Up-regulation in expression of vesicular glutamate transporter 3 in substantia nigra but not in striatum of 6-hydroxydopamine-lesioned rats

Overactivity of the glutamatergic system is suggested to be closely related to the onset and pathogenesis of Parkinson's disease. Vesicular glutamate transporters (VGLUT1, T2 and T3) are a group of glutamate transporters in neurons that are responsible for transporting glutamate into synaptic vesicles and they are key elements for homeostasis of glutamate neurotransmission. The present study was aimed to investigate the expression of VGLUT1, T2 and T3 proteins after the onset of Parkinson's disease. A rat model of Parkinson's disease, the 6-hydroxydopamine-lesioned rat, was employed. Immunocytochemistry revealed that VGLUT1, T2 and T3 immunoreactivity was not modulated in the striatum of the lesioned rat. Western blotting analyses also showed that there was no change in the expression of T1, T2 and T3 proteins in the striatum. In contrast, no VGLUT1 protein was detected in the substantia nigra. After the lesion, levels of VGLUT2 immunoreactivity and protein were not modulated. Significant increase of VGLUT3 immunoreactivity was observed in the perikarya of GABAergic substantia nigra pars reticulata neurons (+14.7%) although VGLUT3 protein was not modulated in the nigral tissues. VGLUT3 in GABAergic neurons is suggested to play a role in GABA synthesis. The present results may therefore implicate that VGLUT1 and T2 are not modulated in the striatum and the substantia nigra of the 6-hydroxydopamine-lesioned rat and only VGLUT3 plays a role in pathogenesis of Parkinson's disease.     

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.     

Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri

A signal peptide (SP)-probe vector pNICE-gfpSP, which employed a green fluorescent protein (Gfp) as the SP-selection marker, was constructed for use in Lactobacillus reuteri. This chimerical plasmid allowed cloning and screening DNA fragments with the SP function by direct visualization of the expressed fluorescence activity around cells. Assay of fluorescent intensity in their culture supernatant with spectrofluorometry further enabled quantifying the secretion efficiency of the identified SP fragment.     

Immunosensor with a controlled orientation of antibodies by using NeutrAvidin-protein A complex at immunoaffinity layer

The orientation of antibody was controlled by using NeutrAvidin-protein A complex on the gold surface of SPR biosensor. The surface density of receptor antibody (anti-hIgG) was compared by treatment of receptor antibody to the layer of avidin, NeutrAvidin, protein A, NeutrAvidin-protein A complex and bare gold surface of SPR biosensor. The ligand antibody (hIgG) was injected to each IA layer and the binding ratio of ligand antibody per unit receptor was estimated as a parameter of orientation control. The NeutrAvidin-protein A complex on gold surface of SPR biosensor showed the highest surface density of receptor antibody as well as the binding ratio of ligand antibody per receptor antibody. The NeutrAvidin-protein A complex was also prepared on biotin-labelled SAM, and the binding ratio of ligand per receptor was found to be significantly improved in comparison to the IA layer prepared by chemical coupling of receptor antibody to the SAM layer. The NeutrAvidin-protein A complex which showed the highest efficiency for the binding of ligand antibodies, was applied for the detection of a cancer marker called CEA. By using NeutrAvidin-protein A complex and sandwich assay for signal amplification, sensitivity was improved to be 1.5-fold higher than bare gold surface and the detection of CEA with the detection limit of 30 ng/ml was achieved.     

Community structure and function in a H2-based membrane biofilm reactor capable of bioreduction of selenate and chromate

Two different H₂-based, denitrifying membrane-biofilm reactors (MBfRs) initially reduced Se(VI) or Cr(VI) stably to Se⁰ or Cr(III). When the oxidized contaminants in the influent were switched, each new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been in its original MBfR. The precipitation of reduced selenium and chromium in the biofilm was verified by scanning electron microscopy and energy dispersive X-ray analysis. These results on selenate and chromate reduction are consistent with the interpretation that the H₂-based biofilm community had a high level of functional diversity. The communities’ structures were assessed by cloning analysis. Dechloromonas spp., a known perchlorate-reducing bacteria, dominated the clones from both reactors during selenate and chromate reductions, which suggests that it may have functional diversity capable of reducing selenate and chromate as secondary and dissimilatory acceptors.     

体外诱导成年大鼠骨髓间充质干细胞分化为 5-羟色胺敏感性神经元

体外诱导成年大鼠骨髓间充质干细胞分化为具有神经元表型和部分功能的细胞。在对Woodbury化学诱导法作改良的基础上,加用全反式视黄酸对骨髓间充质干细胞作预诱导。诱导3h后,细胞开始表现神经元的形态特征,细胞折光性增强,形成收缩的双极或多极胞体和细长突起。细胞可以维持神经元样存活72h以上。诱导5h后,对免疫染色的细胞用DAPI进行复染,(92.4±6.9)%的细胞表达神经元特异性烯醇化酶。诱导24h后,(93.9±5.2)%的细胞表达成熟神经元的标志物神经丝M H。在给予5-羟色胺刺激时可以产生与神经元相似的胞内钙离子峰,且免疫组化证实5-羟色胺1A受体在干细胞上表达微弱,但在分化后的神经元中表达较强。实验不仅从形态、细胞标志物而且从功能上证实诱导后的细胞为5-羟色胺敏感性神经元。     

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development

Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants were viable, but exhibited developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppressed the ERK-dependent differentiation, which was further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulated ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, we further demonstrated that the RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, our study reveals a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila.     

Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.     

Fine-scale genetic structure among genetic individuals of the clone-forming monotypic genus Echinosophora koreensis (Fabaceae)

• Background and Aims For rare endemics or endangered plantspecies that reproduce both sexually and vegetatively it iscritical to understand the extent of clonality because assessmentof clonal extent and distribution has important ecological andevolutionary consequences with conservation implications. Asurvey was undertaken to understand clonal effects on fine-scalegenetic structure (FSGS) in two populations (one from a disturbedand the other from an undisturbed locality) of Echinosophorakoreensis, an endangered small shrub belonging to a monotypicgenus in central Korea that reproduces both sexually and vegetativelyvia rhizomes. • Methods Using inter-simple sequence repeats (ISSRs) asgenetic markers, the spatial distribution of individuals wasevaluated using Ripley's L(d)-statistics and quantified thespatial scale of clonal spread and spatial distribution of ISSRgenotypes using spatial autocorrelation analysis techniques(join-count statistics and kinship coefficient, F_ij) for totalsamples and samples excluding clones. • Key Results A high degree of differentiation betweenpopulations was observed (_ST(g) = 0·184, P < 0·001).Ripley's L(d)-statistics revealed a near random distributionof individuals in a disturbed population, whereas significantaggregation of individuals was found in an undisturbed site.The join-count statistics revealed that most clones significantlyaggregate at 6-m interplant distance. The Sp statistic reflectingpatterns of correlograms revealed a strong pattern of FSGS forall four data sets (Sp = 0·072–0·154), butthese patterns were not significantly different from each other.At small interplant distances (2 m), however, jackknifed 95% CIs revealed that the total samples exhibited significantlyhigher F_ij values than the same samples excluding clones. • Conclusion The strong FSGS from genets is consistentwith two biological and ecological traits of E. koreensis: bee-pollinationand limited seed dispersal. Furthermore, potential clone matesover repeated generations would contribute to the observed highF_ij values among genets at short distance. To ensure long-termex situ genetic variability of the endangered E. koreensis,individuals located at distances of 10–12 m should becollected across entire populations of E. koreensis.     

Mimicking damaged DNA with a small molecule inhibitor of human UNG2

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.