Spectroelectrochemical evaluation of redox potentials of cysteine tryptophylquinone and two hemes c in quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans has a novel cofactor cysteine tryptophylquinone (CTQ) in the smallest gamma subunit and two hemes c in the largest alpha subunit [Datta, S., Mori, Y., Takagi, K., Kawaguchi, K., Chen, Z., Okajima, T., Kuroda, S., Ikeda, T., Kano, K., Tanizawa, K., and Mathews, F. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14268-14273]. The spectral change of QH-AmDH was assigned to the redox reaction of the hemes c alone. The redox potentials of the two hemes c with His and Met as the second axial ligands, respectively, were determined to be 0.149 and 0.235 V versus SHE at pH 7.0 by a mediator-assisted continuous-flow column electrolytic spectroelectrochemistry (MCES). The monomeric gamma subunit of QH-AmDH was isolated from urea-treated QH-AmDH. The fully oxidized and reduced forms of the gamma subunit exhibited a unique absorption band centered at 380 nm and a shoulder band around 315 nm, respectively, at neutral pH. The two-electron redox potential of CTQ in the isolated gamma subunit was evaluated to be 65 mV at pH 7.0 by MCES. The redox reaction was linked to the two-proton transfer at pH <8.6 and to a single-proton transfer at pH >8.6. The pK(a) value (K(a) being the acid dissociation constant) of 8.6 was assigned to one of the phenolic OH groups of the quinol form. Upon deprotonation, the red shift of the shoulder band was observed. The gamma subunit adsorbed on a glassy carbon electrode, and gave a direct but quasi-reversible electrochemical signal. Intra- and interprotein electron transfers of QH-AmDH are discussed from thermodynamic and structural points of view.     

Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans

Here we report the cloning of a full-length cDNA encoding the human ortholog (HSulf-1) of the developmentally regulated putative sulfatases QSulf-1 (Dhoot, G. K., Gustafsson, M. K., Ai, X., Sun, W., Standiford, D. M., and Emerson, C. P., Jr. (2001) Science 293, 1663-1666) and RSulfFP1 (Ohto, T., Uchida, H., Yamazaki, H., Keino-Masu, K., Matsui, A., and Masu, M. (2002) Genes Cells 7, 173-185) as well as a cDNA encoding a closely related protein, designated HSulf-2. We have also obtained cDNAs for the mouse orthologs of both Sulfs. We demonstrate that the proteins encoded by both classes of cDNAs are endoproteolytically processed in the secretory pathway and are released into conditioned medium of transfected CHO cells. We demonstrate that the mammalian Sulfs exhibit arylsulfatase activity with a pH optimum in the neutral range; moreover, they can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Taken together, our results establish that the mammalian Sulfs are extracellular endosulfatases with strong potential for modulating the interactions of heparan sulfate proteoglycans in the extracellular microenvironment.     

Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates

Tetracycline-resistant (Tet(r)) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet(r) gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet(r) genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet(r) strains transferred Tet(r) genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet(r) strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.     

The exocytotic trafficking of TC10 occurs through both classical and nonclassical secretory transport pathways in 3T3L1 adipocytes

To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TC10 or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19 degrees C temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).