Incorporation of β‐amino acids into ascidiacyclamides: Effects on conformation,cytotoxicity and interaction with copper (II) ion

Seven ascidiacyclamide [cyclo(–Ile–oxazoline–d ‐Val–thiazole–)₂] (ASC) analogues incorporating the β‐amino acids βIle, βoxazoline, and/or d ‐βVal were synthesized. We then investigated the effects of the position and number of incorporated β‐amino acids on the structure, cytotoxicity, and copper binding by these seven analogues. The structural analyses revealed that both βIle and d ‐βVal favor a gauche‐type θ torsion angles, while βoxazoline favors a trans‐type θ torsion angle. Expansion of the macrocycle by incorporation of βIle or d ‐βVal readily induced molecular folding. On the other hand, the incorporation of two βoxazoline residues strongly extended the peptide conformation, and the incorporation of one was sufficient for the moderate restriction important for conformational equilibrium and cytotoxicity. Despite expansion of the macrocycles, the structure‐cytotoxicity relationships were largely maintained. In studies of complexation of the analogues with Cu (II) ion, the position and number of incorporated β‐amino acids had a large impact on the structure of the metal complex and may contribute to its stabilization.     

Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues

An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 bp was designed and constructed from 44 oligodeoxynucleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease alpha-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectroscopy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of antithrombin III on glycoprotein Ib/IX/V activation in human platelets: Suppression of thromboxane A(2) generation

We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.     

Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

Biosynthesis of Polyhydroxyalkanaotes by a Novel Facultatively Anaerobic <Emphasis Type="Italic">Vibrio</Emphasis> sp. under Marine Conditions

Marine bacteria have recently attracted attention as potentially useful candidates for the production of practical materials from marine ecosystems, including the oceanic carbon dioxide cycle. The advantages of using marine bacteria for the biosynthesis of poly(hydroxyalkanoate) (PHA), one of the eco-friendly bioplastics, include avoiding contamination with bacteria that lack salt-water resistance, ability to use filtered seawater as a culture medium, and the potential for extracellular production of PHA, all of which would contribute to large-scale industrial production of PHA. A novel marine bacterium, Vibrio sp. strain KN01, was isolated and characterized in PHA productivity using various carbon sources under aerobic and aerobic–anaerobic marine conditions. The PHA contents of all the samples under the aerobic–anaerobic condition, especially when using soybean oil as the sole carbon source, were enhanced by limiting the amount of dissolved oxygen. The PHA accumulated using soybean oil as a sole carbon source under the aerobic–anaerobic condition contained 14% 3-hydroxypropionate (3HP) and 3% 5-hydroxyvalerate (5HV) units in addition to (R)-3-hydroxybutyrate (3HB) units and had a molecular weight of 42 × 10³ g/mol. The present result indicates that the activity of the beta-oxidation pathway under the aerobic–anaerobic condition is reduced due to a reduction in the amount of dissolved oxygen. These findings have potential for use in controlling the biosynthesis of long main-chain PHA by regulating the activity of the beta-oxidation pathway, which also could be regulated by varying the dissolved oxygen concentration.     

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.     

Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species

The signaling pathway that transduces the stimulatory effect of low K⁺ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K⁺ in Madin-Darby canine kidney (MDCK) cells. Low K⁺ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K⁺-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase -subunit 1.9 kb and 900-bp 5'-flanking regions that have a negative regulatory element. Low K⁺ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K⁺. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K⁺ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K⁺. Moreover, catalase and NAC also inhibited low-K⁺-induced increases in the Na,K-ATPase ₁- and ₁-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K⁺ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K⁺-induced increases in the Na,K-ATPase protein contents and cell surface molecules. Madin-Darby canine kidney cells; N-acetylcysteine; catalase