The effect of ethanol on lateral and rotational mobility of plasma membrane vesicles isolated from cultured mar 18.5 hybridoma cells

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of the lateral mobility and the range of the rotational mobility of bulk bilayer structures of the plasma membrane vesicles (ATCC-PMV) isolated from cultured hybridoma cells (ATCC TIB 216). In a concentration-dependent manner, ethanol increased the excimer to monomer fluorescence intensity ratio (I/I) of Py-3-Py in the ATCC-PMV and decreased the anisotropy (r), limiting anisotropy (r) and order parameter (S) of DPH in the ATCC-PMV. This indicates that ethanol increased both the lateral and rotational mobility of the probes in the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was utilized to examine the range of transbilayer asymmetric rotational diffusion of the ATCC-PMV. The anisotropy (r), limiting anisotropy (r ) and order parameter (S) of DPH in the inner monolayer were 0.024, 0.032, and 0.069, respectively, greater than calculated for the outer monolayer of the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was also used to examine the transbilayer asymmetric effects of ethanol on the range of the rotational mobility of the ATCC-PMV. Ethanol had a greater increasing effect on the range of the rotational mobility of the outer monolayer as compared to the inner monolayer of the ATCC-PMV. It has been proven that ethanol exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the ATCC-PMV.This paper was supported in part by a research grant from the Korea Science and Engineering Foundation (KOSEF 88-1013-01) and from the Korea Research Foundation (1991–1993).     

Accelerated organic matter decomposition in thermokarst lakes upon carbon and phosphorus inputs

Mineralization of dissolved organic matter (DOM) in thermokarst lakes plays a non-negligible role in the permafrost carbon (C) cycle, but remains poorly understood due to its complex interactions with external C and nutrient inputs (i.e., aquatic priming and nutrient effects). Based on large-scale lake sampling and laboratory incubations, in combination with ¹³C-stable-isotope labeling, optical spectroscopy, and high-throughput sequencing, we examined large-scale patterns and dominant drivers of priming and nutrient effects of DOM biodegradation across 30 thermokarst lakes along a 1100-km transect on the Tibetan Plateau. We observed that labile C and phosphorus (P) rather than nitrogen (N) inputs stimulated DOM biodegradation, with the priming and P effects being 172% and 451% over unamended control, respectively. We also detected significant interactive effects of labile C and nutrient supply on DOM biodegradation, with the combined labile C and nutrient additions inducing stronger microbial mineralization than C or nutrient treatment alone, illustrating that microbial activity in alpine thermokarst lakes is co-limited by both C and nutrients. We further found that the aquatic priming was mainly driven by DOM quality, with the priming intensity increasing with DOM recalcitrance, reflecting the limitation of external C as energy sources for microbial activity. Greater priming intensity was also associated with higher community-level ribosomal RNA gene operon (rrn) copy number and bacterial diversity as well as increased background soluble reactive P concentration. In contrast, the P effect decreased with DOM recalcitrance as well as with background soluble reactive P and ammonium concentrations, revealing the declining importance of P availability in mediating DOM biodegradation with enhanced C limitation but reduced nutrient limitation. Overall, the stimulation of external C and P inputs on DOM biodegradation in thermokarst lakes would amplify C-climate feedback in this alpine permafrost region.     

Site and mechanism of behavioral tolerance to cocaine: A study of dopamine release in Wistar-Kyoto and spontaneously hypertensive rats

Wistar-Kyoto and spontaneously hypertensive rats received i.v. infusions of cocaine hydrochloride (60 mg/kg per day) for 3, 7, and 14 days, or saline for 7 days. Acute cocaine challenge (40 mg/kg, s.c.) was given to treated and control rats 24 hr after the termination of each infusion period. There were no strain differences in brain levels of cocaine during cocaine infusion, nor after cocaine challenges. There were no strain differences in resting levels of [³H]dopamine release. Release of [³H]dopamine decreased in nuclei accumbens of 7- and 14-day cocaine-infused animals. Release of [³H]dopamine was maximal in both brain regions 2 hr after acute cocaine challenge. After 14 days of cocaine infusion, cocaine challenge in both strains reduced [³H]dopamine release in the nucleus accumbens, but not in the striatum; the reduction being greater in Wistar-Kyoto rats. The behavioral tolerance which accompanies similar cocaine infusion regimens may be related to striatal tolerance to cocaine-induced dopamine release.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.     

The Puccinia striiformis effector Hasp98 facilitates pathogenicity by blocking the kinase activity of wheat TaMAPK4

The obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) employs virulence effectors to disturb host immunity and causes devastating stripe rust disease. However, our understanding of how Pst effectors regulate host defense responses remains limited. In this study, we determined that the Pst effector Hasp98, which is highly expressed in Pst haustoria, inhibits plant immune responses triggered by flg22 or nonpathogenic bacteria. Overexpression of Hasp98 in wheat (Triticum aestivum) suppressed avirulent Pst-triggered immunity, leading to decreased H₂O₂ accumulation and promoting P. striiformis infection, whereas stable silencing of Hasp98 impaired P. striiformis pathogenicity. Hasp98 interacts with the wheat mitogen-activated protein kinase TaMAPK4, a positive regulator of plant resistance to stripe rust. The conserved TEY motif of TaMAPK4 is important for its kinase activity, which is required for the resistance function. We demonstrate that Hasp98 inhibits the kinase activity of TaMAPK4 and that the stable silencing of TaMAPK4 compromises wheat resistance against P. striiformis. These results suggest that Hasp98 acts as a virulence effector to interfere with the MAPK signaling pathway in wheat, thereby promoting P. striiformis infection.     

ARK2 stabilizes the plus-end of microtubules and promotes microtubule bundling in Arabidopsis

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Immune-Enhancing Activity Screening on Extracts from Two Crickets, Gryllus bimaculatus and Teleogryllus emma

This study was performed to explore novel and valuable uses of insect resources, important subjects of the natural compound used in bio‐industries. The whole bodies of two crickets, Gryllus bimaculatus and Teleogryllus emma, selected from medicinal insect species, were carefully ground and treated with 80% EtOH. The insect extracts were solubilized and separated by hexane, butanol, and D.W according to their polarities. Three types of extracts, a D.W fraction (G1) and a boiling extract (G2) of an introduced cricket, G. bimaculatus, and a D.W fraction (T1) of a Korean local cricket, T. emma, were prepared to assay immune stimulating activity of cricket originated compounds. The all of three treated cricket extracts showed to increase IL‐4, IFN‐, and TNF‐α. Among those extract, extract G2, boiled extract from G. bimaculatus, was the best immune–enhancing fraction. The results of this study could be fundamental information for further works to use insects as natural resources having plenty of potentials and varieties.     

Uptake and metabolism of BuCast: a glycoprotein processing inhibitor and a potential anti-HIV drug

We have previously shown (Sunkara et al., 1989; Taylor et al.,1991) that 6-o-butanoyl castanospermine (BuCast) was a 30–50-foldbetter inhibitor of HIV syncytia formalion than castanospermine(Cast). Radiolabeled Cast and BuCast were used to study theuptake and metabolism of these compounds in cultured cells andin mice. BuCast was preferentially taken up by cells comparedto Cast. Approximately 30–50-fold higher radioactivitywas found in cells treated with BuCast compared to cells treatedwith Cast during the initial 4–6h of labeling. HPLC analysisshowed that once BuCast was taken up by cells, it was rapidlyconverted to Cast. Mice given oral doses of BuCast had 5–10-foldhigher levels of Cast in the plasma and tissues as comparedto Cast treated mice. However, when the compounds were giveni.v., the levels of plasma and tissue radioactivity obtainedfrom Cast or BuCast were equivalent suggesting rapid conversionof BuCast to Cast in the blood. In mice orally treated withBuCast, HPLC analysis suggested that only Cast was found inthe plasma and tissues. With multiple dosing of mice, additiveresults were obtained, suggesting that multiple doses may beused to obtain higher concentrations of the compound in thetarget cells. These data suggest that the lipophilic propertiesof butanoyl side chain on the Cast ring makes BuCast significantlybetter absorbed, and this may help to alleviate some of thegut toxicity associated with Cast treatment. HIV AIDS glycoproteins inhibitors