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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 (E 1 C), pregnanediol-3-glucuronide (PdG), estriol-3-glucuronide (E 3 G), and chorionic gonadotropin (CG) were eluted into deionized water from the filter papers and measured using enzyme immunoassays (EIAs). The mean recoveries of E 1 C, PdG, and creatinine from filter papers stored for 1 year were 69.5%, 128.7%, and 83.8%, respectively. The profiles of E 1 C and PdG from preserved filter papers significantly correlated with those derived from a direct analysis of the frozen urine of menstruating chimpanzees. We detected E 3 G and CG from 1-year-old filter papers for urine collected during early pregnancy, but the recovery of E 3 G 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.

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 16 th week of gestation to 25 th 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 ± SD, 8.6 ± 3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ± 2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ± 3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ± 10.7 ng/mL), androsterone sulfate (AnS: 9.2 ± 7.4 ng/mL), estrone sulfate (E1S: 3.0 ± 3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ± 4.0 ng/mL) and estriol (E3: 1.2 ± 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 GSK3β 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 GSK3β 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 GSK3β-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.

Microglia, traditionally known as resident immune cells in the brain, have been recently identified as one of the important cell types engaging in the formation and maintenance of neural circuitry, especially during the development. In this review, I describe the diversity of microglial functions revealed in different phases of development, ranging from neurogenesis to circuit formation. In particular, microglia possess dual functions in supporting the survival of neuronal structures or neural cells themselves, and removing excess neural components. Understanding these processes and underlying molecular mechanisms will provide important insights into the regulation of neural circuitry development. It further implicates the involvement of microglial dysfunction in neurodevelopmental disorders.

Estrogens are potent and efficacious neuroprotectants both in vitro and in vivo in a variety of models of neurotoxicity. We determined the structural requirements for neuroprotection in an in vitro assay using a panel of >70 novel estratrienes, synthesized to reduce or eliminate estrogen receptor (ER) binding. We observed that neuroprotection could be enhanced by as much as 200-fold through modifications that positioned a large bulky group at the C2 or C4 position of the phenolic A ring of the estratriene. Further, substitutions on the B, C or D rings either reduced or did not markedly change neuroprotection. Collectively, there was a negative correlation between binding to ERs and neuroprotection with the more potent compounds showing no ER binding. In an in vivo model for neuroprotection, transient cerebral ischemia, efficacious compounds were active in protection of brain tissue from this pro-oxidant insult. We demonstrated that these non-feminizing estrogens engage in a redox cycle with glutathione, using the hexose monophosphate shunt to apply cytosolic reducing potential to cellular membranes. Together, these results demonstrate that non-feminizing estrogens are neuroprotective and protect brain from the induction of ischemic- and Alzheimer's disease (AD)-like neuropathology in an animal model. These features of non-feminizing estrogens make them attractive compounds for assessment of efficacy in AD and stroke, as they are not expected to show the side effects of chronic estrogen therapy that are mediated by ER actions in the liver, uterus and breast., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The fusion of neurotransmitter-filled vesicles during synaptic transmission is balanced by endocytotic membrane retrieval. Despite extensive research, the speed and mechanisms of synaptic vesicle endocytosis have remained controversial. Here, we establish low-noise time-resolved membrane capacitance measurements that allow monitoring changes in surface membrane area elicited by single action potentials and stronger stimuli with high-temporal resolution at physiological temperature in individual bona-fide mature central synapses. We show that single action potentials trigger very rapid endocytosis, retrieving presynaptic membrane with a time constant of 470 ms. This fast endocytosis is independent of clathrin but mediated by dynamin and actin. In contrast, stronger stimuli evoke a slower mode of endocytosis that is clathrin, dynamin, and actin dependent. Furthermore, the speed of endocytosis is highly temperature dependent with a Q10 of ∼3.5. These results demonstrate that distinct molecular modes of endocytosis with markedly different kinetics operate at central synapses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3' UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

Heng-Gu-Gu-Shang-Yu-He-Ji, also known as OsteoKing, is used as a herbal Traditional Chinese Medicine for the treatment of bone disease, including femoral head necrosis and osteoarthritis. However, whether OsteoKing has anti-osteoporotic properties has remained to be elucidated. The purpose of the present study was therefore to investigate the effects of OsteoKing on ovariectomy-induced osteoporosis in rabbits. Female New Zealand white rabbits were randomly divided into an ovariectomized (OVX) group and a sham-surgery group. The rabbits in the OVX group were subjected to an ovariectomy, while the rabbits in the sham group were subjected to the removal of an area of fat near the two ovaries. Bone mineral density, mechanical properties, serum biochemical parameters and micro-architecture were examined at 150 days post-OVX to characterize the experimental animal model. Once the osteoporotic rabbit model had been established, the rabbits in the OVX group were divided into the following groups: Model group, nilestriol group and 300 and 600 mg/kg OsteoKing groups, containing 16 rabbits in each group. OsteoKing and nilestriol were administered orally. The bone mineral density, mechanical properties, serum biochemical parameters, histology and micro-architecture were examined using dual-energy X-ray absorptiometric analysis, mechanical assessments, enzyme-linked immunosorbent assays, histopathological evaluation and micro-computerized tomography examination following 60 days and 120 days of treatment, respectively. Treatment with OsteoKing led to an elevation in the bone mineral density of the vertebra and serum phosphorus levels, reduced serum concentrations of osteocalcin, procollagen type I N-terminal peptide, tartrate-resistant acid phosphatase 5b and cross-linked N-telopeptide of type I collagen, improved mechanical properties (maximum load, stiffness and energy absorption capacity), and micro-architecture of the lumbar vertebra in the OVX osteoporotic rabbit model following treatment for 120 days. In conclusion, it was demonstrated that OsteoKing is effective in the prevention of estrogen deficiency-associated bone loss and may be a promising drug for the treatment of post-menopausal osteoporosis.

Nilestriol (NIL) has been applied to treat menopausal dysfunctions, yet its mechanism has remained unknown. To understand the relationship between the changes in homeostatic metabolites and ovarian oestrogen deficiency syndromes after NIL treatment, proton Nuclear Magnetic Resonance ((1)H NMR)-based metabonomic technologies were used to analyse a rat model of oestrogen deficiency. An orthogonal partial least-squares regression (OPLS) differentiation model was used on 12-week metabolic analyses of ovariectomised (OVX) rats treated or mock treated with NIL. Furthermore, data analysis using Chenomx software quantified results to identify the most significantly altered metabolite concentrations, allowing for metabolic explanations of the effects of NIL therapies. In this study, PLS results revealed that there are considerably distinct differences between treatment groups. Additionally, a total of 45 metabolites shown to have a high variation between groups were selected for target quantification. Using a one-way LSD ANOVA analysis, 32 metabolite concentrations were significantly altered in the OVX group. A total of 21 metabolites were altered significantly in the NIL-treatment group but later returned to normal. According to the OPLS VIP calculation, the metabolites most affected by NIL treatment were mostly involved in insulin resistance. In addition, abnormal concentration changes in lactate in the NIL-treatment group and 3-indoxylsulfate in the OVX group were observed. To our knowledge, this study is the first to address the molecular mechanism of NIL from a metabonomic perspective, and, more specifically, to establish a catalogue of endo-molecular changes effected by NIL in the regulation of oestrogen deficiency disorder., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Soluble adenylyl cyclase (sAC) has been hypothesized to play a role in insulin secretion. The present study aimed to investigate the interaction between adenosine 3',5'‑cyclic monophosphate (cAMP), volume‑regulated anion channels (VRACs) and the electrogenic sodium bicarbonate (Na+‑HCO3‑) cotransporter, NBCe1, in the regulation of nutrient‑ and hypotonicity‑induced insulin release from rat pancreatic islets and tumoral insulin‑producing BRIN‑BD11 cells. In the islets, 5‑nitro‑2‑(3‑phenylpropylamino)benzoic acid (NPPB) and 5‑chloro‑2‑hydroxy‑3‑(thiophene‑2‑carbonyl)indole‑1‑carboxamide (tenidap) reduced glucose‑stimulated insulin release, however, only NPPB suppressed the enhancing action of cAMP analogs upon such a release. Insulin output from the BRIN‑BD11 cells was stimulated by 2‑ketoisocaproate (KIC) or extracellular hypoosmolarity. cAMP analogs and 3‑isobutyl‑1‑methylxanthine increased the insulin output recorded in the isotonic medium to a greater relative extent than that in the hypotonic medium. The secretory response to KIC or hypotonicity was inhibited by NPPB or tenidap, which both also opposed the enhancing action of cAMP analogs. Inhibitors of mitogen‑activated protein (MAP) kinase decreased insulin output in isotonic and hypotonic media. The inhibitor of sAC, 2‑hydroxyestriol, caused only a modest inhibition of insulin release, whether in the isotonic or hypotonic medium, even when tested at a concentration of 100 µM. The omission of NaHCO3 markedly decreased the secretory response to KIC or extracellular hypotonicity. The omission of Na+ suppressed the secretory response to extracellular hypotonicity. The observations of the present study do not support the hypothesis of a major role for sAC in the regulation of insulin release.

Fertility control has been identified by studies in the laboratory and the field as a more appropriate and long-term control strategy for rodent pests than lethal control. In this study, we investigated the effects of quinestrol on mass of reproductive organs and on social behaviors in female and male Indochinese forest rat (Rattus andamanensis (Blyth, 1860)] using approach of morphometrics and genetics). [corrected]. A total of 16 adult females and 16 adult males were randomly assigned to 4 groups. One male and one female group were fed rice with 0.005% quinestrol by weight for 7 days, and another 2 groups were fed rice only. After 7 days, rats were assigned to 10 min dyadic encounters between groups, and investigation, aggression, defense and attack latency were quantified. All animals were killed on day 10, and reproductive organs were dissected and weighed. Dyadic encounter data showed that there were obvious changes in social behaviors of quinestrol-treated rats. Quinestrol significantly inhibited the investigative behavior of quinestrol-treated males toward control females in Rattus nitidus, but seldom affected investigation between control males and quinestrol-treated females. Aggression of control females toward quinestrol-treated males was higher than that of quinestrol-treated females, and defense of quinestrol-treated males toward control females was more remarkable than that of control males. Quinestrol remarkably decreased wet masses of epididymis and spermotophore in males and ovaries in females, but had no effect on wet masses of testes and uteri after quinestrol treatment. These results indicate that the anti-fertility effects of quinestrol on R. nitidus are attributed to not only suppressing reproductive organs but also impacting social behaviors associated with territory defense and mate choice., (© 2012 Wiley Publishing Asia Pty Ltd, ISZS and IOZ/CAS.)

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.

The past 20 years witnessed an enormous leap in understanding of the central regulation of whole-body energy metabolism. Genetic tools have enabled identification of the region-specific expression of peripheral metabolic hormone receptors and have identified neuronal circuits that mediate the action of these hormones on behavior and peripheral tissue functions. One of the surprising findings of recent years is the observation that brain circuits involved in metabolism regulation remain plastic through adulthood. In this review, we discuss these findings and focus on the role of neurons and glial cells in the dynamic process of plasticity, which is fundamental to the regulation of physiological and pathological metabolic events., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Prune homolog 2 (Drosophila) (PRUNE2) encodes a BCH motif-containing protein that shares homology with the Cayman ataxia-related protein Caytaxin. Caytaxin is a substrate of caspase-3 and is specifically expressed at the presynapse of vesicular-type glutamate transporter (VGLUT)-positive neurons, where it plays a role in glutamate neurotransmission primarily in the cerebellum and hippocampus. Here, we showed that a novel Prune2 isoform contains a BCH motif and localizes predominantly to the synaptic cytosol, similar to Caytaxin. However, the isoform is expressed predominantly in the olfactory bulb and layer Ia of the piriform cortex, where Caytaxin is scarcely expressed. The isoform expression is upregulated during development, similar to that in the presynaptic-localizing proteins Synapsin I and Bassoon. Prune2 and its previously identified isoforms have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein, and an antagonist of cellular transformation. In addition, a novel isoform may develop new roles for Prune2 at the synapse in olfactory systems., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.

Objective: To observe the effect of acupuncture on fracture in the ovariectomized rat and the mechanism., Methods: Sixty SD female rats were randomly divided into 4 groups: normal group (group A), model group (group B), acupuncture group (group C) and Nilestriol group (group D). In all the groups, except the group A which received sham operation, the rats were overiectomized for preparing the osteoporosis model. Three months after the ovariectomy, the left femurs of the rats were closely fractured. Then, the group A and B were treated with oral administration of normal saline solution, 3 mL, weekly. The rats in the group C were treated daily with acupuncture at "Huantiao"(GB 30), "Housanli" (ST 36), "Yanglingquan"(GB 34) and "Weizhong"(BL 40) on the left hind legs; the rats in the group D were given orally Nilestriol solution (0.2 mg/mL) in a dose of 0.6 mL/100 g body weight, weekly. At the 7th, 14th, 21st and 28th days, some rats were sacrificed and their fractural callus and blood samples were taken for histological examinations and immunohistochemical examination of brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB)., Results: HE stained callus slides observed by optical microscope showed that the process of fracture healing in the group A, C, D was faster than that in the group B. Positive immuno-stalning of BDNF and TrkB could be seen in the all groups, mainly on the 7 and 14 days after the fracture. The expression levels from high to low in turn were group A, C, D and B., Conclusion: Expressions of BDNF and TrkB in callus of osteoporotic fracture were less than that of the normal fracture; acupuncture can elevate the expression levels and accelerate the process of fracture healing.

Aim: To investigate the influence of CCE3 on bone mineral element metabolism and relative hormone in ovariectomized rats., Methods: 24 female SD rats were divided randomly into 4 groups (n = 6): (1) Normal control; (2) sham ovariectomy; (3) ovariectomy(OVX); (4) OVX + CCE3., Results: (1) Ca, Mg, S, Co, Mn, Zn reduced obviously and P increased in the bone of the OVX rats. (2) E2, P, TSH, T4, CT, cortisol, GH reduced obviously and IL-6, FSH, LH increased obviously in serum of the OVX rats. (3) CCE3 corrected the changes of Ca, Mg, S, Co, Mn, Zn and P in bone as well as the changes of E2, P, TSH, T4, CT, cortisol, GH and IL-6, FSH, LH in serum of the OVX rats., Conclusion: Changes of relative hormone and IL-6 induced by CCE3 are the important mechanism of the influence of CCE3 on bone mineral element metabolism in ovariectomized rats.

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.

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.

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.

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.

Catechol-O-methyltransferase (COMT) enzyme catalyzes the methylation of the 2- or 4-hydroxyestrogens to 2- or 4-methoxyestrogens. Both the hydroxyestrogens and methoxyestrogens have been shown to block or enhance the effects of estrogen respectively. Our objective was to investigate the potential role of COMT in parturition and cervical ripening using a rat model. Immunohistochemistry was conducted to detect and localize the COMT protein in rat uterine tissues during pregnancy. We measured the longitudinal changes in urinary 2-hydroxyestrogen before, during, and after pregnancy in rats. Animal studies were conducted to determine the effect of treatment with a selective COMT inhibitor on (1) mifepristone-induced preterm birth and (2) cervical resistance to stretch in pregnant rats. The intensity of staining for the COMT protein differed within the luminal epithelium, uterine gland epithelium, endometrium, and myometrium during pregnancy. Levels of staining for the COMT protein in rat myometrium were highest on day 1 and lowest on days 8 and 13, but high levels returned by days 16 and 19 of pregnancy. The levels of urinary 2-hydroxyestrogen gradually increased in the first 2 weeks of pregnancy, peaked from days 16 to 18 of pregnancy, and then gradually returned to pre-pregnancy levels after delivery. The percentage of pups retained in the uterus of pregnant rats treated with both mifepristone and COMT inhibitor (48 +/- 15%) was significantly higher (P < 0.05) when compared with the value of pregnant rats treated with mifepristone alone (12 +/- 4%). The resistance to stretch was significantly higher (P < 0.05) in cervical tissues from the pregnant rats treated with COMT inhibitor (0.28) when compared with cervical tissues taken from rats treated with vehicle control (0.18). Modulation of COMT activity may play a role in the regulation of myometrial contractility and cervical ripening during pregnancy.

Certain estrogen metabolites can act as carcinogens in in vitro and animal experiments. The clinical relevance remains unclear. However, in the presence of factors that could influence estradiol metabolism, such as smoking or genetic polymorphisms, it seems prudent to prefer transdermal therapy to minimize the production of possible toxic metabolites. In addition, various defense mechanisms operate in the physiologic human body that prevent the formation of possible toxic intermediate products of estradiol metabolism, especially during oxidative stress. Only under rare special conditions is it conceivable that the human body cannot react sufficiently.

The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, because hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows stepwise generation from hESCs of cultures composed of approximately 70-80% human neurons that exhibit spontaneous synaptic network activity. Comparison of neurons derived from the well characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are preprogrammed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs.

Cancers of the reproductive tract account for 12% of all malignancies in women. As previous studies have shown that oestrogen metabolites can cause apoptosis, we characterised the effect of oestrogen and oestrogen metabolites on non-cancerous and cancerous human endometrial cells. Herein, we demonstrate that 2-methoxyoestradiol (2ME), but not 17beta-oestradiol, induces apoptosis in cancer cell lines and primary cultured tumours of endometrial origin. In contrast, 2ME had no effect on cell viability of corresponding normal tissue. This ability of 2ME to induce apoptosis does not require oestrogen receptor activation, but is associated with increased entry into the G2/M phases of the cell cycle and the activation of both the intrinsic and the extrinsic apoptotic pathways. The selective behaviour of 2ME on cancerous as opposed to normal tissue may be due to a reduction in 17beta-hydroxysteroid dehydrogenase type II levels in cancer cells and to a differential down-regulation of superoxide dismutase. Furthermore, we demonstrate that pre-treatment with 2ME enhances the sensitivity of reproductive tract cancer cells to the apoptotic drug tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), without the loss in cell viability to normal cells incurred by currently chemotherapeutic drugs. In conclusion, 2ME, alone or in combination with TRAIL, may be an effective treatment for cancers of uterine origin with minimal toxicity to corresponding healthy female reproductive tissue.

Objective: To observe the effect of Gengnianchun Recipe (GNC) on bone mineral density (BMD), bone biomechanical parameters and serum lipid level in the bilaterally ovariectomized (OVX) rats and to explore the prophylactic and therapeutic action of GNC on ovariectomy induced osteoporosis and hyperlipidemia., Methods: OVX SD rats, 10 - 12 months old, were divided into different groups and fed with GNC 2 g/d, GNC 1 g/d and Nilestriol 0.125 mg/week, respectively for 4 months to observe the change of BMD and bone biomechanical parameters of the lumbar vertebrae, and the serum levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), and to compare the effect of the two drugs on the morphology of the uterus., Results: There was marked reduction in BMD and biomechanical parameters in lumbar vertebrae (P < 0.01) and increase of serum TC and LDL-C levels (P < 0.01) in rats after OVX. GNC or Nilestriol significantly improved the decreased BMD and biomechanical parameters of the lumbar vertebrae (P < 0.05 or P < 0.01), and reduced the serum TC and LDL-C levels (P < 0.01). In the Nilestriol group, the wet weight of uterus got increased obviously (P < 0.01), the number of uterine glands increased, uterine columnar epithelium thickened, and the mitotic figures in the epithelial stroma and myointimal cells augmented. But no such effect in wet weight and morphology of uterus was found in the GNC group., Conclusion: GNC could increase the BMD and biomechanical parameters of the lumbar vertebrae, reduce the serum TC and LDL-C levels, yet produce no adverse reaction in stimulating proliferation and hypertrophy of uterus.

This paper describes a way of differentiating between the three isomers of estriol glucuronide by the use of chemical derivatization and liquid chromatography/electrospray tandem mass spectrometry (MS/MS). In their native form, these isomers gave rise to almost identical product ion spectra, involving the neutral loss of 176 Da (i.e. monodehydrated glucuronic acid), which made it impossible to determine the position of conjugation by MS/MS alone. In order to change the fragmentation pathways, positive charges were introduced into the analytes by chemical derivatization. The following reagents were tested: 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2-picolylamine. Interestingly, derivatization using a combination of all three reagents gave a selective fragmentation pattern that could differentiate between the isomers estriol-16-glucuronide and estriol-17-glucuronide. Estriol-3-glucuronide, which lacks a free phenolic group, could be differentiated through a different type of reaction product when exposed to 2-chloro-1-methylpyridinium iodide. Furthermore, in order to assist structural assignment of the fragments, their accurate masses were determined using a hybrid quadrupole time-of-flight mass spectrometer and fragmentation pathways were elucidated by the use of MS3 on an ion trap mass spectrometer., (Copyright 2006 John Wiley & Sons, Ltd.)

Background: It has been suggested that a low level of the 2-hydroxyestrogen metabolites (2-OHE) and a high level of 16alpha-hydroxyestrone (16alpha-OHE1) are associated with an enhanced risk of breast cancer. We examined the association between the metabolite levels and breast cancer in a nested case-control study, which also addressed hormone replacement therapy (HRT) and estrogen receptor status of the tumors., Methods: 24,697 postmenopausal Danish women were enrolled in the "Diet, Cancer and Health" cohort. During follow-up, 426 breast cancer cases were identified and controls were matched by age at diagnosis, baseline age, and HRT use. The concentrations of 2-OHE and 16alpha-OHE1 in spot urine were measured by an enzyme immunoassay. Incidence rate ratios (IRR) and 95% confidence intervals (95% CI) were estimated for total and estrogen receptor-specific breast cancer and were stratified according to HRT use., Results: A higher incidence of estrogen receptor-positive breast cancer with an enhanced 2-OHE level was observed among current HRT users, IRR per doubling = 1.30 (95% CI, 1.02-1.66), whereas no association was seen among nonusers of HRT, IRR per doubling = 1.00 (95% CI, 0.69-1.45). The association between estrogen receptor-positive breast cancer and the 16alpha-OHE1 metabolite level was in the opposite direction but slightly weaker and statistically insignificant. For estrogen receptor-negative breast cancer, no significant associations were seen., Conclusions: The risk of breast cancer, in particular the estrogen receptor-positive type, was enhanced among postmenopausal women using estradiol-based HRT and among those who had a high 2-OHE concentration.

Fast excitatory postsynaptic potentials (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea-pig ileum in vitro. This study used electrophysiological methods to study fEPSPs during stimulus trains to mimic bursts of synaptic activity in vitro. The amplitude of fEPSPs or fast excitatory postsynaptic currents (EPSCs) declined (rundown) during stimulus trains at frequencies of 0.5, 5, 10 and 20 Hz. At 0.5 Hz, fEPSP or fEPSC amplitude declined by 50% after the first stimulus but remained constant for the remainder of the train. At 5, 10 and 20 Hz, synaptic responses ran down completely with time constants of 0.35, 0.21 and 0.11 s, respectively. Recovery from rundown occurred with a time constant of 7 s. Mecamylamine, a nicotinic cholinergic receptor antagonist, or PPADS, a P2X receptor antagonist, reduced fEPSP amplitude, but they had no effect on rundown. Responses caused by trains of ionophoretically applied ATP or ACh (to mimic fEPSPs) did not rundown. Blockade of presynaptic inhibitory muscarinic, adenosine A1, opioid, alpha2-adrenergic and 5-HT1A receptors or pertussis toxin (PTX) treatment did not alter rundown. Antidromic action potentials followed a 10-Hz stimulus train. Iberiotoxin (100 nM), a blocker of large conductance calcium activated K+ (BK) channels, did not alter rundown. These data suggest that synaptic rundown is not due to: (a) action potential failure; (b) nicotinic or P2X receptor desensitization; (c) presynaptic inhibition mediated by pertussis-toxin sensitive G-proteins, or (d) BK channel activation. Synaptic rundown is likely due to depletion of a readily releasable pool (RRP) of neurotransmitter.

klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) beta-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl beta-D-glucuronide in contrast to bovine liver beta-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of beta-glucuronidase. A number of natural beta-glucuronides were screened by competitive inhibition for KLFc beta-glucuronidase. We found that steroid beta-glucuronides such as beta-estradiol 3-beta-D-glucuronide, estrone 3-beta-D-glucuronide, and estriol 3beta-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel beta-glucuronidase and that steroid beta-glucuronides are potential candidates for the natural substrate(s) of Klotho.

2-Methoxyoestradiol (2-MeOE2) is an endogenous oestrogen metabolite that inhibits the proliferation of cancer cells in vitro, and it is also antiangiogenic. In vivo 2-MeOE2, when administered at relatively high doses, inhibits the growth of tumours derived from breast cancer cells, sarcomas and melanomas. Sulphamoylated derivatives of 2-MeOE2 are more potent inhibitors of in vitro breast cancer cell growth than 2-MeOE2. In the present study, we have compared the pharmacokinetic profiles and metabolism of 2-MeOE2 and its sulphamoylated derivative, 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE), in adult female rats. Their ability to inhibit tumour growth was compared in nude mice bearing xenografts derived from MDA-MB-435 (oestrogen receptor negative) melanoma cancer cells. After a single oral 10 mg kg(-1) dose of 2-MeOE2bisMATE, significant concentrations of this compound were still detectable at 24 h. In contrast, no 2-MeOE2 or metabolites were detected in plasma at any time after a 10 mg kg(-1) oral dose. Thus, the bioavailability of 2-MeOE2 is very low, whereas for 2-MeOE2bisMATE it was 85%. No significant metabolites of 2-MeOE2bisMATE were detected in plasma after oral or intravenous dosing, showing that this drug is resistant to metabolism. In the tumour efficacy model, oral administration of 2-MeOE2bisMATE, at 20 mg kg(-1) day(-1) daily for 28 days, almost completely inhibited tumour growth. Inhibition of tumour growth was maintained for a further 28 days after the cessation of dosing. At this dose level, 2-MeOE2 did not inhibit tumour growth. The resistance to metabolism shown by 2-MeOE2bisMATE and its ability to inhibit tumour growth in vivo suggest that this compound should have considerable potential for development as a novel anticancer drug.

Background: Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria., Methods: Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0-8 days at room temperature and for 0-10 freeze-thaw cycles., Results: Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C-estradiol; r = 0.98 for PDG-progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles., Conclusions: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

Using column-switching liquid chromatography/tandem mass spectrometry (LC-MS/MS), we developed an improved analytical method of urinary estriol glucuronides. This new method is derived predominantly from maternal and fetal precursors in pregnancy. We used in the following procedure: first, we filtered urine samples with a membrane filter. Next, we directly injected the 50 microL aliquot of urine samples onto a pre-column. Then, after activating the column-switching valve, we backflushed the loaded samples onto the C(18) analytical column. Urine samples can be assayed within 20 min without any sample preparation steps. We monitored separated estriol glucuronides by negative electrospray ionization (ESI) and selected-reaction monitoring (SRM). The calibration range of estriol-3-glucuronide (E3-3G) and estriol-16-glucuronide (E3-16G) was 0.1-20 microg/mL and the linearity of the method was 0.9984 for E3-3G and 0.9987 for E3-16G. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng/mL (E3-3G) and 5 ng/mL (E3-16G). The analytical recovery was over 85% and, in general, inter-day and intra-day variability for precision and accuracy were less than 10%. When applied to a pregnancy urine sample to biomedical monitoring of the function of the maternal/fetal unit, the proposed method allowed rapid and sensitive screening for the detection of E3-3G and E3-16G., (Copyright 2003 John Wiley & Sons, Ltd.)

The primary hydroxy functions of 16alpha-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17beta-ol (3a) and 16beta-hydroxymethyl-3-methoxy-13alpha-estra-1,3,5(10)-trien-17alpha-ol (4a) were stereoselectively transformed into good leaving groups. On alkaline methanolysis of the 16-halomethyl or 16-tolylsulfonyloxymethyl derivatives, a new D-seco-13alpha-estrone derivative was obtained in high yield.

There is currently considerable interest in the use of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) for the treatment and prevention of breast cancer. We have previously shown that sulphamoylation of 2-MeOE2 and related derivatives greatly enhances their ability to inhibit the proliferation of ER+ and ER- breast cancer cells. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-ethyloestradiol-bis-sulphamate (2-EtE2bisMATE) with that of 2-MeOE2 to inhibit the proliferation of breast cancer cells when grown on three different substrata: plastic, collagen I and Matrigel. The human breast cell line MCF-7 was utilised for these studies together with its doxorubicin resistant variant, MCF-7 DOX40 and mitoxantrone resistant variant, MCF-7 MR, as a longitudinal model of in vitro drug resistance. On a plastic substratum all three cell lines were sensitive to the effects of 2-MeOE2bisMATE and 2-EtE2bisMATE whereas MCF-7 cells and the MCF-MR variant cells were resistant to the effects of 2-MeOE2 at 1 microM. The sensitivity of the cell lines to those compounds also remained significant when grown on more physiological substrata. All of the drugs tested arrested cells in the G2/M phase of the cell cycle. The finding that breast cancer cells that are resistant to conventional chemotherapeutic agents remain sensitive to 2-substituted oestrogen sulphamates offers considerable potential for the treatment of women with drug-resistant breast cancer.

Objectives: To summarise the experience and evaluate the performance of the Ontario maternal serum screening (MSS) programme., Setting: The Ontario MSS programme between October 1993 and September 2000., Methods: This study used information collected in the Ontario MSS database, which contains data on each screened pregnancy. In the Ontario MSS programme, women are screened between 15 and 20 weeks of gestation. The risk cut-off for Down's syndrome was >or= 1 in 385 at term and women with a serum alpha-fetoprotein >or= 2.2 multiples of the unaffected population median were defined as screen-positive for open neural tube defects., Results: Between 1 October 1993 and 30 September 2000, 428410 women residing in Ontario were screened for open neural tube defects, and 423895 women were screened for Down's syndrome and trisomy 18. Approximately 48% of all pregnant women in the province had MSS. The uptake rate of amniocentesis following a positive Down's syndrome screening was 67%. Of 717 cases of Down's syndrome ascertained in the screened population, 531 were detected by MSS, giving a term detection rate (DR) of 70.6%, with a false-positive rate (FPR) of 7.2%. For neural tube defects, the DR was 72.7%, with a FPR of 2.0%. The screen also detected 50% of cases of trisomy 18 at term, with a FPR of 0.2%. Coincidentally, 113 cases of chromosome aneuploidies other than Down's syndrome and trisomy 18 were detected., Discussion: In the Ontario MSS programme, MSS performed as expected in the detection of Down's syndrome, open neural tube defects and trisomy 18. MSS is an effective and practical method for large-scale second trimester screening for Down's syndrome, open neural tube defects and trisomy 18, and the MSS database is an extremely useful tool in monitoring the performance of this screen.