Chronic methamphetamine exposure significantly decreases microglia activation in the arcuate nucleus.

Methamphetamine is a powerful psychostimulant drug and its use and abuse necessitates a better understanding of its neurobiobehavioral effects. The acute effects of binge dosing of methamphetamine on the neurons in the CNS are well studied. However, the long-term effects of chronic, low-dose methamphetamine are less well characterized, especially in other cell types and areas outside of the major dopamine pathways. Mice were administered 5mg/kg/day methamphetamine for ten days and brain tissue was analyzed using histochemistry and image analysis. Increased microglia activity in the striatum confirmed toxic effects of methamphetamine in this brain region using this dosing paradigm. A significant decrease in microglia activity in the arcuate nucleus of the hypothalamus was observed with no effect noted on dopamine neurons in the arcuate nucleus. Given the importance of this area in homeostatic and neuroendocrine regulation, the current study highlights the need to more fully understand the systemic effects of chronic, low-dose methamphetamine use. The novel finding of microglia downregulation after chronic methamphetamine could lead to advances in understanding neuroinflammatory responses towards addiction treatment and protection from psychostimulant-induced neurotoxicity.

Quantitative proteomic profiling of paired cancerous and normal colon epithelial cells isolated freshly from colorectal cancer patients.

PURPOSE: The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC. EXPERIMENTAL DESIGN: CEC and NEC cells are respectively isolated from five different tumor and normal locations in the resected colon tissue from each patient (N = 12 patients) using an optimized epithelial cell adhesion molecule (EpCAM)-based enrichment approach. An ion current-based quantitative method is employed to perform comparative proteomic analysis for each patient. RESULTS: A total of 458 altered proteins that are common among >75% of patients are observed and selected for further investigation. Besides known findings such as deregulation of mitochondrial function, tricarboxylic acid cycle, and RNA post-transcriptional modification, functional analysis further revealed RAN signaling pathway, small nucleolar ribonucleoproteins (snoRNPs), and infection by RNA viruses are altered in CEC cells. A selection of the altered proteins of interest is validated by immunohistochemistry analyses. CONCLUSION AND CLINICAL RELEVANCE: The informative comparison between matched CEC and NEC enhances our understanding of molecular mechanisms of CRC development and provides biomarker candidates and new pathways for therapeutic intervention.

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions.

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.

Discovery of S-[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2,3-dihydroxypropoxy)phenyl]methanone (RO3201195), an orally bioavailable and highly selective inhibitor of p38 MAP kinase.

A novel class of highly selective inhibitors of p38 MAP kinase was discovered from high throughput screening. The synthesis and optimization of a series of 5-amino-N-phenyl-1H-pyrazol-4-yl-3-phenylmethanones is described. An X-ray crystal structure of this series bound in the ATP binding pocket of unphosphorylated p38alpha established the presence of a unique hydrogen bond between the exocyclic amine of the inhibitor and threonine 106 which likely contributes to the selectivity for p38. The crystallographic information was used to optimize the potency and physicochemical properties of the series. The incorporation of the 2,3-dihydroxypropoxy moiety on the pyrazole scaffold resulted in a compound with excellent drug-like properties including high oral bioavailability. These efforts identified 63 (RO3201195) as an orally bioavailable and highly selective inhibitor of p38 which was selected for advancement into Phase I clinical trials.

Plasma neurohormone levels correlate with left ventricular functional and morphological improvement in LVAD patients.

OBJECTIVE: Brain natriuretic peptide (BNP) and endothelin-1 (ET-1) have been shown to be markers of left ventricular (LV) function. To determine the feasibility of using serial assays of these neurohormones in the assessment of cardiac status in the left ventricular assist device (LVAD) setting, we examined the relationship between LV function, myocardial morphology, and plasma levels of these hormones in LVAD recipients. METHODS: Plasma BNP and ET-1 levels were serially assayed in 19 end-stage congestive heart failure (CHF) patients before and after LVAD implantation with various devices (i.e., MicroMed DeBakeyVAD/DVAD, Novacor/NVAD, TCI Heartmate/TCI, Thoratec/TVAD). Echocardiography performed correspondingly at the time points of the hormonal assays and immunohistochemical collagen staining of left ventricular tissue samples, derived from six non-failing hearts as well as from LVAD patients at the time of device insertion and removal, were then contrasted. Patients were grouped according to device used and etiology of heart disease (ischemic or dilated cardiomyopathy, ICM/DCM). RESULTS: LVAD therapy significantly improved LV ejection fraction (EF%: 21 +/- 3.8% to 28.11 +/- 3.57%), cardiac output (CO: 3.49 +/- 1.3 to 7.3 +/- 0.2 l/m), and left ventricular end-diastolic diameter (LVEDD: 6.68 +/- 0.92 versus 4.79 +/- 1.54 cm, P < 0.0001) in all patients. Absolute BNP and ET-1 plasma levels remained significantly lower in all patients after LVAD implantation (both P < 0.001). The NVAD group exhibited the most BNP reduction and EF% increase (P < 0.0004 and P < 0.038, respectively). Average collagen levels were reduced in all patients (P < 0.0005). Among the devices, the NVAD group demonstrated the most evident change (P < 0.0036), while there was comparable reduction in the DCM and ICM groups (both P < 0.03). In general, postoperative BNP and ET-1 trends exhibited a notable parallelism with both manifesting bi-phasic tendencies and an inverse proportionality to corresponding EF% measurements. CONCLUSIONS: Device selection appears to influence the cardiac morphological and neurohormonal expressive tendencies exhibited by recipients. Plasma BNP and ET-1 levels correlate with both LV function and myocardial morphological improvement. Alterations in the levels of these hormones during LVAD support may be real-time indicators of prevailing myocardial autocrine/paracrine activity and as such may be of potential use in future algorithms of cardiac assessment and therapeutic decision-making with regard to transplant urgency and/or possible device explantation.

Design and synthesis of 4-azaindoles as inhibitors of p38 MAP kinase.

Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.