池原 早苗

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Abstract In bronchial asthma patients, mucous cell metaplasia (MCM) and fibrosis occur in the bronchial epithelium and interstitium, respectively. The mucus and collagen fibers are identified by Periodic acid-Schiff stain (PAS) or Sirius red stain on optical microscopy. On a scanning electron microscope (SEM) observation, formalin-fixed-paraffin-embedded specimens have high insulation, thereby attenuating the scattered electron signals leading to insufficient contrast. Moreover, there were no staining methods for SEM observation, which characterizes the changes in epithelium and interstitium by enhancing the scattered electrons. In this study, we established a method of coating osmium thin film on pathological tissue specimens using plasma chemical vapor deposition technology. This method ensured the intensity of scattered electron signals and enabled SEM observation. Furthermore, we found that morphological changes in MCM and interstitial fibrosis could be characterized by Grocott stain, which we optimized to evaluate pathological remodeling in bronchial asthma. Using these techniques, we compared asthma-induced mice with Amphiregulin (Areg) knockout mice, and found that Areg induce MCM, but the production of Grocott-stain-positive substrate in the interstitium is Areg-independent. The method developed in this study provides an understanding of the pathological spatial information linked to the ultrastructural changes in cells and interstitium due to disease-related signaling abnormalities.

The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.

To understand the aggregation mechanism of serum protein dispersed in the solution of distilled water induced by a low temperature atmospheric pressure plasma, we compared the relationship between the amount of aggregation and experimental condition dependencies such as gas species, gas flow rate, and the distance between the plasma device and the solution. In this experiment, pure argon, helium, and/or a mixture of helium and argon gases were used. From statistical analyses of various experimental conditions, it was found that a monotonic supply of total absolute charge in one period was important for protein aggregation. When the coefficient of variation (standard deviations/averaged total absolute charges during one period) is small, the amount of protein aggregation becomes larger. Excess or scarcity of averaged total absolute charges does not appear to relate to protein aggregation phenomena; however, the coefficient of variation strongly relates to the mechanism of protein aggregation. Therefore, a possible serum protein aggregation process by low-temperature plasma treatment was proposed in which the serum protein, which had negative charges in the solution, was locally aggregated through the trigger of charge neutralization induced by a stable charge supply, and/or positively charged aggregated protein on the solution surface attracted serum protein in the solution to the surface.

Trypanosoma brucei (Tb) is a pathogenic protozoan causing sleeping sickness in humans. Despite little knowledge of how the produced reactive oxygen species (ROS) kills this protozoan, the research on the killing mechanism using chemical compounds and the phagosome in the macrophages has suggested that the protozoan is highly susceptible to the increased oxidative stress. Because the prescribed drug can react with various kinds of molecules and the second produced intermediate compounds, in this study, we clarified the immediate killing effect on Tb in the condition of increased oxidative stress using a low-temperature plasma at atmospheric pressure (LTP) equipment. Results Show the significant growth inhibition of Tb in the LTP-treated medium, the loss of morphological homeostasis with twisted to puffed appearance, and demonstrated the swelled changes on mitochondria and endoplasmic reticulum. In conclusion, this study revealed how the increased oxidative stress kills Tb using LTP technology. (c) 2021 The Japan Society of Applied Physics

Striation phenomena in a plasma flare produced by an atmospheric pressure plasma jet had been observed. In this study, we measured the striation structure in a neon plasma flare by optical emission spectroscopy. As a result, it shows that the emission from the plasma flare is synchronized with the plasma current. We discuss the temperature estimated from the Boltzmann plot using the two emission lines of Ne I at 692.9 and 724.5nm whose upper levels are Ne [3p (2p(6))] and Ne [3p (2p(10))], respectively. This temperature has peaks with a spatial interval of about 0.5mm, and the peak positions correspond to the spatial variation of the emission intensity. We also discuss the mechanism of the striation phenomena with the estimated temperature, and collisional relaxation between excited and neutral atoms.

© 2018 IOP Publishing Ltd. In a low-temperature atmospheric pressure plasma jet using helium gas, emission-propagation phenomena, such as streamers and striations were measured using a high-speed intensified charge-coupled device camera. A particular focus was placed on the study of the dependence of the phenomena on the distance between the nozzle of the plasma device and a target plate. When the distance decreased, a transition from the positive streamer to a spatially continuous emission resulted. A further distance reduction resulted in a new propagation mode in which the positive and negative streamers appeared alternately with different current waveforms over two cycles of applied voltage. This phenomenon may be related to residual charges of the preceding cycle when streamer propagation begins. Striation structures were observed in the tail of the positive streamer head and in the successive spatially continuous-emission region. These structures can be measured only within a shorter period than one voltage cycle.

Low temperature plasma (LTP) coagulation equipment, which causing burn injuries to patients, has been introducing into minimally invasive surgery. The mechanism by which this equip-ment stops bleeding is to directly occupy the injury with the formed blood clots, and different from the mechanism of the common electrical hemostatic devices that cauterize the tissues around the bleeding to stem the blood flow. A noteworthy point is that LTP treatment with our equipment is not confined only to the blood coagulation system, but it has significant effects on the other blood components to form clots with or without hemolysis, and that there is a plasma current threshold that determines whether the treatment makes stable clots. In this review, we introduce the clinical benefits of LTP current and describe the clot formation it facilitates.

A method for blood coagulation using low-energy atmospheric-pressure plasma (LEAPP) is confirmed as an alternative procedure to reduce tissue damage caused by heat. Blood coagulation using LEAPP behaves differently depending on working gas species; helium is more effective than argon in promoting fast coagulation. To analyse the difference in reactive species produced by helium and argon plasma, spectroscopic measurements were conducted without and with a target material. To compare emissions, blood coagulation experiments using LEAPP for both plasmas were performed under almost identical conditions. Although many kinds of reactive species such as hydroxyl radicals and excited nitrogen molecules were observed with similar intensity in both plasmas, intensities of nitrogen ion molecules and nitric oxide molecules were extremely strong in the helium plasma. It is considered that nitrogen ion molecules were mainly produced by penning ionization by helium metastable. Near the target, a significant increase in the emissions of reactive species is observed. There is a possibility that electron acceleration was induced in a local electric field formed on the surface. However, in argon plasma, emissions from nitrogen ion were not measured even near the target surface. These differences between the two plasmas may be producing the difference in blood coagulation behaviour. To control the surrounding gas of the plasma, a gas-component-controllable chamber was assembled. Filling the chamber with O-2/He or N-2/He gas mixtures selectively produces either reactive oxygen species or reactive nitrogen species. Through selective treatments, this chamber would be useful in studying the effects of specific reactive species on blood coagulation.

Low-temperature plasma is useful for the care of wounded skin. It accelerates wound healing. However, the mechanism of this effect has not been fully elucidated yet. Galectin-1 is reported to accelerate wound healing via the Smad signaling pathway. In the present study to clarify whether or not galectins were expressed during the process of wound healing in the plasma-treated skin, we examined the effect of low-temperature plasma on galectin expression in the healing skin. We compared the effects of low-temperature plasma on the expression of galectin-1, -2, and -3 in the healing skin with those of electrocoagulation conducted with a high-frequency electrical coagulator. Immediately after the start of low temperature plasma treatment following the incision made in the skin, a membrane-like structure was formed on the surface of the wound. Immunoelectron microscopy showed that these galectins were localized in the membrane-like structure of the plasma-treated skin. The expressions of these galectins were increased by the low-temperature plasma treatment, whereas they were inhibited by the electrocoagulation. These results suggest that galectins were involved in the wound healing of low temperature plasma-treated skin. Galectins will thus be good markers for further examination of the effects of low-temperature plasma on the healing of wounded skin. (C) 2016 Elsevier Inc. All rights reserved.

Low-temperature plasma (LTP) treatment promotes blood clot formation by stimulation of the both platelet aggregation and coagulation factors. However, the appearance of a membrane-like structure in clots after the treatment is controversial. Based on our previous report that demonstrated characteristics of the form of coagulation of serum proteins induced by LTP treatment, we sought to determine whether treatment with two plasma instruments, namely BPC-HP1 and PN-110/120TPG, formed clots only from red blood cells (RBCs). LTP treatment with each device formed clots from whole blood, whereas LTP treatment with BPC-HP1 formed clots in phosphate-buffered saline (PBS) containing 2 x 10(9)/mL RBCs. Light microscopic analysis results showed that hemolysis formed clots consisting of materials with membrane-like structures from both whole blood and PBS-suspended RBCs. Moreover, electron microscopic analysis results showed a monotonous material with high electron density in the formed clots, presenting a membrane-like structure. Hemolysis disappeared with the decrease in the current through the targets contacting with the plasma flare and clot formation ceased. Taken together, our results and those of earlier studies present two types of blood clot formation, namely presence or absence of hemolysis capability depending on the current through the targets. (C) 2016 Elsevier Inc. All rights reserved.

To understand the mechanism of turbulent enhancement phenomena of a neutral gas flow containing plasma ejected from the nozzle of plasma equipment, the schlieren optical method was performed to visualize the neutral gas behavior. It was confirmed that the turbulent starting point became closer to the nozzle exit, as the amplitude of discharge voltage electric field) increased. To study the effect of electric field on turbulent enhancement, two sets of external electrodes were arranged in parallel, and the gas from the nozzle was allowed to flow between the upper and lower electrodes. It was found that the neutral gas flow was bent, and the bending angle increased as the amplitude of the external electric field increased. The results obtained using a simple model analysis roughly coincide with experimental data. These results indicate that momentum transport from drifted ions induced by the electric field to neutral particles is an important factor that enhances turbulence. (C) 2016 The Japan Society of Applied Physics

The objective of this study is to determine whether there is a pathway different from the natural blood coagulation process involved in the plasma-induced activation of platelets and coagulation factors. Plasma treatment of bleeding wounds generated glomerular structures, which could be detected in high-magnification scanning electron microscopy images, and plasma flares produced aggregation on the surface of solutions containing bovine serum albumin, ovalbumin (OVA), or similar protein material. Ultramicroscopic analysis of the plasma-generated aggregation of OVA indicated a possible mechanism to explain the particle formation and fusion as a monotonous solid appearance. This study suggests that plasma coagulation involves aggregations of materials in blood in addition to the activation of platelets and coagulation factors by plasma treatment.

Low energy ionized gas coagulation equipment (LE-IGCE) using low temperature nonthermal atmospheric pressure plasmas has been attracting special attention with the popularization of minimally invasive surgery. In this work, the thermal characteristics of a commercial high energy ionized gas coagulation equipment (HE-IGCE) and a specially designed LE-IGCE have been studied using an infrared camera and optical emission spectroscopy during their treatments. The four substrates used for the treatments were pork meats, copper (Cu) plates, wet tissues, and glass plates. In the HE-IGCE treatment, the surface temperature of the pork meat rose to 350�C, and the rotational temperatures of nitrogen molecules in plasmas were 450 to 1630 K, depending on which substrate was used during HE-IGCE treatments. In the LE-IGCE treatment, the surface temperature of pork meats was lower than 40�C, and the rotational temperatures of nitrogen molecules were lower than 350 K. The results show that the LE-IGCE can maintain the temperature of biological tissue below the threshold that would cause irreversible tissue damage.

The premalignant lesions of pancreatic cancer, pancreatic intraepithelial neoplasia (PanIN), have a high frequency of mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS), and genetic alterations in the retinoblastoma (Rb)-E2 factor (E2F) and transformed 3T3 cell double minute 2 (MDM2)-p53 pathways accelerate development of pancreatic ductal adenocarcinoma. The viral oncoprotein SV40 large T antigen (TAg) can inhibit the effects of the Rb family of molecules and of p53 on these pathways, and targeted expression of TAg in mouse pancreas is associated with the development of endocrine or acinar cell tumours. In this study, to determine whether the viral oncoprotein promotes pancreatic duct carcinogenesis initiated by oncogenic KRAS, we generated mice expressing temperature-sensitive SV40 large T antigen (tsTAg) on pancreatic epithelial cells in the presence or absence of KrasG12D. Mice with pancreas-specific tsTAg expression developed acinar cell dysplasia by 22 weeks without PanIN formation, while mice expressing both tsTAg and KrasG12D developed highly aggressive adenocarcinoma with a ductal cell phenotype within a short period, and died within 3 weeks. The tumours resembled human pancreatic ductal adenocarcinoma (PDAC) at the histological level, and oncogenic Kras and tsTAg synergistically activated E2f and Sre transcription in established PDAC cell lines. These results suggest that tsTAg synergistically promotes KrasG12D-associated PDAC formation, and our study identifies a new mouse model of PDAC that may allow a better understanding of the mechanism of carcinogenesis in pancreatic carcinoma, which shows a catastrophic clinical course.

We recently established a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs) which are probably taken up by macrophages (M phi) to carry anti-cancer drugs to milky spots known as preferential metastatic sites of gastric cancers [Y. Ikehara, T. Niwa, L. Biao, S.K. Ikehara, N. Ohashi, T. Kobayashi, Y. Shimizu, N. Kojima, H. Nakanishi, A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle, Cancer Res. 66 (2006) 8740-8748]. In the present study, we applied this intraperitoneal DDS for systemic cancer immunotherapy employing ovalbumin (OVA) as a model antigen. The cells taking up the OMLs containing FITC-OVA injected into the peritoneal cavity were predominantly M(, as they showed adhesive characteristics and expressed F4/80 and CD11b almost exclusively. The phagocytic cells also took up bare OVA directly to the same extent as OML-enclosed OVA (OML-OVA), as it is a highly mannosilated protein. The phagocytic cells taking up OML-OVA, however, could activate OVA-specific CD8(+) (from OT-I: H-2K(b)/OVA(257-264)-specific)and CD4(+) (from OT-II: H-2A(b)/OVA(323-339)-specific) T cells much more effectively in vitro than those taking up bare OVA. Furthermore, only the mice pre-immunized with OML-OVA rejected E.G7-OVA (OVA-transfected EL4) but not EL4. These results indicate that the OMLs can also be used as an effective antigen delivery system for cancer immunotherapy activating both CTL and Th subsets. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor ( EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.

beta 1,4-N-acetylgalactosan nyltransferase III (beta 4GaINAc-T3), which was recently cloned and identified, exhibits GaINAc transferase activity toward a GlcNAcP residue with beta 1,4-linkage, forming the N,N'-diacetyllactosediamine, GatNAc beta 1,4GIcNAc (LacdiNAc or LDN). Though LacdiNAc has not been found in the gastric mucosa, a large amount of transcript was detected in our previous study. To increase our knowledge of beta 4GaINAc-T3 expression and its product LacdiNAc, we examined the exact localization of beta 4GaINAc-T3 in human gastric mucosa using a newly developed antibody, monoclonal antibody (mAb) K1356. This antibody specifically detected the enzyme that transfected the beta 4GaINAc-T3 gene into MKN45 cells, and the terminal beta GaINAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). beta 4GaINAc-T3 was localized in the supra-miclear region of surface mucous cells in gastric mucosa, and WFA positively stained the mucins secreted by the cells. In contrast, in the cells of the glandular compartment in the fundic glands and a few cells in the pyloric glands, beta 4GaINAc-T3 was observed in the basolateral position of the nucleus, where no WFA reactivity was detected. The anti-Tn (GaINAc alpha-O-Ser/Thr) antibody staining did not overlap with the WFA staining. By measuring the binding activity of WFA using automated frontal affinity chromatography (FAC), we found WFA to bind most strongly LacdiNAc among the sugar chains examined. Neither beta 4GaINAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-miclear expression of beta 4GaINAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells and that LacdiNAc and beta 4GaINAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa.

The lymphoid tissue in the omentum, at the so-called milky spots, is known as an initial place for disseminated cancer cells to develop into solid tumors. In the present study, i.p. macrophages significantly took up oligomannose-coated liposomes (OMLs) that were injected into the peritoneal cavity, and then gradually accumulated in the omentum and the other lymphoid tissues within 24 hours of i.p. injection of OMLs. When 5-fluorouracil (5-FU) was encapsulated in the OMLs, > 60% of administered 5-FU accumulated in the omentum. Treatment of macrophages at 39 degrees C for 30 minutes led to the release of 5-FU from the macrophages, suggesting that controlled release from macrophages could be achieved by mild hyperthermia. We encased magnetic nanoparticles, which are known to convert electromagnetic energy to heat in the OMLs to achieve in vivo hyperthermia at the site. Using this system in a mouse i.p. metastasis model, we successfully controlled tumor development by coadministration of OML-encased 5-FU and OML-encased magnetic nanoparticles, followed by treatment with an alternating magnetic field. No apparent reduction was seen in tumor growth with the administration of OML-encased magnetic nanoparticles or OML-encased 5-FU alone. Thus, we have established the use of i.p. macrophages as a novel drug delivery system for the control of cancer metastatic to milky spots.

Proinflammatory cytokine gene polymorphisms have been demonstrated to associate with gastric cancer risk, of which IL1B-31T/C and -511C/T changes have been well investigated due to the possibility that they may alter the IL1B transcription. The signal transduction target upon interleukin 1 beta (IL1 beta) stimulation, the nuclear factor of kappa B (NF kappa B) activation, supports cancer development, signal transduction in which is mediated by FS-7 cell-associated cell surface antigen (FAS) signaling. Based on recent papers describing the prognostic roles of the polymorphisms and the NF kappa B functions on cancer development, we sought to determine if Japanese gastric cancer patients were affected by the IL1B-31/-511 and FAS-670 polymorphisms. A case-control study was conducted on incident gastric adenocarcinoma patients (n=271) and age-gender frequency-matched control subjects (n=271). We observed strong linkage disequilibrium between the T allele at -511 and the C allele at -31 and between the C allele at -511 and the T allele at -31 in IL1B in both the cases and controls (R-2 = 0.94). Neither IL1B-31, -511 nor FAS-670 polymorphisms showed significantly different risks of gastric adenocarcinoma. Though FAS-670 polymorphisms did not show any significant difference, the proportion of subjects with IL1B-31TT (or IL1B-511CC) increased according to stage (trend P=0.019). In particular, subjects with stage IV had a two times higher probability of having either IL1B-31TT (or IL1B-511CC) genotype compared with stage I subjects. These observations suggest that IL1B-31TT and IL1B-511CC are associated with disease progression.

Siglec-7 (p70/AIRM) and Siglec-9 are "CD33"-related siglecs expressed on natural killer (NK) cells and subsets of peripheral T cells. Like other inhibitory NK cell receptors, they contain immunoglobulin receptor family tyrosine-based inhibitory motifs in their cytoplasmic domains, and Siglec-7 has been demonstrated to negatively regulate NK cell activation. Based on reports of the presence of these siglecs on T cells, we sought to determine if they are capable of modulating T cell receptor (TCR) signaling using Jurkat T cells stably and transiently transfected with Siglec-7 or Siglec-9. Following either pervanadate stimulation or TCR engagement, both Siglecs exhibited increased tyrosine phosphorylation and recruitment of SHP-1. Effects of Siglec-7 and -9 were also evident in downstream events in the signaling pathway. Both siglecs reduced phosphorylation of Tyr(319) on ZAP-70, known to play a pivotal role in up-regulation of gene transcription following TCR stimulation. There was also a corresponding decreased transcriptional activity of nuclear factor of activated T cells ( NFAT) as determined using a luciferase reporter gene. Like all siglecs, Siglec-7 and -9 recognize sialic acid-containing glycans of glycoproteins and glycolipids as ligands. Mutation of the conserved Arg in the ligand binding site of Siglec-7 (Arg(124)) or Siglec-9 (Arg(120)) resulted in reduced inhibitory function in the NFAT/luciferase transcription assay, suggesting that ligand binding is required for optimal inhibition of TCR signaling. The combined results demonstrate that both Siglec-7 and Siglec-9 are capable of negative regulation of TCR signaling and that ligand binding is required for optimal activity.

ST8Sia II (STX) and ST8Sia IV (PST) are polysialic acid (polySia) syntheses that catalyze polySia formation of neural cell adhesion molecule (NCAM) in vivo and in vitro. It still remains unclear how these structurally similar enzymes act differently in vivo. In the present study, we performed the enzymatic characterization of ST8Sia II and IV; both ST8Sia II and IV have pH optima of 5.8-6.1 and have no requirement of metal ions. Because the pH dependence of ST8Sia II and IV enzyme activities and the pK profile of His residues are similar, we hypothesized that a histidine residue would be involved in their catalytic activity. There is a conserved His residue (cf. His(348) in ST8Sia II and His(331) in ST8Sia IV, respectively) within the sialyl motif VS in all sialyltransferase genes cloned to date. Mutant ST8Sia II and IV enzymes in which this His residue was changed to Lys showed no detectable enzyme activity, even though they were folded correctly and could bind to CDP-hexanolamine, suggesting the importance of the His residue for their catalytic activity. Next, the degrees of polymerization of polySia in NCAM catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger polySia formation of NCAM than ST8Sia II. We also analyzed the (auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II or IV itself was sialylated under conditions for polysialylation, the disialylated compound was the major product, even though polysialylated compounds were also observed. These results suggested that both ST8Sia II and IV catalyze polySia synthesis toward preferred acceptor substrates such as NCAM, whereas they mainly catalyze disialylation, similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.