Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.     

Implications of interveinal distance for quantum yield in C<Subscript>4</Subscript> grasses: a modeling and meta-analysis

The distance between veins has the potential to affect photosynthesis in C₄ grasses because photon capture and photosynthetic carbon reduction are primarily restricted to vascular bundle sheath cells (BSC). For example, BSC density should increase as interveinal distance (IVD) decreases, and thus IVD may influence photon capture and photosynthesis in C₄ grasses. The objective of this study is to evaluate the potential importance of IVD to the function of C₄ grasses, and a literature survey is conducted to test the hypothesis that quantum yield of photosynthesis () increases with decreasing IVD. First, a meta-analysis of and IVD values obtained for 12 C₄ grass species supports this hypothesis as and IVD are significantly negatively correlated (r=–0.61). Second, a regression of carbon isotope discrimination () versus IVD was conducted and the regression equation was used in a simple biochemical model that relates to and leakage of CO₂ from the BSC. The modeling analysis also supports the hypothesis that decreases with increasing IVD in C₄ grasses. C₄ grasses are virtually absent from shaded habitats, and the biochemical model is employed to examine the implications of IVD for shade-tolerance in C₄ grasses. The model predicts that only those species with uncommonly small IVD values are able to tolerate prolonged shade.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Activation of Toll-like Receptor 4 (TLR4) Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress

Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study we hypothesized that Toll-like receptor 4 (TLR4) activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by a high fat diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of endoplasmic reticulum (ER) stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced up-regulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data indicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective brown adipose tissue activation.     

Coumarins from Malaysian Micromelum minutum

In a continuation of our study of the Rutaceae, detailed chemical investigation on Micromelum minutum (Rutaceae) collected from Sepilok, Sabah, Malaysia gave four new coumarins. The structures of the coumarins have been fully characterised by spectroscopic methods as 3",4"-dihydrocapnolactone 1, 2',3'-epoxyisocapnolactone 2, 8-hydroxyisocapnolactone-2',3'-diol 3 and 8-hydroxy-3",4"-dihydrocapnolactone-2',3'-diol 4.     

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1^−/− mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1^−/− mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1^−/− mouse with ²H₇-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of ²H₇-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1^−/− mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Two forms of NADP-dependent malic enzyme in expanding maize leaves

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a K_m (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a K_m (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar K_m (NADP⁺), and the typical increase of K_m for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min^-1 mg protein^-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.Abbreviation MES 2 (N-morpholino)ethane sulfonic acid Work supported by grants from the Consiglio Nazionale delle Ricerche for years 1975 and 1976     

Rain use efficiency as affected by climate warming and biofuel harvest: results from a 12‐year field experiment

The efficiency of a terrestrial ecosystem to use rainfall in production is critical in regulating the ecological functions of the earth system under global change. However, it remains unclear how rain use efficiency (RUE) will be altered by changes in climate and human activities such as biofuel harvest. In this study, we used RUE data from a long‐term experiment in a tallgrass prairie to analyze the effects of warming and biofuel harvest (clipping). From 2000 to 2011, experimental warming enhanced RUE in most years, with larger positive effects in normal and wet than dry hydrological years. Clipping decreased RUE in dry and normal hydrological years, but had no impact on RUE in wet years. The observed RUE responses resulted from treatment‐induced changes in both biologically ineffective (i.e., runoff and soil evaporation) and effective (i.e., transpiration) parts of precipitation. For example, litter cover was increased in warming plots, but reduced by clipping, leading to negative and positive effects on runoff and soil evaporation, respectively. The dominance of C₄ species, which usually have higher water use efficiency than C₃ species, was enhanced by warming, but reduced by clipping. Moreover, RUE was positively correlated with ratios of rainfall in the late growing season (June–August), when the growth of C₄ plants was most active, relative to that in the other seasons. Our results indicate that RUE is positively influenced by climate warming, but negatively affected by biofuel harvest in tallgrass prairie of the Great Plains. These findings highlight the important roles of plant community structure and temporal distribution of precipitation in regulating ecosystem RUE.     

一步法扩增克隆IBDV上海超强毒VP2-4-3基因

分离、纯化了鸡传染性法氏囊病病毒超强毒 (vvIBDV) 上海株SH95的病毒核酸dsRNA,应用随机引物将RNA反转录成cDNA,以此为模板一步扩增出A片段前体融合蛋白基因即VP2-4-3基因,将其克隆入pGEM-T载体,并进行序列分析,其与超强毒株HK46的核苷酸序列的同源性达98%,整个基因有5个氨基酸差异,同源性达99.51%(1007/1012).