Acupuncture combined with mifepristone improves sex hormones and inflammatory factors in patients with uterine fibroids.

OBJECTIVE: To analyze the therapeutic effect of acupuncture combined with mifepristone on uterine fibroids and its influence on sex hormones and inflammatory factors. METHODS: Data of 102 patients with uterine fibroids admitted to Shanxi Provincial Hospital of Chinese Medicine from January 2019 to January 2022 were retrospectively analyzed. Among them, there were 50 patients treated with mifepristone alone (control group) and 52 patients undergoing combined treatment of acupuncture and mifepristone (observation group). After 2 months of continuous treatment, the therapeutic efficacy, volume of uterine fibroids and uterus, levels of inflammatory factors (C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α)), as well as levels of estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), along with hemodynamic levels and incidence of adverse reactions were recorded and compared between the two groups. Logistic analysis was employed to identify the independent risk factors for the recurrence of uterine fibroids in patients. RESULTS: Compared with the control group, the observation group was identified with significantly higher overall response rate (P < 0.05). The uterine fibroid volume and uterine volume significantly improved in both groups after treatment, and the improvements were more prominent in the observation group than in the control group (P < 0.05). After treatment, the serum CRP and TNF-α were both evidently decreased in the two groups, while levels of E2, FSH, LH and peak blood flow velocity were significantly ameliorated, and the improvements in the observation group were more significant than those in the control group (P < 0.05). There was no significant difference in the incidence of adverse reactions between the two groups (P > 0.05). Alcohol intake and treatment regime were independent risk factors for the recurrence of uterine fibroids in patients. CONCLUSION: Combining acupuncture with mifepristone can significantly improve uterine fibroids, estrogen and progesterone levels, as well as reduce inflammation, with a high level of safety, making it a promising treatment for clinical use.

Migalastat Tissue Distribution: Extrapolation From Mice to Humans Using Pharmacokinetic Modeling and Comparison With Agalsidase Beta Tissue Distribution in Mice.

Approved therapies for Fabry disease (FD) include migalastat, an oral pharmacological chaperone, and agalsidase beta and agalsidase alfa, 2 forms of enzyme replacement therapy. Broad tissue distribution may be beneficial for clinical efficacy in FD, which has severe manifestations in multiple organs. Here, migalastat and agalsidase beta biodistribution were assessed in mice and modeled using physiologically based pharmacokinetic (PBPK) analysis, and migalastat biodistribution was subsequently extrapolated to humans. In mice, migalastat concentration was highest in kidneys and the small intestine, 2 FD-relevant organs. Agalsidase beta was predominantly sequestered in the liver and spleen (organs unaffected in FD). PBPK modeling predicted that migalastat 123 mg every other day resulted in concentrations exceeding the in vitro half-maximal effective concentration in kidneys, small intestine, skin, heart, and liver in human subjects. However, extrapolation of mouse agalsidase beta concentrations to humans was unsuccessful. In conclusion, migalastat may distribute to tissues that are inaccessible to intravenous agalsidase beta in mice, and extrapolation of mouse migalastat concentrations to humans showed adequate tissue penetration, particularly in FD-relevant organs.

Isothiazolinone Detection in Dish Soap and Personal Care Products: Comparison of Lovibond Isothiazolinone Test Kit and Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Isothiazolinones are commonly used preservatives, which may cause allergic contact dermatitis. The Lovibond Isothiazolinone Test Kit (LITK) has been reported to successfully identify clinically relevant, occult isothiazolinones in patient personal care products. OBJECTIVE: The aim of the study was to analyze dish soaps and personal care products that do not declare isothiazolinones ("no-ISO") for the presence of isothiazolinones via 2 methods: LITK and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). METHODS: No-ISO dish soaps (n = 9), a convenience sample of patient products (n = 6), and controls (positive [isothiazolinone declared], n = 5; negative, n = 2) were tested with LITK (X3) and UHPLC-MS/MS. RESULTS: Several no-ISO dish soaps and personal products were positive for isothiazolinones (LITK, n = 12; UHPLC-MS/MS, n = 3). Ultrahigh-performance liquid chromatography-tandem mass spectrometry specifically identified methylisothiazolinone alone in 1 no-ISO dish soap, methylchloroisothiazolinone in another, and both in a third. Using UHPLC-MS/MS as the criterion standard, we observed the accuracy of LITK for 9 dish soaps was poor (sensitivity, 66.7%; specificity, 20%) and very poor for 6 personal care products (sensitivity, 0%; specificity, 0%). CONCLUSIONS: Personal products may contain undeclared isothiazolinones. The current study found that LITK had poor accuracy for testing dish soap and personal care products. Clinicians should be aware of these factors when managing patients with contact allergy to isothiazolinones.

Who listens and who doesn't? Factors associated with adherence to antibiotic stewardship intervention in a Singaporean tertiary hospital.

INTRODUCTION: Antibiotic stewardship programmes (ASPs) can improve patient outcomes by prospective audit and feedback with interventions. However, adherence to ASP interventions is not mandatory. Identifying factors associated with improved adherence may help to enhance ASP recommendations and activities. METHODS: A retrospective cohort study was conducted, comprising all ASP interventions performed as part of the prospective audit and feedback strategy in our institution (an acute tertiary-care hospital in Singapore) from January 2016 to July 2018. Adherence to ASP intervention was ascertained based on documented compliance with the recommended interventions within 48h. Factors associated with adherence to ASP interventions, such as patient demographics, clinical condition, type of infection, and characteristics of ASP interventions were identified using the χ2 test for categorical variables. On multivariate analysis, factors independently associated with adherence to ASP intervention were identified using logistic regression. RESULTS: Adherence to ASP intervention was 81.9% (5758/7028). On univariate and multivariate analysis, interventions coupled with direct communication via phone call (adjusted odds ratio [aOR] 1.61, 95% CI 1.23-2.08) were associated with higher odds of adherence, whereas admission to a surgical unit, intervention involving carbapenem use, and recommendation to de-escalate or discontinue antibiotics were associated with lower odds of adherence to ASP interventions. CONCLUSION: Although adherence rates to ASP interventions were relatively high, interventions made to the surgical unit and recommendations related to carbapenem use were not so well received. Interventions communicated verbally via phone call were well received, highlighting the need for a close working relationship between ASP teams and hospital physicians.

Improved efficacy of a next-generation ERT in murine Pompe disease.

Pompe disease is a rare inherited disorder of lysosomal glycogen metabolism due to acid α-glucosidase (GAA) deficiency. Enzyme replacement therapy (ERT) using alglucosidase alfa, a recombinant human GAA (rhGAA), is the only approved treatment for Pompe disease. Although alglucosidase alfa has provided clinical benefits, its poor targeting to key disease-relevant skeletal muscles results in suboptimal efficacy. We are developing an rhGAA, ATB200 (Amicus proprietary rhGAA), with high levels of mannose-6-phosphate that are required for efficient cellular uptake and lysosomal trafficking. When administered in combination with the pharmacological chaperone AT2221 (miglustat), which stabilizes the enzyme and improves its pharmacokinetic properties, ATB200/AT2221 was substantially more potent than alglucosidase alfa in a mouse model of Pompe disease. The new investigational therapy is more effective at reversing the primary abnormality - intralysosomal glycogen accumulation - in multiple muscles. Furthermore, unlike the current standard of care, ATB200/AT2221 dramatically reduces autophagic buildup, a major secondary defect in the diseased muscles. The reversal of lysosomal and autophagic pathologies leads to improved muscle function. These data demonstrate the superiority of ATB200/AT2221 over the currently approved ERT in the murine model.

Isothiazolinone Content of US Consumer Adhesives: Ultrahigh-Performance Liquid Chromatographic Mass Spectrometry Analysis.

BACKGROUND: There are limited data regarding the prevalence and concentration of isothiazolinone preservatives in consumer adhesives. OBJECTIVES: The aim of this study was to determine the prevalence and concentration of 5 specific isothiazolinones (methylisothiazolinone [MI], methylchloroisothiazolinone [MCI], benzisothiazolinone [BIT], butyl BIT, and octylisothiazolinone) in US adhesives. METHODS: Thirty-eight consumer adhesives were analyzed using ultrahigh-performance liquid chromatographic-mass spectrometry. Fisher exact tests were used to test for isothiazolinone content and: 1) glue format (2) application purpose and 3) extraction method. RESULTS: Nineteen adhesives (50%) had at least 1 isothiazolinone, and 15 contained 2 isothiazolinones. Frequencies and concentrations were as follows: MI (44.7%; 4-133 ppm), MCI (31.6%; 7-27 ppm), BIT (15.8%; 10-86 ppm), and octylisothiazolinone (2.6%; 1 ppm). Butyl BIT was not detected in any of the adhesives. Format (stick vs liquid) was not statistically associated with isothiazolinone presence. At least half of adhesives in the following application purposes had at least 1 isothiazolinone: shoe, craft, fabric, and school. All-purpose glues had a statistically significant lower concentration of MI and MCI, whereas craft glues were associated with higher concentrations of MI and MCI. Compared with other glues, fabric adhesives were associated with a higher risk of containing BIT. CONCLUSIONS: Half of the tested adhesives contained at least 1 isothiazolinone. Methylisothiazolinone and MCI were the most common. Consumers and dermatologists should be aware of adhesives as a source of isothiazolinones.

Isothiazolinone in Residential Interior Wall Paint: A High-Performance Liquid Chromatographic-Mass Spectrometry Analysis.

BACKGROUND: There is limited information regarding isothiazolinone content in residential wall paints in the United States. OBJECTIVE: The aim of this study was to evaluate the prevalence of 5 isothiazolinones-methylisothiazolinone (MI), methylchloroisothiazolinone, benzisothiazolinone (BIT), butyl BIT, and octylisothiazolinone-in US residential wall paints. METHODS: Forty-seven paints were obtained from retailers in Minneapolis/St Paul, Minnesota. Paint samples were assessed for the presence of the 5 isothiazolinones using high-performance liquid chromatographic-mass spectrometry. RESULTS: At least 1 isothiazolinone was detected in all 47 paints. However, no paint contained butyl BIT, and only 1 paint had octylisothiazolinone. The MI and BIT were found in 96% and 94% of the paints, respectively. Methylisothiazolinone ranged in concentration from 17 to 358 ppm, whereas BIT varied from 29 to 1111 ppm. Methylchloroisothiazolinone was found solely in oil-based paints. Isothiazolinones were declared in 15% of Safety Data Sheets but did not correlate with high-performance liquid chromatographic-mass spectrometry. One "preservative-free" paint had BIT at 71.5 ppm. Paint sheen was not statistically associated with BIT or MI concentrations. Unpigmented paints and paints with volatile organic compound claims had significantly lower concentrations of MI, but not BIT. CONCLUSIONS: All paints contained at least 1 isothiazolinone. Methylisothiazolinone and BIT were the most common. Safety Data Sheets are insufficient for ascertaining isothiazolinone content in US paints.

Coformulation of a Novel Human α-Galactosidase A With the Pharmacological Chaperone AT1001 Leads to Improved Substrate Reduction in Fabry Mice.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene that encodes α-galactosidase A and is characterized by pathological accumulation of globotriaosylceramide and globotriaosylsphingosine. Earlier, the authors demonstrated that oral coadministration of the pharmacological chaperone AT1001 (migalastat HCl; 1-deoxygalactonojirimycin HCl) prior to intravenous administration of enzyme replacement therapy improved the pharmacological properties of the enzyme. In this study, the authors investigated the effects of coformulating AT1001 with a proprietary recombinant human α-galactosidase A (ATB100) into a single intravenous formulation. AT1001 increased the physical stability and reduced aggregation of ATB100 at neutral pH in vitro, and increased the potency for ATB100-mediated globotriaosylceramide reduction in cultured Fabry fibroblasts. In Fabry mice, AT1001 coformulation increased the total exposure of active enzyme, and increased ATB100 levels in cardiomyocytes, cardiac vascular endothelial cells, renal distal tubular epithelial cells, and glomerular cells, cell types that do not show substantial uptake with enzyme replacement therapy alone. Notably, AT1001 coformulation also leads to greater tissue globotriaosylceramide reduction when compared with ATB100 alone, which was positively correlated with reductions in plasma globotriaosylsphingosine. Collectively, these data indicate that intravenous administration of ATB100 coformulated with AT1001 may provide an improved therapy for Fabry disease and thus warrants further investigation.

The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.

Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.

The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease.

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.

Lysosomal dysfunction in a mouse model of Sandhoff disease leads to accumulation of ganglioside-bound amyloid-ß peptide.

Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-ß peptide (Aß)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of ß-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aß-like immunoreactivity (iAß-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aß. In addition, we observed increased levels of Aß40 and Aß42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aß (GAß) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aß were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAß-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAß-LIR may be associated with the lysosomal-autophagic turnover of Aß and fragments of APP-containing Aß epitopes. Importantly, intraneuronal GAß immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.

Co-administration with the pharmacological chaperone AT1001 increases recombinant human α-galactosidase A tissue uptake and improves substrate reduction in Fabry mice.

Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by mutations in the gene (GLA) that encodes the lysosomal hydrolase α-galactosidase A (α-Gal A), and is characterized by pathological accumulation of the substrate, globotriaosylceramide (GL-3). Regular infusion of recombinant human α-Gal A (rhα-Gal A), termed enzyme replacement therapy (ERT), is the primary treatment for Fabry disease. However, rhα-Gal A has low physical stability, a short circulating half-life, and variable uptake into different disease-relevant tissues. We hypothesized that coadministration of the orally available, small molecule pharmacological chaperone AT1001 (GR181413A, 1-deoxygalactonojirimycin, migalastat hydrochloride) may improve the pharmacological properties of rhα-Gal A via binding and stabilization. AT1001 prevented rhα-Gal A denaturation and activity loss in vitro at neutral pH and 37 °C. Coincubation of Fabry fibroblasts with rhα-Gal A and AT1001 resulted in up to fourfold higher cellular α-Gal A and ~30% greater GL-3 reduction compared to rhα-Gal A alone. Furthermore, coadministration of AT1001 to rats increased the circulating half-life of rhα-Gal A by >2.5-fold, and in GLA knockout mice resulted in up to fivefold higher α-Gal A levels and fourfold greater GL-3 reduction than rhα-Gal A alone. Collectively, these data highlight the potentially beneficial effects of AT1001 on rhα-Gal A, thus warranting clinical investigation.

In vitro dermal penetration of 4-chloro-3-methylphenol from commercial metal working fluid and aqueous vehicles.

The biocide 4-chloro-3-methylphenol (CMP, CAS number 59-50-7) is a common additive to metal-working fluids (MWF) and building materials. National Institute for Occupational Safety and Health (NIOSH) researchers previously identified and quantified CMP in a commercial water-soluble MWF, TRIM VX, and demonstrated irritancy and sensitization potential of both TRIM VX and CMP alone after dermal exposure in a murine model. In the current study, the in vitro human epidermal permeability of CMP contained in a working dilution of TRIM VX (20% in water) was evaluated and, for comparison, permeability from an aqueous buffer was also assessed. CMP penetration was also measured from transient exposures to 20% TRIM VX. To address differences in penetration rates from 20% TRIM VX and from buffer, the role of thermodynamic activity of CMP in the 2 vehicles on dermal penetration was investigated. Static headspace gas chromatography was used to measure vapor pressures and infer fractional thermodynamic activities of CMP in the mixtures. Permeability coefficient (k(p)) of CMP from 20% TRIM VX was (4.1 +/- 0.8) x 10(-3) cm/h (mean +/- SD, n = 5), and CMP was found at a concentration of 3555 +/- 191 microg/ml in this donor. In contrast, k(p) was 0.18 +/- 0.03 cm/h (n = 5) at a similar concentration (3919 +/- 240) from buffer donor. Steady-state fluxes from 20% TRIM VX and buffer were comparable when expressed as functions of thermodynamic activity of CMP in the donor, rather than as concentrations. Transient (20 or 40 min) exposures of epidermal membranes to 20% TRIM VX (n = 4) resulted in total penetration of 4.2 +/- 1.2 and 7.3 +/- 0.8 microg/cm(2), respectively; these amounts are comparable to amounts predicted using a simple algebraic equation.

The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of beta-glucosidase.

Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid beta-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5- and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2- to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease.

The pharmacological chaperone 1-deoxygalactonojirimycin reduces tissue globotriaosylceramide levels in a mouse model of Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (alpha-Gal A) activity and subsequent accumulation of the substrate globotriaosylceramide (GL-3), which contributes to disease pathology. The pharmacological chaperone (PC) DGJ (1-deoxygalactonojirimycin) binds and stabilizes alpha-Gal A, increasing enzyme levels in cultured cells and in vivo. The ability of DGJ to reduce GL-3 in vivo was investigated using transgenic (Tg) mice that express a mutant form of human alpha-Gal A (R301Q) on a knockout background (Tg/KO), which leads to GL-3 accumulation in disease-relevant tissues. Four-week daily oral administration of DGJ to Tg/KO mice resulted in significant and dose-dependent increases in alpha-Gal A activity, with concomitant GL-3 reduction in skin, heart, kidney, brain, and plasma; 24-week administration resulted in even greater reductions. Compared to daily administration, less frequent DGJ administration, including repeated cycles of 4 days with DGJ followed by 3 days without or every other day with DGJ, resulted in even greater GL-3 reductions that were comparable to those obtained with Fabrazyme. Collectively, these data indicate that oral administration of DGJ increases mutant alpha-Gal A activity and reduces GL-3 in disease-relevant tissues in Tg/KO mice, and thus merits further evaluation as a treatment for Fabry disease.

Dissecting self-renewal in stem cells with RNA interference.

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

Inactivation of acetylcholinesterase by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride.

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to reversibly inhibit the activity of acetylcholinesterase. The inactivation of the enzyme was detected by monitoring the accumulation of yellow color produced from the reaction between thiocholine and dithiobisnitrobenzoate ion. The kinetic parameter, Km for the substrate (acetylthiocholine), was found to be 0.216 mM and Ki for MPTP inactivation of acetylcholinesterase was found to be 2.14 mM. The inactivation of enzyme by MPTP was found to be dose-dependent. It was found that MPTP is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The dilution assays indicate that MPTP is a reversible inhibitor for AChE. These data suggest that once MPTP enters the basal ganglia of the brain, it can inactivate the acetylcholinesterase enzyme and thereby increase the acetylcholine level in the basal ganglia of brain, leading to potential cell dysfunction. It appears that the nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may, in part, be mediated via the acetylcholinesterase inactivation.

Effect of chlorogenic acid on hydroxyl radical.

Chlorogenic acid (CGA) is considered to act as an antioxidant. However, the inhibitory effects of CGA on specific radical species are not well understood. Electron spin resonance (ESR) in combination with spin trapping techniques was utilized to detect free radicals. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin trapping reagent while the Fenton reaction was used as a source of hydroxyl radical (*OH). We found that CGA scavenges *OH in a dose-dependent manner. The kinetic parameters, IC50 and Vmax, for CGA scavenging of *OH were 110 and 1.27 microM/sec, respectively. The rate constant for the scavenging of *OH by CGA was 7.73 x 10(9) M(-1) sec(-1). Our studies suggest that the antioxidant properties of CGA may involve a direct scavenging effect of CGA on *OH.

Determination of alachlor and its metabolites in rat plasma and urine by liquid chromatography-electrospray ionization mass spectrometry.

A method based on liquid chromatography (LC) in combination with mass spectrometry (MS) for the analysis of alachlor (ALA) and its metabolites, 2-chloro-N-[2,6-diethylphenyl]acetamide (CDEPA) and 2,6-diethylaniline (DEA), in rat plasma and urine has been developed. 13C-labeled ALA was used as the internal standard for quantitation. The analyte in plasma or urine was isolated using a Waters Oasis HLB extraction plate. The mass spectrometer was operated in the ESI MS-SIM mode with a programming procedure. The retention times for ALA, CDEPA and DEA were 1.84, 3.11 and 4.12 min, respectively. The limits of quantification (LOQ) for ALA, CDEPA and DEA were 2.3, 0.8 and 0.8 ng per injection, respectively. The linear fit of analyte to mass response had an R2 of 0.99. Reproducibility of the sample handling and LC-MS analysis had a RSD of < or = 10%. The average recoveries for these analytes in rat plasma were better than 90%. Similar results were obtained with rat urine.