Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Bone morphogenetic protein 2 may be a good predictor of success in oocyte fertilization during assisted reproductive technology

The aim of this study was to determine whether the bone-morphogenetic proreins-2 (BMP-2) or -7 (BMP-7) levels in follicular fluid (FF) are associated with success in oocyte fertilization during assisted reproductive technology (ART) treatment. Twenty-four cycles in 24 patients who underwent oocyte retrieval in Sugiyama clinic were included in this retrospective study. The patients were divided into two groups according to the success or failure of fertilization. FF samples were obtained from a single follicle in each patient, and the levels of BMP-2, BMP-7, Anti-Müllerian hormone (AMH), estradiol and progesterone from FF were measured, and evaluated in relation to the ART outcomes. The BMP-2 levels correlated positively with the AMH levels in FF (r(2) = 0.4928), but there was no statistically significant difference between BMP-7 and AMH levels. The BMP-2 and BMP-7 levels had no relation with either progesterone or estradiol levels, but BMP-2 levels in the fertilized group were significantly higher than those in the unfertilized group (P < 0.05). The BMP-2 levels in FF positively correlated with the AMH levels in FF, and those in the fertilized group were significantly higher than in the unfertilized group. Therefore, the BMP-2 levels in FF could be a predictive marker for fertilization.     

Structure–activity relationships of bacterial outer-membrane permeabilizers based on polymyxin B heptapeptides

A series of cationic cyclic heptapeptides based on polymyxin B have been synthesized for use as permeabilizers of the outer membrane of Gram-negative bacteria. Only analogs with the Dab²-d-Phe³-Leu⁴-Xxx⁵ sequence (Xxx = Dab or Orn) showed a synergistic bactericidal effect when combined with conventional antibiotics, indicating that the Dab² residue plays a critical role in permeation of the outer membrane of Gram-negative bacteria.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

One residue substitution in PcyA leads to unexpected changes in tetrapyrrole substrate binding

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the sequential reduction of the vinyl group of the D-ring and A-ring of biliverdin IXα (BV), using reducing equivalents provided by ferredoxin. This reaction produces phycocyanobilin, a pigment used for light-harvesting and light-sensing in red algae and cyanobacteria. The crystal structure of PcyA-BV reveals that BV is specifically bound in the PcyA active pocket through extensive hydrophobic and hydrophilic interactions. During the course of a mutational study of PcyA, we observed that mutation of the V225 position, apart from the processing sites, conferred an unusual property on PcyA; V225D mutant protein could bind BV and its analog BV13, but these complexes showed a distinct UV-vis absorption spectrum from that of the wild-type PcyA-BV complex. The crystal structures of BV- and BV13-bound forms of V225D protein revealed that gross structural changes occurred near the substrate-binding pocket, and that the BV/BV13 binding manner in the pocket was dramatically altered. Protein folding in V225D-BV/BV13 was more similar to that of substrate-free PcyA than that in PcyA-BV; the “induced-fit” did not occur when BV/BV13 was bound to the V225D protein. The unexpected structural change presented here provides a cautionary note about interpreting functional data derived from a mutated protein in the absence of its exact structure.     

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.     

Chemosystematics of tea trees based on tea leaf polyphenols as phenetic markers

This study examined the polyphenols of tea leaves as chemotaxonomic markers to investigate the phenetic relationship between 89 wild (the small-leaved C. sinensis var. sinensis and large-leaved C. sinensis var. assamica), hybrid, and cultivated tea trees from China and Japan. (?)-Epigallocatechin 3-O-gallate, EGCG (1); (?)-epigallocatechin, EGC (2); (?)-epicatechin 3-O-gallate, ECG (3); (?)-epicatechin, EC (4); (+)-catechin, CA (5); strictinin, STR (6); and gallic acid, GA (7) were used as polyphenolic markers. Of the 13 polyphenol patterns observed, Principal Component Analysis (PCA) indicated that the structure-types of the flavonoid B-rings, such as the pyrogallol-(EGCG (1) and EGC (2)) and catechol-(ECG (3) and EC (4)) types, greatly influenced the classification. Ward’s minimum-variance cluster analysis was used to produce a dendrogram that consisted of three sub-clusters. One sub-cluster (A) was composed of old tea trees ‘Gushu’ cha (C. sinensis var. assamica) and cv ‘Taidi’ cha, suggesting that relatively primitive tea trees contain greater amounts of compounds 3 and 4 and lower amounts of compounds 1 and 2. The other two sub-clusters B and C, made up of Chinese hybrids (sub-cluster B) and Japanese and Taiwanese tea trees (sub-cluster C), had lower contents of 3 and 4 than sub-cluster A. Therefore, PCA and cluster analysis indicated that the greater the amounts of 1 and 2 (and the lower of 3 and 4), the more recent the origin of the tea line. Based on morphological characteristics, geographical information, and the historical information on tea trees, these results show good agreement with the current theory of tea tree origins, and this suggests that the Xishuangbanna district and Puer City are among the original sites of the tea tree species.     

Oviposition-stimulatory activity of phenanthroindolizidine alkaloids of host-plant origin to a danaid butterfly, Ideopsis similis

Oviposition response of Ideopsis similis (L.) (Lepidoptera: Danaidae) was examined for 12 phenanthroindolizidine alkaloids present in its host plant, Tylophora tanakae (Maxim.) (Asclepiadaceae). At least five alkaloids, i.e. (+)‐isotylocrebrine (3,4,6,7‐tetramethoxyphenanthroindolizidine; l ), (+)‐3‐demethyliso‐ tylocrebrine ( 3 ), (+)‐isotylocrebrine N‐oxide ( 5 ), (+)‐6‐demethyltylocrebrine ( 8 ) and (–)‐7‐demethyltylophorine ( 10 ), were found to individually stimulate oviposition by females. Of these, compounds 1, 3 and 10 were regarded as key components most responsible for host recognition or preference. However, female egg‐laying was much higher in response to a mixture of the five alkaloids. In two‐choice bioassays, more eggs were deposited on samples comprising the five alkaloids than on samples consisting of a single alkaloid. This suggests strongly that host selection by the butterfly is mediated by the synergistic action of several phenanthroindolizidine alkaloids present in the host plant.     

Protein kinase C-delta phosphorylates Ebp1 and prevents its proteolytic degradation, enhancing cell survival

ErbB3-binding protein (Ebp1) promotes cell survival by preventing apoptotic DNA fragmentation through a complex with active nuclear Akt. Ebp1 phosphorylation by protein kinase C (PKC)-delta mediates its binding to nuclear Akt. In this study, we show that Ebp1 itself acts as a substrate of active caspase 3 during the programmed cell death. PKC-delta phosphorylation on Ebp1 protects it from apoptotic degradation initiated in cell-free apoptotic solution. Moreover, Ebp1 is evidently cleaved in PKC-delta-deficient cells but not in wild-type cells. Ebp1 translated from first ATG is resistant to apoptotic cleavage; by contrast, Ebp1 from second and third ATG demonstrates robust degradation, and PKC phosphorylation on S360 suppresses its cleavage by active caspase 3. Ebp1 can be digested at both D53 and D196 sites, but cleavage at D196 appears to be a prerequisite for its further degradation at D53 site. Compared with wild-type Ebp1, D196A mutant markedly protects cells from apoptosis. Thus, PKC-delta antagonizes apoptosis through phosphorylating Ebp1 and protects it from apoptotic degradation.