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.     

Correlation between fibronectin and its receptor in chick myoblast differentiation

Alterations in the amount of fibronectin and in the number of its receptors during myoblast differentiation of chicken embryo were investigated. The amount of fibronectin in the cell surface pool as measured by immunoblotting decreased during myogenesis To identify and characterize the fibronectin receptors on the myoblasts, the interactions of the 28,000 dalton (28 kDa) amino terminal fragment and 85,000 dalton (85 kDa) cell-binding fragment of fibronectin with my-oblasts were examined. The binding of the 28 kDa fragment was found to be time-dependent and reached a maximum level within 60 min. The unlabeled 28 kDa fragment inhibited the binding of the radioiodinated 28 kDa fragment, whereas the unlabeled 85 kDa fragment and antibody to integrin did not inhibit it, suggesting that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. There was a single class of 3.4 × 10⁵ binding sites per cell with an apparent dissociation constant of 1.4 × 10^?7 M on 30 hr old myoblasts. The specific binding of the radioiodinated 28 kDa fragment to myoblasts decreased as the fusion proceeded. This decrease of binding was consistent with the decrease in the amount of fibronectin. Furthermore, the levels of fibronectin and binding of the radioiodinated 28 kDa fragment in the fusion-blocked myoblasts by EGTA treatment appeared to remain constant. These results suggest that the decrease and/or loss of fibronectin during myoblast fusion is closely correlated with the alteration of fibronectin receptors and with the fusion of myoblasts.     

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     

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation

Summary The unusual amino acid hypusine [N -(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to its-amino group to form the intermediate, deoxyhypusine [N -(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105–3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305–311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.     

Cell separation from high cell density broths of Alcaligenes eutrophus by using a coagulant

Summary Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it.     

Flow cytometric analysis of antibody producing cells using double immunofluorescent staining

Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)₂ specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)₂ specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion.     

SH－SY5Y细胞胞内钙库特性研究

运用单细胞显微荧光测量技术测量了单个ＳＨ—ＳＹ５Ｙ细胞内游离钙离子浓度的变化。首次报道了ＳＨ—ＳＹ５Ｙ细胞内存在毒蕈碱敏感而非咖啡因敏感的钙库,并研究了它的动力学特征。Ｎ￣￣Ｃ￣ｈｅｔｗＭａｄＵｇｈ＊ＴＯｗｈｏｍＣｂ￣ｐｏｎｄｅｎｅｅｓｈｏｕｌｄｂｅ￣．     

硫酸盐还原一甲烷化两相厌氧消化系统运行工艺条件的研究

以合成废水为基质,研究了采用硫酸盐还原-甲烷化两相厌氧新型工艺处理含高浓度硫酸盐有机废水的系统运行工艺条件.结果表明,酸化-硫酸盐还原反应器的适宜pH为6.5-7.0;500mg/l的S~(2-)使SRB的硫酸盐还原活性下降;208mg/l的[H_2S]_L抑制MPB活性的95.4%;推导出估算气提塔出水回流比R的模型;以得到的工艺条件为依据处理了含19200mg/1的SO_4~(2-)和29400mg/l COD的味精废水.