No abstract

No abstract

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

(8,9). A number of investigators have found that the level of MAO B in preparations exhibiting high affinity sites is only 5-10% of the total enzyme present (9 -11). It is not known whether this high affinity binding also results in enzyme inhibition. To date, no molecular explanation has been found to resolve these observations except to propose that the nanomolar binding site is separate from the active site and is possessed by a subpopulation of enzyme occurring by an unknown mechanism. No evidence exists for any altered enzyme forms (alternate splicing or posttranslational modification(s)), which might account for the observed substoichiometric levels of high affinity binding sites on MAO B.The Eli Lilly group (11-13) observed that inhibition of human MAO B by tranylcypromine increases the level of high affinity I 2 -binding sites from 5-10% to ϳ90% of the total enzyme. This potentiation is observed with MAO B in human platelets, in membrane preparations from human cortex, and from medulla preparations as well as with membrane particles of human recombinant MAO B (but not with human MAO A) expressed in insect cells (13). These data suggest that inhibition of MAO B by tranylcypromine alters the enzyme to a form that * This work was supported, in whole or in part, by National Institutes of Health Grant GM 29433 (to D. E. E.) and a predoctoral fellowship from the National Institutes of Health through NINDS Award F31NS063648 (to E. M. M.). This work was also supported by grants from the Fondazione Cariplo (to D. E. E. and A. M.), , and Canadian Institutes of Health Research (Grant MOP77529) to A. H. The atomic coordinates and structure factors (codes 2XFU, 2XCG, 2XFN, 2XFO, 2XFP, and 2XFQ) The abbreviations used are: MAO, monoamine oxidase; 2-BFI, 2-(2-benzofuranyl)-2-imidazoline.

The exposure sources of perfluorooctane sulfonate (PFOS) in humans and wildlife are not well characterized. Human biomonitoring data show that PFOS profiles may consist of up to approximately 50% branched isomers, despite the fact that historical direct manufacturing of PFOS generally resulted in products containing no more than approximately 30% branched isomers. These observations cannot be explained based on what is known about the pharmacokinetics of branched PFOS isomers; thus, here we examined the relative isomer-specific biotransformation rates of a model PFOS-precursor (N-ethylperfluorooctane sulfonamide, NEtFOSA) with human microsomes and recombinant human cytochrome P450s (CYPs) 2C9 and 2C19. Using solid phase microextraction-gas chromatography-electron capture detection to monitor NEtFOSA disappearance, and liquid chromatography-tandem mass spectrometry to monitor product formation, we showed that, in general, human microsomes and CYP isozymes transformed the branched isomers more rapidly than linear NEtFOSA. Among branched isomers, perfluoroalkyl branching geometry significantly influenced the rate of biotransformation. As a result, PFOS isomer patterns in biota exposed predominantly to precursors could be much different than expected from the isomer pattern of the precursor. While these data are suggestive that the relatively high abundance of branched PFOS isomers present in some humans, or wildlife, may be explained by substantial exposure to PFOS-precursors, in vivo studies with other relevant PFOS-precursors are warranted to validate this as a biomarker of exposure source.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by motor, cognitive and psychiatric problems. Previous studies indicated that levels of brain gangliosides are lower than normal in HD models and that administration of exogenous ganglioside GM1 corrects motor dysfunction in the YAC128 mouse model of HD. In this study, we provide evidence that intraventricular administration of GM1 has profound disease‐modifying effects across HD mouse models with different genetic background. GM1 administration results in decreased levels of mutant huntingtin, the protein that causes HD, and in a wide array of beneficial effects that include changes in levels of DARPP32, ferritin, Iba1 and GFAP, modulation of dopamine and serotonin metabolism, and restoration of normal levels of glutamate, GABA, L‐Ser and D‐Ser. Treatment with GM1 slows down neurodegeneration, white matter atrophy and body weight loss in R6/2 mice. Motor functions are significantly improved in R6/2 mice and restored to normal in Q140 mice, including gait abnormalities that are often resistant to treatments. Psychiatric‐like and cognitive dysfunctions are also ameliorated by GM1 administration in Q140 and YAC128 mice. The widespread benefits of GM1 administration, at molecular, cellular and behavioural levels, indicate that this ganglioside has strong therapeutic and disease‐modifying potential in HD.

This article is a clinical guide which discusses the “state-of-the-art” usage of the classic monoamine oxidase inhibitor (MAOI) antidepressants (phenelzine, tranylcypromine, and isocarboxazid) in modern psychiatric practice. The guide is for all clinicians, including those who may not be experienced MAOI prescribers. It discusses indications, drug-drug interactions, side-effect management, and the safety of various augmentation strategies. There is a clear and broad consensus (more than 70 international expert endorsers), based on 6 decades of experience, for the recommendations herein exposited. They are based on empirical evidence and expert opinion—this guide is presented as a new specialist-consensus standard. The guide provides practical clinical advice, and is the basis for the rational use of these drugs, particularly because it improves and updates knowledge, and corrects the various misconceptions that have hitherto been prominent in the literature, partly due to insufficient knowledge of pharmacology. The guide suggests that MAOIs should always be considered in cases of treatment-resistant depression (including those melancholic in nature), and prior to electroconvulsive therapy—while taking into account of patient preference. In selected cases, they may be considered earlier in the treatment algorithm than has previously been customary, and should not be regarded as drugs of last resort; they may prove decisively effective when many other treatments have failed. The guide clarifies key points on the concomitant use of incorrectly proscribed drugs such as methylphenidate and some tricyclic antidepressants. It also illustrates the straightforward “bridging” methods that may be used to transition simply and safely from other antidepressants to MAOIs.