Proton nuclear magnetic resonance (1H NMR) spectroscopy is a noninvasive technique that can provide information on a wide range of metabolites. Marked abnormalities of 1H NMR brain spectra have been reported in patients with neurological disorders, but their neurochemical implications may be difficult to appreciate because NMR data are obtained from heterogeneous tissue regions composed of several cell populations. The purpose of this study was to examine the 1H NMR profile of major neural cell types. This information may be helpful in understanding the metabolic abnormalities detected by 1H NMR spectroscopy. Extracts of cultured cerebellar granule neurons, cortical astrocytes, oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, oligodendrocytes, and meningeal cells were analyzed. The purity of the cultured cells was > 95% with all the cell lineages, except for neurons (approximately 90%). Although several constituents (creatine, choline-containing compounds, lactate, acetate, succinate, alanine, glutamate) were ubiquitously detectable with 1H NMR, each cell type had distinctive qualitative and/or quantitative features. Our most unexpected finding was a large amount of N-acetyl-aspartate (NAA) in O-2A progenitors. This compound, consistently detected by 1H NMR in vivo, was previously thought to ne present only in neurons. The finding that meningeal cells have an alanine:creatine ratio three to four times higher than astrocytes, neurons, or oligodendrocytes is in agreement with observations that meningiomas express a higher alanine:creatine ratio than gliomas. The data suggest that each individual cell type has a characteristic metabolic pattern that can be discriminated by 1H NMR, even by looking at only a few metabolites (e.g., NAA, glycine, beta-hydroxybutyrate).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in serotonin neurotransmission have also been implicated in the etiology and treatment of impulse control disorders, depression, and anxiety. We have investigated the effect of enhancing serotonin function on fundamental brain processes that we have proposed are abnormal in these disorders. In all, 12 male volunteers received citalopram (7.5 mg intravenously) and placebo pretreatment in a single-blind crossover design before undertaking Go/No-go, Loss/No-loss, and covert (aversive) face emotion recognition tasks during functional magnetic resonance imaging (fMRI). Blood oxygenation level dependent responses were analyzed using Statistical Parametric Mapping (SPM2). The tasks activated prefrontal and subcortical regions generally consistent with literature with lateral orbitofrontal cortex (BA47) common to the three tasks. Citalopram pretreatment enhanced the right BA47 responses to the No-go condition, but attenuated this response to aversive faces. Attenuations were seen following citalopram in the medial orbitofrontal (BA11) responses to the No-go and No-loss (ie relative reward compared with Loss) conditions. The right amygdala response to aversive faces was attenuated by citalopram. These results support the involvement of serotonin in modulating basic processes involved in psychiatric disorders but argue for a process-specific, rather than general effect. The technique of combining drug challenge with fMRI (pharmacoMRI) has promise for investigating human psychiatric disorders.

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3epsilon, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) zeta was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRzeta, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRzeta in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRzeta when both were coexpressed in 293T cells, establishing TCRzeta as a direct substrate of PTPN22.

SummaryGenetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine.

Full bibliographic details must be given when referring to, or quoting from full items including the author's name, the title of the work, publication details where relevant (place, publisher, date), pagination, and for theses or dissertations the awarding institution, the degree type awarded, and the date of the award.