Tonic extracellular glutamate and ischaemia: glutamate antiporter system xc - regulates anoxic depolarization in hippocampus.

In stroke, the sudden deprivation of oxygen to neurons triggers a profuse release of glutamate that induces anoxic depolarization (AD) and leads to rapid cell death. Importantly, the latency of the glutamate-driven AD event largely dictates subsequent tissue damage. Although the contribution of synaptic glutamate during ischaemia is well-studied, the role of tonic (ambient) glutamate has received far less scrutiny. The majority of tonic, non-synaptic glutamate in the brain is governed by the cystine/glutamate antiporter, system xc - . Employing hippocampal slice electrophysiology, we showed that transgenic mice lacking a functional system xc - display longer latencies to AD and altered depolarizing waves compared to wild-type mice after total oxygen deprivation. Experiments which pharmacologically inhibited system xc - , as well as those manipulating tonic glutamate levels and those antagonizing glutamate receptors, revealed that the antiporter's putative effect on ambient glutamate precipitates the ischaemic cascade. As such, the current study yields novel insight into the pathogenesis of acute stroke and may direct future therapeutic interventions. KEY POINTS: Ischaemic stroke remains the leading cause of adult disability in the world, but efforts to reduce stroke severity have been plagued by failed translational attempts to mitigate glutamate excitotoxicity. Elucidating the ischaemic cascade, which within minutes leads to irreversible tissue damage induced by anoxic depolarization, must be a principal focus. Data presented here show that tonic, extrasynaptic glutamate supplied by system xc - synergizes with ischaemia-induced synaptic glutamate release to propagate AD and exacerbate depolarizing waves. Exploiting the role of system xc - and its obligate release of ambient glutamate could, therefore, be a novel therapeutic direction to attenuate the deleterious effects of acute stroke.

Enhancing Geranylgeraniol Production by Metabolic Engineering and Utilization of Isoprenol as a Substrate in Saccharomyces cerevisiae.

The amount of geranylgeranyl diphosphate (GGPP) is vital for microbial production of geranylgeraniol (GGOH) in Saccharomyces cerevisiae. In this study, a GGPP synthase with stronger catalytic ability was used to increase the supply of GGPP, and an engineered strain producing 374.02 mg/L GGOH at the shake flask level was constructed. Then, by increasing the metabolic flux of the mevalonate (MVA) pathway and the supply of isopentenyl pyrophosphate (IPP), the titer was further increased to 772.98 mg/L at the shake flask level, and we achieved the highest GGOH titer to date of 5.07 g/L in a 5 L bioreactor. This is the first report on the utilization of isoprenol for increasing the amount of IPP and enhancing GGOH production in S. cerevisiae. In the future, these strategies and engineered strains can be used to enhance the production of other terpenoids in S. cerevisiae.

Overproduction of α-Farnesene in Saccharomyces cerevisiae by Farnesene Synthase Screening and Metabolic Engineering.

Maximizing the flux of farnesyl diphosphate (FPP) to farnesene biosynthesis is the main challenge of farnesene overproduction in Saccharomyces cerevisiae. In this study, we screened α-farnesene synthase from soybean (Fsso) with a higher catalytic ability. Combining the overexpression of the mevalonate (MVA) pathway with the expression of Fsso, an engineered yeast strain producing 190.5 mg/L α-farnesene was screened with poor growth. By decreasing the copies of 3-hydroxy-3-methylglutaryl-coenzyme (HMGR) overexpressed, the titer was increased to 417.8 mg/L. Then, the coexpression of Fsso and HMGR under the control of the GAL promoter and inactivation of lipid phosphate phosphatase encoded by DPP1 promoted the titer to 1163.7 mg/L. The titer was further increased to 1477.2 mg/L at the shake flask level with better growth by the construction of a prototrophic strain. Finally, the highest α-farnesene production of 10.4 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Synaptic and Network Contributions to Anoxic Depolarization in Mouse Hippocampal Slices.

Ischemic stroke remains the third leading cause of death and leading cause of adult disability worldwide. A key event in the pathophysiology of stroke is the anoxic depolarization (AD) of neurons in the ischemic core. Previous studies have established that both the latency to AD and the time spent in AD prior to re-oxygenation are predictors of neuronal death. The present studies used hippocampal slices from male and female mice to investigate the electrophysiological events that affect latency to AD after oxygen deprivation. The results confirm that the epoch between AD and re-oxygenation largely determines the magnitude of synaptic recovery after anoxic challenge. Using a selective antagonist of adenosine A1 receptors, we also confirmed that adenosine released during anoxia (ANOX) suppresses synaptic glutamate release; however, this action has no effect on AD latency or the potential for post-anoxic recovery of synaptic transmission. In contrast, antagonism of AMPA- and NMDA-type glutamate receptors significantly prolongs the latency to AD and alters the speed and synchrony of associated depolarizing waves. Experiments using slices with fields Cornu ammonis 3 (CA3) and Cornu ammonis 1 (CA1) disconnected showed that AD latency is longer in CA1 than in CA3; however, the early AD in CA3 is propagated to CA1 in intact slices. Finally, AD latency in CA1 was found to be longer in slices from female mice than in those from age-matched male mice. The results have implications for stroke prevention and for understanding brain adaptations in hypoxia-tolerant animals.

Immunohistochemical characterization of benign activation of junctional melanocytes and melanoma in situ of the nail unit.

BACKGROUND: Immunohistochemical (IHC) stains that distinguish benign, pigmented nail lesions from malignancy are needed. Candidate markers of malignant transformation include p16, HMB45, and Ki-67, with p16 being of particular interest. There is limited knowledge about the spectrum of p16 expression in pigmented lesions, especially junctional melanocytic proliferations of the nail. The objective of this study was to determine if any of these markers demonstrate diagnostic utility in distinguishing between benign activation of junctional melanocytes (BAM) and melanoma in situ (MIS) of the nail unit. METHODS: In this retrospective study, ten cases of BAM and eight cases of MIS were identified. Archival slides available for review included H&E (hematoxylin and eosin), Fontana-Masson, and MelanA (Mart1) IHC slides. IHC studies for p16, HMB45, and dual-color Ki-67/MelanA (Mart1) were then performed. RESULTS: None of the tested IHC stains distinguished BAM from MIS. p16 IHC expression was uniformly negative with the exception of two cases of MIS. HMB45 was positive in all BAM and MIS cases. Ki-67/MelanA showed positive Ki-67 staining of MelanA-positive melanocytes in two cases of MIS, and all other cases of MIS and BAM were negative for Ki-67. The two positive p16 and two positive Ki-67/MelanA cases were non-overlapping. CONCLUSION: p16, HMB45, and Ki-67/MelanA IHC studies show no apparent utility in distinguishing BAM from MIS in the nail unit.

Preparation of immobilized lipase by modified polyacrylonitrile hollow membrane using nitrile-click chemistry.

The application of immobilized lipase in the enzymatic production of biodiesel has shown numerous advantages. In this study, surface of Polyacrylonitrile (PAN) hollow membrane was first modified using nitrile-click chemistry in order to fit for interaction with enzyme proteins. Then sodium alginate (SA) was introduced and the membrane was post-treated by CaCl2. When the prepared PAN-PEI-SA-CaCl2 was used for lipase immobilization, the protein loading was 36.90 mg/g, and the enzyme activity reached up to 54.47 U/g, which was 2.5 times as much as that of Novozym® 435. As a result, the constructed immobilized lipase obtained a maximum biodiesel yield of 78.5%, which was 2.4 times that of the Novozym® 435 in transesterification reactions. Moreover, the biodiesel yield decreased by only 11% after the immobilized enzyme was continuously used for 20 times. This study exhibits that this technic has broad application prospects in the field of conversion of biomass resources.

A deuterated analog of dasatinib disrupts cell cycle progression and displays anti-non-small cell lung cancer activity in vitro and in vivo.

The pan-Src family kinase inhibitor dasatinib has been approved for chronic myeloid leukemia treatment but displays limited activity in lung cancer patients. In this study, we used a deuterium substitution strategy to develop a class of novel chemicals based on dasatinib and found that these compounds maintain inhibition on c-Src activity and display anti-non-small cell lung cancer activity in vitro and in vivo. BRP800, one of these compounds, was chosen for further studies. BRP800 mainly displayed antiproliferative but not proapoptotic activity. Molecularly, BRP800 did not show significant effects on the expression of antiapoptotic genes, such as Bcl-2 and Mcl1, or on the activation of apoptotic enzymes, such as caspase-3, -8 or 9. However, BRP800 decreased expression of cell cycle promoting genes such as cyclins D1, D3, E, A and CDK4 and 6, and increased the expression of cell cycle negative regulators including p21, p27 and p53. Consistent with these findings, BRP800 arrested cells at the G0/G1 phase in a concentration-dependent manner, and the G0/G1 fraction was increased from 64% in control to 85% in BRP800-treated cells. We also evaluated the effects of BRP800 on NSCLC xenografts using H460 as a model in nude mice. Compared with the known NSCLC drug docetaxel, BRP800 displayed potent and similar antitumor activity but with less toxicity. These findings suggest that the deuterated analog of dasatinib is antiproliferative by inhibiting c-Src and disrupting cell cycle progression, and could be further developed as a novel drug for non-small lung cancer treatment.

Executive function on the Psychology Experiment Building Language tests.

The measurement of executive function has a long history in clinical and experimental neuropsychology. The goal of the present report was to determine the profile of behavior across the lifespan on four computerized measures of executive function contained in the recently developed Psychology Experiment Building Language (PEBL) test battery http://pebl.sourceforge.net/ and evaluate whether this pattern is comparable to data previously obtained with the non-PEBL versions of these tests. Participants (N = 1,223; ages, 5-89 years) completed the PEBL Trail Making Test (pTMT), the Wisconsin Card Sort Test (pWCST; Berg, Journal of General Psychology, 39, 15-22, 1948; Grant & Berg, Journal of Experimental Psychology, 38, 404-411, 1948), the Tower of London (pToL), or a time estimation task (Time-Wall). Age-related effects were found over all four tests, especially as age increased from young childhood through adulthood. For several tests and measures (including pToL and pTMT), age-related slowing was found as age increased in adulthood. Together, these findings indicate that the PEBL tests provide valid and versatile new research tools for measuring executive functions.

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing.

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.