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.

No abstract

Although being an atmospheric pressure ion source, electrospray ionization (ESI) has rarely been used directly for ambient imaging mass spectrometry because the sample has to be introduced as liquid solution through the capillary. Instead of capillary, probe electrospray ionization (PESI), which has been developed recently, uses a solid needle as the sampling probe, as well as the electrospray emitter, and has been applied not only for liquid solutions but also for the direct sampling on wet samples. Biological tissues are composed of cells that contain 70-90% water, and when the surface is probed by the needle tip, the biological fluid adhering to the needle can be electrosprayed directly or assisted by additional solvent added onto the needle surface. Here, we demonstrate ambient imaging mass spectrometry of mouse brain section using PESI, incorporated with an auxiliary heated capillary sprayer. The solvent vapor generated from the sprayer condensed on the needle tip, re-dissolving the adhered sample, and at the same time, providing an indirect means for needle cleaning. The histological sections were prepared by fixation using paraformaldehyde, and the spatial analysis was automated by maintaining an equal sampling depth into the sample in addition to raster scan. Phospholipids and galactosylceramides were readily detected from the mouse brain section in the positive ion mode, and were mapped with 60 microm lateral resolution to form mass spectrometric images.

Thermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057-1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca 2؉ as a cofactor. Removal of the Ca 2؉ from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca 2؉ to the Ca 2؉ -depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Q y transition between 915 nm for the native and 880 nm for the Ca 2؉ -depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15°C higher and the enthalpy change is about 28% larger than the Ca 2؉ -depleted LH1-RC. Substitution of the Ca 2؉ with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca 2؉ ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources.Purple sulfur photosynthetic bacterium, Thermochromatium tepidum, was originally isolated from a hot springs in Yellowstone National Park and can grow anaerobically at optimum temperatures of 48 -50°C with an upper limit of 58°C (1). This is the highest temperature of all known purple bacteria (2). A number of soluble proteins purified from this organism have been shown to be thermostable with respect to their mesophilic counterparts. Ribulose-1,5-bisphosphate carboxylase/oxygenase, a key enzyme of the Calvin cycle, from Tch. tepidum was reported to be most catalytically active at 50°C, and it remained active at 60°C over 20 min, whereas the same enzyme from the closely related mesophilic bacterium Allochromatium vinosum completely lost its activity over the same period (3, 4). Similar behavior was observed for the high potential iron-sulfur proteins from the two bacteria (5, 6), and was attributed to subtle differences in the amino acid sequence and structure (7).The thermal stability mechanism of the membrane proteins is somewhat complicated because of complex protein-protein and protein-detergent/lipid interactions (8). In the case of reaction center (RC) 3 from Tch. tepidum, the thermal stability was shown to be strongly dependent on the type and compo...

Technologies to microfabricate electrochemical sensors and systems are rapidly advancing and will surely have an impact on critical fields such as medicine, environment, and information processing. It is a good opportunity to review the state of the art and trends in the technologies developed in this field during the last two decades of the 20th century and prepare for the coming decades. Here, developed electrochemical microsensors and microsystems will be reviewed in terms of techniques and performance along with the problems left as issues for the next century.