Crystal structures of wild-type and mutant plastocyanins from a higher plant, Silene

Plastocyanin functions as an electron carrier between the cytochrome b6f complex and photosystem I. The crystal structures of the wild-type and E43K/D44K double mutant from the higher plant, Silene, have been determined at 2.0 and 1.75 A resolution, respectively. The wild-type plastocyanin comprises two monomers per asymmetric unit, one of which shows the unusually great distance between the copper ion and the Ndelta1 atom of H87 because of the hydrogen bond network formation between H87 and symmetry-related G10. The root mean square deviation for Ca atoms between the wild-type and mutant plastocyanins is 0.44 A, however, the electrostatic potential maps of their molecular surfaces are remarkably different. The low electron-transfer rate in the E43K/D44K mutant results from the hindrance of electrostatic interactions, not from the structural change due to the mutation.     

Genomic sequence and organization of the family of CNR/Pcdhalpha genes in rat

CNR/Pcdhalpha family proteins are known as synaptic cadherins and Reelin receptors. Here we report the complete genomic sequence and organization of the rat CNR. The rat CNR cluster encodes 15 variable and 3 constant exons. The genomic organizations of the rat, mouse, and human CNR/Pcdhalpha are orthologous. The percentage identity of the coding regions between the rat and the mouse is 93.6% on average at the nucleic acid level, and between rat and human it is 82.8%. The rat CNRs (v1-v13) also contain an RGD motif in the extracellular cadherin 1 domains and cysteine repeats that are characteristic of the transmembrane and cytoplasmic domains of CNR proteins. The number of variable exons in the rat CNR cluster is identical to that of the human. The rat CNR cluster has one more variable exon than is found in laboratory mouse strains, because in the mouse a variable exon located between v7 and v8 is divided by the insertion of a retrotransposon. This exon is not disrupted in the rat, in which it is transcribed. By in silico analysis, CNR/Pcdhalpha was also mapped to rat chromosome 18, but the orientation was opposite for the mouse CNR/Pcdhalpha gene cluster. The relative expression profiles of the rat CNRs (v1-v13) show that all the CNRs are transcribed, but there are variations in the expression ratios among the CNRs.     

Development of an efficient enzymatic production of gamma-D-glutamyl-L-tryptophan (SCV-07), a prospective medicine for tuberculosis, with bacterial gamma-glutamyltranspeptidase

Gamma-D-Glutamyl-L-tryptophan (SCV-07) is a prospective medicine for the treatment of tuberculosis, according to the phase two clinical trial. Because gamma-D-glutamyl-L-tryptophan has several reactive groups in its molecule, consists of D- and L-amino acids, and is connected by gamma-glutamyl linkage, its chemical synthesis is complicated. An efficient enzymatic method to synthesize gamma-D-glutamyl-L-tryptophan from D-glutamine and L-tryptophan employing bacterial gamma-glutamyltranspeptidase was developed. The optimum reaction conditions were 50 mM D-glutamine, 50 mM L-tryptophan, and 0.2 U ml(-1) gamma-glutamyltranspeptidase, pH 9-9.5, and incubation at 37 degrees C for 5 h. After a 5 h incubation, 33 mM gamma-D-glutamyl-L-tryptophan was obtained, the conversion rate being 66%. The product was purified by Dowex 1 x 8 column and was considered to be gamma-D-glutamyl-L-tryptophan.     

Pseudodeflectusin, a novel isochroman derivative from Aspergillus pseudodeflectus a parasite of the sea weed, Sargassum fusiform, as a selective human cancer cytotoxin

A new isochroman derivative named pseudodeflectusin was isolated from a culture broth of Aspergillus pseudodeflectus. The structure was determined by spectroscopic means as 9-hydroxy-7-methyl-2-(methylethylidine)-furano[3,2-H]isochroman-3-one. This compound exhibited cytotoxicity for several human cancer cell lines from the stomach (NUGC-3), cervix (HeLa-S3), and peripheral blood (HL-60), but did not affect those from the lung (A549) or colon (DLD-1). The LD50 value of this compound for HL-60 cells was 39 microM.     

Thienopyridine and benzofuran derivatives as potent anti-tumor agents possessing different structure-activity relationships

(3-Amino-6-thiophen-2-yl-thieno[2,3-b]pyridin-2-yl)phenylmethanone (3) was discovered as a new type of cytotoxic agent selective against a tumorigenic cell line. The molecular structure of a previously reported compound, (4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)phenylmethanone (2), had remarkably similar bioisosteric substructures to that of compound 3. Although the relationship between the molecular structure and biological activity of each derivative synthesized from these two hit compounds (2 and 3) were studied, unexpectedly no correlation was observed. However, after further synthetic study from 3, one of the most potent derivative (10k) having a different SAR profile from 2, was discovered.     

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.     

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.