DR, which precedes retinal microvasculopathy and RGCs apoptosis. Moreover, intravitreal administration of an siRNA targeting to tau or a specific inhibitor of GSK3ß reverses synapse loss and restores visual function of RGCs by attenuating tau hyperphosphorylation within a certain time frame of DR. The cellular mechanisms by which hyperphosphorylated tau induces synapse loss of RGCs upon glucolipotoxicity include i) destabilizing microtubule tracks and impairing microtubule-dependent synaptic targeting of cargoes such as mRNA and mitochondria; ii) disrupting synaptic energy production through mitochondria in a GSK3ß-dependent manner. CONCLUSIONS: Our study proposes mild retinal tauopathy as a new pathophysiological model for DR and tau as a novel therapeutic target to counter diabetic RGCs neurodegeneration occurring before retinal vasculature abnormalities.