We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208 -9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.Heparan sulfate proteoglycans (HSPGs) 1 are ubiquitously present on cell surface and in extracellular matrix including basement membrane and have divergent structures and functions (1-3). The heparan sulfate (HS) chains in HSPGs are known to interact with a variety of proteins such as heparinbinding growth factors, extracellular matrix components, protease inhibitors, protease, and lipoprotein lipase (4 -8). These interactions are implicated not only in various dynamic cellular behaviors including cell proliferation, differentiation, adhesion, migration, and morphology during development (9 -16), but also in various physiological phenomena such as inflammation (17), blood coagulation (18 -20), and tumor cell invasion and malignancy (21-23). Moreover, the pathogens such as bacteria, parasites, and viruses are known to infect host cells through the interactions between the cell surface HS on host cell and the coat proteins or cell surface proteins of pathogens (24,25).Recently, genetic screens and analyses are suggesting not only in Drosophila but also in mammals that these interactions also play pivotal roles in embryonic development. For example, the sugarless mutant (10 -12), which is deficient in UDP-glucose dehydrogenas...

Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 degrees C and exhibits an unusual Qy absorption at 915 nm for the core light-harvesting complex (LH1), an approximately 35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Qy transition is strongly dependent on metal cations and Ca2+ is involved in the unusual red shift. Removal of the Ca2+ resulted in formation of a species with the LH1 Qy absorption at 880 nm, and addition of the Ca2+ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca2+ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca2+/alphabeta-subunit = 1:1 and the binding constant was in 10(5) m(-1) order of magnitude, which is comparable with those for EF-hand Ca2+-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca2+ binding were small and occurred slowly, with a typical time constant of approximately 6 min. Replacement of the Ca2+ with other metal cations caused blue shifts of the Qy bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca2+-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the alpha-polypeptide and the N-terminal region of the beta-polypeptide.

Adherent cells, cell sheets, and spheroids were harvested noninvasively from a culture surface by means of electrochemical desorption of a self-assembled monolayer (SAM) of alkanethiol. The SAM surface was made adhesive by the covalent bonding of Arg-Gly-Asp (RGD)-peptides to the alkanethiol molecules. The application of a negative electrical potential caused the reductive desorption of the SAM, resulting in the detachment of the cells. Using this approach greater than 90% of adherent cells detached within 5 min. Furthermore, this approach was used to obtain two-dimensional (2D) cell sheets. The detached cell sheets consisted of viable cells, which could be easily attached to other cell sheets in succession to form a multilayered cell sheet. Moreover, spheroids of hepatocytes of a uniform diameter were formed in an array of cylindrical cavities at a density of 280 spheroids/cm(2) and were harvested by applying a negative electrical potential. This cell manipulation technology could potentially be a useful tool for the fabrication and assembly of building blocks such as cell sheets and spheroids for regenerative medicine and tissue engineering applications.

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, alpha-1-fetoprotein and beta2-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.