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What is known and objective Oxaliplatin is a platinum drug used for treating digestive cancers that can lead to drug-induced thrombocytopenia (DITP). We report a case of oxaliplatin-induced anaphylaxis and DITP, complicated by idiosyncratic drug-induced liver injury (IDILI). Case summary A 46-year-old woman with rectal cancer developed anaphylaxis shortly after oxaliplatin administration (post-operative CapeOX), presenting with low platelet count (0.2 × 104 /µL) and elevated aspartate aminotransferase (1091 IU/L) and alanine aminotransferase (1010 IU/L) by day 10. Following 50 mg/d prednisolone administration from day 9, she left the hospital on day 36 after recovering. What is new and conclusion This is the first case report of oxaliplatin-induced IDILI and its effective treatment with steroids.

The influence of non-spectral matrix effects on the determination of twenty-two trace elements (Rb, Sr, Y, Cs, Ba, lanthanides, Pb, Th and U) in rock samples using ICP-MS was investigated. Three types of multi-element solutions were synthesised containing the twenty-two trace elements, In, Tl and ten major rock-forming elements with varying mass fractions mimicking the compositions of basalt, peridotite and dolomite. The synthetic solutions were conditioned to have dilution factors (DF) of 1000–10000. The extent of sensitivity suppression relative to the DF = 10000 solution became more significant for smaller DF solutions, which was not constant across different elements in a single solution but displayed general dependence on m/z. This indicates that at least two internal standards (e.g., In and Tl) are required for the correction of sensitivity variation. On the basis of the results, a new isotope dilution-internal standardisation method for the determination of twenty-two trace elements with ICP-MS was developed, in which the sensitivity variation was corrected by monitoring two enriched isotopes, 113In and 203Tl. This method, coupled with the quantitative correction of interference from oxides and hydroxides, achieved precise determination of twenty-two trace elements in some rock reference materials with reproducibilities of ±2% for basaltic to andesitic samples.

We conducted calibration tests and field deployments of a newly developed micro CTD data logger on Japanese sea bass Lateolabrax japonicus to measure fine-scale movement and reconstruct micro-salinity profiles of estuarine habitat. In June, July and November 2013 and 2014, Japanese sea bass were caught in the Ohno (33°12’N, 131°36’E) and Oita (33°16’N, 131°41’E) Rivers in Oita Prefecture, Japan. In seawater tanks, the measurement drift for salinity was negligible over a 2 wk period (n = 8). Moreover, in the estuary, the values of standard seawater measured at pre- and post-deployment of sensors on fish were consistent to the first decimal place; therefore, sensor drift was negligible (n = 4, duration: 53-129 h). In situ measurements of depth, temperature, and salinity (conductivity) successfully revealed the microenvironment, and showed that Japanese sea bass often ascended from deep seawater to shallow freshwater at night. In all fish, over 99% of these excursions were completed within 10 min. The maximum duration of these excursions was about 6 h. This might be an effective strategy for temporal utilization of hypo-osmotic environments for foraging without physiological and energetic costs. This is the first report to clearly validate the accuracy of salinity measurement without sensor drift in free-ranging fish. The device and methods presented here can be applied to other euryhaline fish.

Understanding the energy expenditure of top predators is important because a collapse in them could trigger trophic cascades through ecosystems. One such top predator, Japanese sea bass Lateolabrax japonicus, helps to balance the structure of the coastal marine ecosystem through predation. In this study, accelerometry was applied to the Japanese sea bass to estimate its energy expenditure under natural conditions. We attached accelerometers to five wild fish and measured metabolic rates such as the oxygen consumption rate ( $${\dot{\text{V}}\text{O}}_{ 2}$$ , mg O2 kg−1 min−1) using a swim tunnel. Body beat frequency (BBF) was measured using the accelerometer. The BBF was correlated with the tail beat frequency (TBF) by analyzing video recordings. $${\dot{\text{V}}\text{O}}_{ 2}$$ was related to swimming speed (U), TBF, and BBF. We estimated the standard (45.9 kJ kg−1 day−1) and active (124.0 kJ kg−1 day−1) metabolic rates when fish were not swimming and when they were swimming at the optimum swimming speed, respectively. The energy required to compensate for the above metabolic rates are between 83.3 and 275.6 kJ kg−1 day−1 using an assimilation efficiency of 0.7 and assuming that the growth rate is zero. These costs were comparable to consuming one or two prey fish per day (e.g., Japanese sardine: mean total length 155 ± SD 6 mm).

This chapter focuses on recent developments in the understanding of the structure and functionality relationships of soy proteins. Seed proteins play an important role in food consumption worldwide. Soy proteins have been utilized for many kinds of traditional foods. The use of soy protein products as functional ingredients is gaining increasing acceptance in food manufacturing from the standpoints of human nutrition and health. The seed proteins of legumes, including soybeans, are albumins and globulins. Globulins, the dominant storage proteins, account for about 50–90% of seed proteins. Storage globulins are grouped into two types according to their sedimentation coefficients: 7S globulins and 11S globulin. The ratio of 11S to 7S globulins varies among cultivars. It is about 0.5–1.7 in soybean. The 11S globulins, legumin-type proteins, are widely distributed in many legume and nonlegume seeds and are generally simple proteins, although there are some exceptions; for example, lupin 11S globulin is a glycosylated protein.

INTRODUCTIONSkeletal muscle metastases from carcinomas, especially to intercostal muscles, are rare. Most metastatic chest wall tumors from hepatocellular carcinoma (HCC) result from disseminations through needle tracts of intrahepatic HCC treatments.PRESENTATION OF CASEWe report the case of a 65-year-old man with chronic viral hepatitis B whose intrahepatic lesions were stabilized by repeated radiofrequency ablations and transcatheter arterial chemoembolization. Follow-up computed tomography demonstrated a well-enhanced mass in the right chest wall. Because α-fetoprotein and des-γ-carboxy prothrombin levels were elevated and no other tumors were detected, we diagnosed the mass as an extrahepatic metastasis from the HCC and resected it along with the surrounding ribs. There was no involvement of the bone, pleura, and lung.DISCUSSIONThe tumor was microscopically diagnosed as an intercostal muscle tumor metastasized from HCC, which has not been documented previously. The resection rate of extrahepatic tumors of HCC is low in literature. No other apparent extrahepatic recurrence has been observed for more than 20 months after the surgery.CONCLUSIONWe report the case of HCC patient who underwent surgical resection of an intercostal muscle tumor that had metastasized from HCC. Pathological examination of the tumor revealed the tumor cells in the blood vessels, and we speculate it hematogeneous metastasis.

We will consider the characteristics of LED backlights discussing merits and demerits of those characteristics which we will summarize. We have also evaluated combination with multi-primary color panels. By combining with multi-primary color panels, it will be possible to create new effects in Local Dimming technology.

症例は80歳，男性．嘔吐を主訴に近医受診．腸閉塞の診断で当院消化器内科に紹介され入院となったが，保存的加療で軽快しないため入院3日目に腹部CT検査を施行した．肝表面・下腹部にfree airを認め，S状結腸のhigh densityな物体と，これを中心とした小腸・結腸の壁肥厚がみられた．家人が6カ月前に義歯がなくなっていることに気付いていたが放置しており，同部位での義歯誤飲による腸管穿孔の診断で緊急手術施行した．穿孔部位はS状結腸の腸間膜対側で，穿孔部を含めたS状結腸を切除し，機能的端々吻合で再建した．経過は良好で術後22日目に退院した．高齢化社会を迎えるにあたり，本症例のように自覚なしに誤飲し，穿孔しても症状をうまく伝えられない患者が増えると考えられる．義歯誤飲後長期間を経た後にも腸管穿孔の原因となり得ることに留意すべきである．

症例は69歳，男性．耕運機と木の間に腹部を挟まれ腹痛のため搬入された．腹部造影CTで腸間膜出血と診断され開腹すると，回盲部より約50cm口側から，口側約70cmにわたる小腸間膜の断裂と動脈性の出血がみられ回腸を切除し器械による機能的端々吻合で再建した．術中血圧は100前後で安定していたが閉腹時心室細動となり蘇生術を要した．経口摂取に伴い発熱が持続し腹部CTで腸壁の壊死が疑われ改善しないため，術後43日目に再開腹すると吻合部縫合不全を認め腸管切除を行った．縫合不全部周囲の小腸は病理学的に虚血性腸炎と診断され，特に肛門側の腸壁は著明に肥厚し内腔がほとんどなかったため，縫合不全には吻合部の虚血のみならず肛門側腸管の狭窄による通過障害が関与したと考えられた．腹部外傷後遅発性小腸狭窄の報告は多いが小腸切除後に吻合部の虚血性狭窄を認めた報告はなく，初回手術時の心室細動が原因と推察され示唆に富む症例と考えられた．

We have studied the effect of fluorine substitution on growth mode and electronic structure of hexa-peri-hexabenzocoronene (C42H18, HBC). Fluorinated-HBC, 2,5,8,11,14,17-hexafluoro-hexa-peri-hexabenzocoronene (C42F6H12, 6F-HBC), grew epitaxially on a Cu(111) single-crystal surface, similar to HBC. Higher crystalline films with rather flat surfaces were obtained for 6F-HBC than for HBC due to the attractive intermolecular C−H···F−C interaction. Electron spectroscopy measurements of HBC and 6F-HBC films indicated that fluorination causes energy shifts of molecular orbitals to higher binding energies and increase of the work function. Thickness dependence spectroscopic measurements revealed that the interface dipole and the ionization potential are drastically increased in 6F-HBC/Cu(111), which is favorable for n-type electric conduction through the decrease of electron injection barrier to LUMO.

症例は72歳，女性．20年前に右卵巣破裂にて子宮全摘術および両側付属器切除術，10年前に骨盤腔の腫瘍（病理診断は滑膜肉腫）に対して腫瘍摘出術の既往がある．下腹部違和感を主訴に外来受診し，画像検査にて腹腔内腫瘍を認めたため，滑膜肉腫の再発と考え腫瘍摘出術を行った．しかし病理検査の詳細な検討によって今回の腫瘍組織は20年前の卵巣破裂の原因であった卵巣顆粒膜細胞腫と同様の組織であることが判明し，最終的には今回の腫瘍は卵巣顆粒膜細胞腫が10年後に腹腔内再発し，再発巣摘出後更に10年後に腹腔内に再再発したものと考えられた．婦人科疾患の既往がある患者の腹腔内腫瘍においてはたとえ年数がたっていても顆粒膜細胞腫の再発などの婦人科疾患に関連した病変も鑑別診断に入れるべきである．

症例は58歳，女性．右下腹部痛を主訴に他院を受診し，急性虫垂炎と診断され保存的加療を受けた．症状は消失し，退院となったが，しばらくして右下腹部の違和感をみとめたため，虫垂炎の再発を心配し，当院を受診した．外来にてフォローを行ったところ，症状は消失し，炎症反応も消失したが，虫垂の腫脹は改善しなかった．FDG-PETを行ったところ，陽性所見をみとめたため，手術を施行した．術中迅速病理診断で腺癌と診断され，また虫垂先端がS状結腸に穿通していたため，右半結腸切除（D3）+S状結腸部分切除を行った．病理検査において虫垂粘液嚢胞腺癌と診断された．虫垂炎の保存的治療後も悪性腫瘍の可能性を考えたフォローアップが必要であり，その際FDG-PETは有用な検査であると考えられた．

A fluorinated-graphene, 2,5,8,11,14,17-hexafluoro-hexa-peri-hexabenzocoronene (6F-HBC), has been synthesized. 6F-HBC was deposited by vacuum sublimation as an active layer in an organic field-effect transistor (OFET). The OFET with 6F-HBC performed as an n-type semiconductor, while that with hexa-peri-hexabenzocoronene (HBC) performed as a p-type semiconductor. The electron field-effect mobility and on/off ratio for 6F-HBC were 1.6 × 10 - 2 cm 2 / Vs and 10 4 , respectively. Hexafluoro-substituting reduces both the highest occupied molecular orbital and lowest unoccupied molecular orbital levels by 0.5 eV, which is suitable for electron injection from the electrode. 6F-HBC has a face-to-face structure which is a preferable crystal structure for carrier transport.

症例は42歳の女性で, 下腹部痛と嘔吐を主訴に当院救急外来を受診した. 腹部膨満と下腹部圧痛, 反跳痛を認めた. 腹部CTで便を多量に含んだ結腸の著明な拡張が下腹部正中に見られ, ダグラス窩に中等量の腹水を認めた. 上腸間膜動脈が上腸間膜静脈の右側に位置しており, 腸回転異常がベースにあるイレウスが示唆された. 全身状態が安定しており保存的加療でいったん症状が軽快したので下部消化管内視鏡検査を行うと, 上行結腸と思われる部位に捻れたような狭窄を認め, 造影で上行結腸の狭窄であると確認された. 経口摂取に伴い症状が再燃したため開腹すると, 回腸末端から上行結腸が大網の裂け目に陥入しておりこれを解除した. Nonro-tationの腸回転異常症で, これに伴う腸管の配置異常と固定不全が原因の内ヘルニアと考えられ, 腸回転異常症患者のイレウスとして典型的なものではなく, 示唆に富む症例であった.

Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in a wide variety of tumor cells, while it has no toxicity for the majority of normal cells.Therefore, TRAIL may be a suitable agent for anticancer therapy. We previously reported that a number of pancreatic cancer cell lines show resistance to TRAIL-induced apoptosis via overexpression of XIAP and FLIP. The present study was conducted to further examine TRAIL-based therapeutic strategies by aiming to restore functional apoptotic pathways in resistant pancreatic cancer cells. Methods In various pancreatic cancer cell lines, TRAIL-induced apoptosis was evaluated in the presence or absence of an XIAP-inhibitor (Smac peptide). Second, TRAIL-induced apoptosis was evaluated in TRAIL-resistant AsPC-1 cells with or without FLIP antisense. Third, the combined effect of Smac peptide and FLIP antisense was tested, and the activation of apoptosis-related caspases and poly (ADP-ribose) polymerase was evaluated. Finally, TRAIL-induced apoptosis was evaluated in the presence or absence of FLIP antisense and an XIAP inhibitor (embelin). Results Smac peptide enhanced TRAIL-induced apoptosis in a dose-dependent manner for several pancreatic cancer cell lines, but showed no effect on TRAIL-resistant AsPC-1 cells. Smac peptide alone had no influence on cell viability. TRAIL-induced apoptosis was restored in TRAIL-resistant AsPC-1 cells by exposure to FLIP antisense, which suppressed the expression of FLIP. The effect of TRAIL was augmented by the combination of FLIP antisense and Smac peptide. Similarly, TRAIL-induced apoptosis was restored by the combination of FLIP antisense and embelin. Activation of apoptotic caspases and cleavage of poly (ADP-ribose) polymerase was observed after sensitization of TRAIL-resistant pancreatic cancer cells. Conclusions Pancreatic cancer cells gain resistance to TRAIL-induced apoptosis via expression of the antiapoptotic proteins XIAP and FLIP. Smac peptide and FLIP antisense could restore the apoptotic effect of TRAIL. An XIAP inhibitor, embelin, enhanced the effect of TRAIL in the presence of FLIP antisense. These findings may provide useful information for the development of TRAIL-based therapeutic strategies by restoring functional apoptotic pathways in resistant pancreatic cancer cells. In addition, a low molecular weight XIAP inhibitor like embelin could be a lead compound for the development of effective XIAP inhibitors.

The effects of heat treatment on the formation of chlorophyll (Chl) derivatives and Chl-degrading enzyme activities in stored broccoli (Brassica oleracea L. Italica Group ‘Ryokutei’) florets were determined. The Chl a level barely changed during heat treatment, but each Chl a derivative level changed. Chlorophyllide (Chlide) a, pheophorbide (Pheide) a, C132-hydroxychlorophyll (C132-OHChl) a, Chl a′, an isomer of Chl a, and pheophytin (Phy) a were detected as a Chl derivative during heat treatment and during storage after treatment. Chlide a, Pheide a, and C132-OHChl a levels decreased during 2-h heat treatment (50°C), whereas Chl a′ and Phy a levels increased. Chl-degrading enzyme activities, in particular, Mg-dechelation activity, were effectively suppressed after 2 h of heat treatment. The content of Chls a and b in control broccoli florets decreased greatly during storage at 15°C, while the content in heat-treated broccoli florets hardly changed at all. In Chl derivatives, the Pheide a level in broccoli florets treated with or without heat treatment, especially the former, increased appreciably during storage. The Chlide a level in control florets decreased markedly during storage. On the other hand, the Chlide a level in heat-treated broccoli florets did not change during storage. Mg-dechelation activity in control florets markedly increased after 4 days of storage at 15°C, but the enhancement of the activity was suppressed by heat treatment. These findings suggest that Chl derivatives, especially Pheide a, are accumulated as intermediates in heat-treated broccoli florets, and Mg-dechelating action, in conjunction with that of chlorophyllase and Chl-degrading peroxidase, could be involved in Chl degradation in stored broccoli florets.

症例は76歳, 女性. 右側腹部痛と血便を主訴に救急搬送された. 血液検査上は白血球の上昇のみであったが, 腹部CT検査上肝左葉外側区域に門脈ガスを認め, 回腸末端から横行結腸にかけて全周性壁肥厚と周囲脂肪組織濃度の上昇をみた. 全身状態は安定しており腹部所見の増悪がないため保存的加療を選択した. 下痢はあるものの血便は止まり, 入院2日後の腹部CT検査で門脈ガスの著明な減少を認めた. 圧痛が右下腹部に限局したため, 入院後3週間目に下部消化管内視鏡検査を行ったところ上行結腸中央に狭窄部を認め, その口側の観察は不可能なため造影し, 盲腸, 回腸末端の著明な狭窄をみたので腹腔鏡補助下結腸切除術を行った. 術後経過は良好で術後9日目に退院した. 病理組織診断は, 潰瘍型虚血性腸炎による腸管狭窄であった. 門脈ガス血症を伴う虚血性腸炎に対する手術適応について検討した.

The chemokine receptor CXCR4 is highly expressed in tumor cells and plays an important role in tumor metastasis. The aim of this study was to develop a radiopharmaceutical for the imaging of CXCR4-expressing tumors in vivo. Based on structure-activity relationships, we designed a 14-residue peptidic CXCR4 inhibitor, Ac-TZ14011, as a precursor for radiolabeled peptides. For 111In-labeling, diethylenetriaminepentaacetic acid (DTPA) was attached to the side chain of d-Lys(8) which is distant from the residues indispensable for the antagonistic activity. In-DTPA-Ac-TZ14011 inhibited the binding of a natural ligand, stromal cell-derived factor-1alpha, to CXCR4 in a concentration-dependent manner with an IC50 of 7.9 nM (Ac-TZ14011: 1.2 nM). In biodistribution experiments, more 111In-DTPA-Ac-TZ14011 accumulated in the CXCR4-expressing tumor than in blood or muscle. Furthermore, the tumor-to-blood and tumor-to-muscle ratios were significantly reduced by coinjection of Ac-TZ14011, indicating a CXCR4-mediated accumulation in tumor. These findings suggested that 111In-DTPA-Ac-TZ14011 would be a potential agent for the imaging of CXCR4 expression in metastatic tumors in vivo.

Focal adhesion kinase (FAK) is a non-receptor, cytoplasmic protein tyrosine kinase that is involved in the regulation of cellular signaling, migration, apoptosis, and cell cycle progression. Previous reports have shown that FAK is expressed in various kinds of cancer tissues and cancer cell lines; however, no information is available about human pancreatic carcinoma specimens. Tissue such specimens were obtained from 50 patients who underwent pancreatic resection for pancreatic invasive ductal carcinoma at our institute from 1996 to 2002. Immunohistochemical analysis of FAK was performed in the resected specimens. Focal adhesion kinase expression in seven human pancreatic cancer cell lines was analyzed by reverse transcription polymerase chain reaction (PCR) analysis and Western blot analysis. Focal adhesion kinase expression was detected in 24 of 50 cases (48%). There was a statistically significant correlation between FAK expression and tumor size (P = 0.004), although FAK expression did not significantly correlate with other factors such as tumor histological grade, lymph node metastasis, distant metastasis, histological stage, and overall survival. Reverse transcription PCR analysis and Western blot analysis showed that FAK was expressed in all seven pancreatic cancer cell lines. Focal adhesion kinase expression was not directly related to clinicopathological factors except tumor size in pancreatic carcinoma. Focal adhesion kinase expression may not be a prognostic marker for pancreatic cancer patients.

Mammalian target of rapamycin (mTOR) is considered to be a major effector of cell growth and proliferation that controls protein synthesis through a large number of downstream targets. We investigated the expression of the phosphatidylinositol 3'-kinase (PI3K)/mTOR signaling pathway in human pancreatic cancer cells and tissues, and the in vivo antitumor effects of the mTOR inhibitor CCI-779 with/without gemcitabine in xenograft models of human pancreatic cancer. We found that the Akt, mTOR and p70 S6 kinase (S6K1) from the PI3K/mTOR signaling pathway were activated in all of the pancreatic cancer cell lines examined. When surgically resected tissue specimens of pancreatic ductal adenocarcinoma were examined, phosphorylation of Akt, mTOR and S6K1 was detected in 50, 55 and 65% of the specimens, respectively. Although CCI-779 had no additive or synergistic antiproliferative effect when combined with gemcitabine in vitro, it showed significant antitumor activity in the AsPC-1 subcutaneous xenograft model as both a single agent and in combination with gemictabine. Furthermore, in the Suit-2 peritoneal dissemination xenograft model, the combination of these 2 drugs achieved significantly better survival when compared with CCI-779 or gemcitabine alone. These results demonstrate promising activity of the mTOR inhibitor CCI-779 against human pancreatic cancer, and suggest that the inhibition of mTOR signaling can be exploited as a potentially tumor-selective therapeutic strategy.

Background Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutation of either of 2 tumor suppressor genes, TSC1 or TSC2, which encode hamartin and tuberin, respectively. Several studies have shown that tuberin functions independently of hamartin and inhibits signaling pathways via the mammalian target of rapamycin, a critical regulator of cell proliferation. Recent studies have revealed that the signaling pathways regulating the mammalian target of rapamycin such as Akt and S6K1 are frequently activated in pancreatic cancer. We hypothesized that tuberin might be involved in the proliferation and survival of pancreatic cancer cells. Methods We immunohistochemically examined the expression of tuberin in 42 pancreatic cancerous and noncancerous pancreatic tissue specimens using an antituberin antibody. The correlations between tuberin expression and various clinicopathologic features, including survival, were evaluated. Reverse transcriptase-polymerase chain reaction was performed to evaluate the level of tuberin expression in paired samples of pancreatic cancer and noncancerous tissue. Results Twenty-four of the 42 pancreatic cancer samples (57%) were negative for tuberin expression. The patients with tuberin-negative tumors had a significantly higher incidence of pT3 or pT4 disease (primary tumor extent by the TNM classification) than those with tuberin-positive tumors ( P = .024). Female patients had a significantly higher incidence of tuberin-positive tumors than male patients ( P = .014). The survival rate of the tuberin-positive group tended to be better than that of the tuberin-negative group, but there was no significant difference ( P = .4). Expression of TSC2 in cancer tissue was lower than in the corresponding noncancerous tissue for 7 of the 9 samples examined. Conclusions This study demonstrates that reduced expression of tuberin might be involved in the progression of pancreatic cancer. Accordingly, tuberin may provide a new therapeutic target in patients with this type of cancer.

Background Tumor necrosis factor–related, apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in a wide variety of tumor cells, but it does not cause toxicity in the majority of normal cells. Therefore, TRAIL could become a suitable agent for anticancer therapies. However, a number of tumor cell lines are known to be resistant to TRAIL-induced apoptosis. The purpose of this study was to determine the mechanisms of resistance to TRAIL in pancreatic cancer cells. Methods In human pancreatic cancer cell lines, the sensitivity to TRAIL-induced apoptosis was tested. The expression of TRAIL receptors (DR4, DR5, DcR1, and DcR2) and the expression of death signal–transducing proteins were investigated. In the TRAIL-resistant pancreatic cancer cells, effects of cycloheximide, a protein synthesis inhibitor, on death signal–transducing proteins were tested. Finally, the effects of the combined treatment with cycloheximide and TRAIL on the induction of apoptosis and on the expression of death signal–transducing proteins were examined. Results Pancreatic cancer cells responded to TRAIL in a different way. Resistant cell lines, AsPC-1, Suit-2, and CFPAC-1, expressed higher levels of FLIP-S protein, one of the splice variants of FLIP. Cycloheximide reduced the expression of FLIP in the resistant cells. Combined treatment with cycloheximide and TRAIL induced cleaved forms of caspases and simultaneously restored the sensitivity to TRAIL-induced apoptosis in the resistant cells. Conclusions Pancreatic cancer cells are resistant to TRAIL-induced apoptosis via strong expression of the anti-apoptotic protein FLIP-S. Suppression of FLIP-S by cycloheximide restored sensitivity to TRAIL-induced apoptosis in resistant cancer cells. These findings may provide useful information for the development of TRAIL-based therapeutic strategies aimed at restoring the functionality of apoptotic pathways in pancreatic cancer cells.

Objectives:Cadherins are cell surface glycoproteins that mediate Ca2+-dependent, homophilic cell-cell adhesion. The classic cadherins interact with either β-catenin or γ-catenin, which is bound to α-catenin that links the complex to the actin cytoskeleton. It has been reported that alteration in cad

To clarify the involvement of G protein in denatonium signal transduction, we carried out a whole-cell patch-clamp analysis with isolated taste cells in mice. Two different responses were observed by applying GDP-beta-S, a G-protein inhibitor. One response to denatonium was reduced by GDP-beta-S (G-protein-dependent), whereas the other was not affected (G-protein-independent). These different patterns were also observed by concurrently inhibiting the phospholipase C beta2 and phosphodiesterase pathways via G protein. These data suggest dual, G-protein-dependent and -independent mechanisms for denatonium. Moreover, the denatonium responses were not attenuated by singly inhibiting the phospholipase C beta2 or phosphodiesterase pathway, implying that both pathways were involved in G-protein-dependent transduction. In the G-protein-independent cells, the response was abolished by the depletion of calcium ions within the intracellular store. These results suggest that Ca2+ release from the intracellular store is an important factor. Our data demonstrate multiple transduction pathways for denatonium in mammalian taste cells.

The effect of protein and peptide addition on the oxidation of eicosapentaenoic acid ethyl ester (EPE) encapsulated by maltodextrin (MD) was investigated. The encapsulated lipid (powder lipid) was prepared in two steps, i.e., mixing of EPE with MD solutions (+/- protein and peptides) to produce emulsions and freeze-drying of the resultant emulsions. EPE oxidation in MD powder progressed more rapidly in the humid state [relative humidity (RH) = 70%] than in the dry state (RH = 10%). The addition of soy protein, soy peptide, and gelatin peptides improved the oxidation stability of EPE encapsulated by MD, and the inhibition of lipid oxidation by the protein and the peptides was more dramatic in the humid state. Especially, the oxidation of EPE was almost perfectly suppressed when the lipid was encapsulated with MD + soy peptide during storage in the humid state for 7 days. Several physical properties such as the lipid particle size of the emulsions, the fraction of nonencapsulated lipids, scanning electron microscopy images of powder lipids, and the mobility of the MD matrix were investigated to find the modification of encapsulation behavior by the addition of the protein and peptides, but no significant change was observed. On the other hand, the protein and peptides exhibited a strong radical scavenging activity in the powder systems as well as in the solution systems. These results suggest that a chemical mechanism such as radical scavenging ability plays an important role in the suppression of EPE oxidation in MD powder by soy proteins, soy peptides, and gelatin peptides.

Background Recent studies have demonstrated that transforming growth factor beta 1 (TGF-β1) expression is markedly enhanced in invasive ductal pancreatic adenocarcinomas, although the precise role of TGF-β1 in pancreatic carcinogenesis remains unclear. We analyzed TGF-β1 expression in pancreatic intraepithelial neoplasias (PanINs) and the effects of chronic TGF-β1 exposure on conditionally immortalized pancreatic epithelial (IMPE) cells. Methods Sixty-one PanIN lesions were immunohistochemically stained with a polyclonal rabbit antibody against human TGF-β1. Growth-inhibitory effects of short-term exposure to TGF-β1 were examined in IMPE cells. IMPE cells resistant to TGF-β1 (IMPE-Tr cells) were generated by continuous exposure to 1 ng/mL of TGF-β1 for more than 50 days. Phenotypic alterations of IMPE-Tr cells were examined by soft agar and Matrigel assay and Western blot analysis. IMPE and IMPE-Tr cells were injected subcutaneously into nude mice for an in vivo tumorigenicity assay. Results Forty-six percent of PanINs (28/61) were positive for TGF-β1 expression, whereas all the epithelia of normal pancreatic ducts were negative. TGF-β1 treatment showed the marked growth-inhibitory effects (>75%) in IMPE cells, whereas its effects were not observed in IMPE-Tr cells. IMPE-Tr cells were more spindle shaped compared with IMPE cells. In soft agar and Matrigel, formations of many colonies were observed in IMPE-Tr cells, but not in IMPE cells. Interestingly, the expression of p21WAF1/CIP1 was induced by short-term exposure to TGF-β1 in IMPE cells, whereas the induction was decreased in IMPE-Tr cells. All of the IMPE-Tr cell-injected mice (5/5) had subcutaneous tumors, although no tumor was found in the IMPE cell-injected mice. Conclusions TGF-β1 expression in PanINs and neoplastic transformation of IMPE cells by long-term exposure to TGF-β1 suggest that TGF-β1 may act as a tumor promoter in the early stage of pancreatic carcinogenesis.

Purpose: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors. Experimental Design: We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)β, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines. Results: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change. Conclusion: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

Pancreatic ductal adenocarcinomas arise through the accumulation of certain genetic alterations including ras, p16, p53, and DPC4. We found that activation of ras and inactivation of p53 could cooperatively induce in vitro tumorigenicity in conditionally immortalized pancreatic epithelial (IMPE) cells. IMPE cells were established from transgenic mice bearing a temperature-sensitive mutant SV40 Large T (LT) antigen. IMPE cells grew continuously under permissive conditions (33 degrees C with interferon-gamma), but rapidly suffered growth arrest under non-permissive conditions (39 degrees C without interferon-gamma). The cells showed strong expression of E-cadherin and beta-catenin as epithelial markers, and cytokeratin 19, a specific ductal cell marker. Cell proliferation under permissive conditions was associated with down-regulation of p21 expression through inactivation of p53 after overexpression of LT antigen. Intriguingly, the shift from the permissive to non-permissive culture conditions caused G2/M arrest of IMPE cells. Although the cells did not form colonies when cultured in soft agar without activation of ras, cells with ras activation via an adenovirus vector formed colonies under permissive conditions. These findings suggest that activation of ras and inactivation of p53 can cooperatively induce anchorage-independent growth of IMPE cells. This cell line might be useful for studying the processes involved in pancreatocarcinogenesis.

The effects of heat treatment on the interaction of lipid and zein in a dry powder system were investigated. Linolenic acid ethyl ester (LAE) was mixed with the zein powder. The glass transition temperature (T(g)) for the dry powder zein was shown to be approximately 107 degrees C by differential scanning calorimetry. The thermogram of the zein-LAE mixed powder showed an exothermic transition near the T(g) of zein. Fourier transform infrared (FT-IR) spectroscopy was used for detecting the structural changes in zein by heat treatment, that is, elevating the temperature from 25 to 160 degrees C. The heat treatment of the powdery zein with and without LAE caused increases in the alpha-helix, beta-turn, and beta-sheet, concomitant with decreases in the intermolecular hydrogen-bonded beta-sheet and random coil. Such changes in the secondary structure were more drastic for the powder with LAE. The heating of the zein-LAE mixed powder also caused decreases in the peaks originating from LAE in the FT-IR spectra. These results suggest that the heat treatment induced the interaction of the zein and LAE in the powdery system. The influence of heating on the antioxidative activity of dry powder zein was studied by measurements of the peroxide value. When zein-LAE mixed powder was heated before storage, the oxidation of LAE was inhibited for 7 days, whereas LAE was oxidized within 1 day in the absence of heat treatment.

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p

The inhibitory effects of polysaccharides on lipid oxidation in emulsions were investigated. Methyl linoleate was emulsified by β-casein or surfactants to prepare emulsions, and the oxidation was induced by an azo-compound or FeSO4 addition. The initial oxidation was followed by measuring the oxygen consumption in the emulsions for 30 min, and the extent of the lipid oxidation over a long time (∼48 h) was evaluated by the determination of the unoxidized lipid by gas chromatography and the thiobarbituric acid test. Peptide-bound polysaccharides, such as gum arabic and soluble soybean polysaccharides (SSPS), showed an ability to inhibit lipid oxidation, whereas maltodextrin and pullulan exhibited no inhibitory effects. Especially, SSPS almost perfectly suppressed the lipid oxidation from the initial to the late stages, irrespective of the emulsifiers and oxidation-inducing reagents. The mechanism whereby the peptide-bound polysaccharides inhibited the lipid oxidation in the emulsions is considered in terms of the chemical composition and other properties of the polysaccharides.

Many cancers are resistant to Fas-mediated apoptosis despite the expression of Fas. To investigate the mechanisms by which Fas signals are attenuated, we focused on decoy receptor 3 (DcR3). DcR3 is a soluble receptor against Fas ligand belonging to the tumor necrosis factor receptor superfamily and overexpresses in some forms of cancers. Exogenous DcR3 inhibits Fas-mediated apoptosis in Fas-sensitive Jurkat cells. In our study, we examined the expression and function of DcR3 in pancreatic cancers. TaqMan RT-PCR showed that DcR3 mRNA was highly expressed in pancreatic cancer cell lines (71%) and tissues (67%). Its expression significantly correlated with cancer invasion to veins. Western blotting showed that the DcR3 protein was produced and secreted in 4 of 6 cell lines. The protein expressions were compatible with the mRNA expression. Five of 7 pancreatic cancer cell lines became sensitive to agonistic anti-Fas antibody (CH-11) to various extents, without Fas upregulation, when exposed to CH-11 for 48 hr after pretreatment with IFNγ. Four of 7 pancreatic cancer cell lines were inhibited from growing, compared to control cells, when cocultured with membrane-bounded Fas ligand (mFasL) transfected lymphomas for 48 hr after pretreatment with IFNγ. DcR3 reduced this growth inhibition when added exogenously. Regression analysis showed that the DcR3 expression significantly correlated with the sensitivity to mFasL, and not to CH-11. These results suggest that DcR3 is highly expressed in many pancreatic cancers and endogenous DcR3 blocks the growth inhibition signals mediated by mFasL. DcR3 can be a candidate target molecule for the therapeutic intervention. © 2003 Wiley-Liss, Inc.

Ion-exchange HPLC systems using POROS 20 HQ and Mono Q HR 10/10 columns were applied to isolate glycinin subunits under denaturing conditions. Analyses by SDS-PAGE, N-terminal amino acid sequence, and sucrose density gradient centrifugation showed that the pseudoglycinin from the highly purified homo-subunit, A3B4, was reconstituted. The A3B4 pseudoglycinin was similar to the native glycinin with respect to molecular size, subunit structure, and secondary structure. The hexameric pseudoglycinin dissociated into trimers after long storage at pH 7.6.

It has been found that commercial water-soluble soybean polysaccharides (SSPS) can make cooked rice and noodles non-sticky and prevent rice grains and noodles from adhering to each other. We studied in detail the phenomenon of rice cooked with SSPS. We assumed that the phenomenon resulted from the interaction between SSPS and starch during cooking, and studied the effects of SSPS on the gelatinizing behavior of rice starch by using a Rapid-Visco-Analyzer. The addition of SSPS reduced the viscosity of the gelatinized starch. This lower final viscosity of the rice starch was more distinct from than that of potato starch. These results imply that the properties of SSPS in forming a non-sticky condition might result from a decrease in the viscosity of the gelatinized starch.

The effects of water activity (A(w)) and lipid addition on the secondary structure of powdery zein were investigated using Fourier transform infrared spectroscopy. Two fatty acid esters, i.e., the linolenic and eicosapentaenoic acid ethyl esters (LAE and EPE), were mixed with the zein powder. The powders were stored in the "dry" state (with silica gel) and the "humid" state (A(w) = 0.9). The powdery zein without the lipids was shown to have a high content of the intermolecular hydrogen-bonded beta-sheet in the "dry" state, indicating the presence of protein aggregates. An increase in A(w) induced a decrease in this beta-sheet, concomitant with increases in the alpha-helix and beta-turn structures. The addition of LAE caused decreases in the alpha-helix and intermolecular hydrogen-bonded beta-sheet of zein when the powder was stored in the "humid" state, suggesting the strong interaction of LAE and zein molecules. However, LAE did not affect the secondary structure of zein in the "dry" state. The addition of EPE hardly influenced the secondary structure of zein, irrespective of A(w). These results are discussed in relation to the antioxidative activity of zein in the powder system, which had studied previously.

Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.

A 4 polypeptide-containing (Shirotsurunoko and York) and A 4 polypeptide-lacking (Raiden and Suzuyutaka) soybean cultivars were used to investigate the heterogeneity of glycinin molecular species. Purification of glycinin by DEAE-Toyopearl column chromatography afforded molecular species eluting before the glycinin fraction. Analysis of this fraction by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and sucrose density gradient centrifugation indicated that this protein consisted of A 1 and A 2 polypeptides. The A 4 -containing soybean cultivars contained less of this protein than the A 4 -lacking soybean cultivars, as exhibited by the size of the early peak appearing during column chromatography. Alkaline PAGE and N-terminal amino acid sequence analysis confirmed that the A 1 - and A 2 -rich molecular species in the A 4 polypeptide-lacking cultivars consisted of the A 1a and A 2 polypeptides. Estimation of the molecular mass by gel permeation chromatography and multi-angle laser light scattering (GPC–MALLS) indicated that the A 1a - and A 2 -rich molecular species were similar to a monomer of glycinin.

Reactions of cucumber root lipoxygenase (CRLOX) were investigated using l -α-phosphatidylcholine dilinolenoyl (PCdiLNA) vesicles as substrates at pH 5.5. The progress of the enzyme reaction was followed by measuring oxygen consumption using an oxygen electrode. The CRLOX activity was drastically changed according to the time of sonication treatment for the preparation of substrate vesicles. Dynamic light scattering (DLS) data suggested that a large unilamellar vesicle was most susceptible to the reaction of CRLOX. The addition of free fatty acid (FFA) such as oleic acid to PCdiLNA vesicles enhanced the enzymatic activity effectively, whereas the addition of phosphatidic acid (PA) slightly suppressed the activity. In order to reveal the factors affecting the enzymatic activity, measurements of DLS, ζ-potential, 31P-NMR and electron microscopy observations were made for PCdiLNA vesicle dispersions, which contained FFA and PA. The particle size and ζ-potential of the vesicles were found to be closely related to the enzymatic reaction. The electron microscopy observations and 31P-NMR spectra indicated that FFA and PA differently modified the structure of PCdiLNA vesicles, thereby modulating the enzymatic activity. These results suggest that CRLOX strictly can recognize the physical states of a PCdiLNA assemblage when catalyzing oxygenation of the phospholipids.

Heat-induced gels were prepared from glycinins of various soybean cultivars at protein concentrations of 18 to 20%. Textural properties of the gels measured by a compression-decompression test were evaluated by the three-dimensional representation of the gels through factor analysis of the instrumental data and calculation of factor scores for each gel. Differences in gel texture were clearly observed among the soybean cultivars,with Shirotsurunoko gel being the most fracturable and Yamabe-A3 gel the most unfracturable. The most elastic was the gel from Hill and Matsuura gel exhibited the lowest elasticity. The existence of A4 polypeptide also contributed to the textural features of the gels. The gels of A4-containing cultivars were more unfracturable and less elastic compared to those of A4-lacking cultivars. Physical properties of the gels, gel network structure, and polypeptide composition of the glycinin were related each other to some extent. The compressibility which corresponded to the textural attribute of fracturability was related to regularity and/or pore size of network structure of the gels. The acidic polypeptide of A4 seemed to be responsible for whether the gel network was aggregate or strand type, thereby relating to the physical properties of compressibility and resiliency of the gels. The results obtained here suggest that polypeptide composition of the glycinin affects the properties of gel networks and thereby contributes to different physical and textural properties of the gels.

The effcts of deamidation of fragmentation on the antioxidative activity of α-zein were investigated individually. Heat treatment at 70°C in 70% ethanol-0.05M HCl caused deamidation of α-zein without fragmentation. Antioxidative activity of α-zein was improved by this deamidation. α-Zein with deamidation treatment for 12 hours protected 80% of docosahexaenoic acid ethyl ester from autoxidation until 7 days. Model peptide of α-zein had a lower antioxidative activity than α-zein. Fragmentation by N-btromosuccinimide decreased the antioxidative activity of α-zein.

Monosodium glutamate (MSG) elicits a unique taste in humans called 'umami' that is potentiated synergistically by the 5′-ribonucleotides IMP and GMP.Recent studies suggest that several mechanisms are involved in the transduction of umami taste. We have previously shown that MSG (10 mM) induced three different responses in mouse taste cells under whole-cell voltage-clamp in Na+ free Ringer solution; transient inward current, sustained inward current and outward current. Two, and occasionally all three types of currents, were observed in the same cell. We report here responses of mouse taste cells to the metabotropic glutamate receptor agonist, L-AP4 (1 mM), a mixture of MSG (10 mM) and IMP (0.5 mM), and a mixture of L-AP4 (1 mM) and IMP (0.5 mM) under whole-cell voltage-clamp. The mixture of MSG and IMP induced three different types of responses, similar to MSG alone. But the amplitudes of both the transient and sustained inward currents were larger than observed with MSG alone. The outward currents were similar in amplitude to those elicited by MSG alone. L-AP4 induced only outward currents in taste cells, while the mixture of L-AP4 and IMP induced both sustained inward currents and outward currents. These results suggest that umami taste reception may involve the coactivation of mGluR4 and one or more types of ionotropic glutamate receptors. Presumably the net transduction current resulting from activation of these receptors mediates the umami taste response, while an additional receptor or receptor site which is related to sustained inward current response, is involved in the synergistic effect of ribonucleotides.

We have previously reported that monosodium glutamate (MSG) stimulation elicited three types responses (transient inward current, sustained inward current and outward current) while L-AP4 (a potent mGluR4 agonist) evoked only outward currents in C57BL/6J mouse taste cells. The outward current responses to MSG and L-AP4 appeared to be mediated by metabotropic glutamate type 4 receptors (mGluR4). In this study, we examined whether agonists of ionotropic glutamate receptor agonists (NMDA, AMPA and ibotenic acid) also elicit responses in mouse taste cells. NMDA (1 mM) elicited transient inward currents, similar to those often observed with MSG, indicating the presence of NMDA receptor/channels that are permeable to Ca2+ ions and are activated by MSG in some taste cells. The sustained inward current response to MSG appeared to result from activation of a nonselective cation conductance, but it is not known if this response is coupled to ionotropic or metabotropic receptors. AMPA (1 mM) elicited small outward currents in all responding cells. Ibotenic acid, which produces a considerably stronger umami taste than MSG in humans, elicited two types of responses in isolated cells; transient inward currents and sustained inward current, suggesting that inward, as well as outward, currents are related to umami transduction. Also, we confirmed that MSG, AP4 (2 mM) and NMDA all can increase [Ca2+]i in taste cells. These results indicate that some cells have both metabotropic and ionotropic receptors, while other taste cells have only one or the other type of receptor.

The effects of fat crystallization induced by thermal treatment on the rheological properties of creams and physical phenomena at the oil droplet surfaces were investigated. Creams A or B were prepared from commercial proprietary fats A or B (vegetable oils with different triaclyglycerol composition) and aqueous solution containing proteins. Thermal treatment of the creams at the “critical temperatures” (temperatures inducing a small percentage of solid fats in the oil droplets) caused a rapid increase of solid fat contents in the following cooling process. The thermal treatment of cream B at the “critical temperature” caused an increase of viscosity of the cream and an increase of protein surface coverage during the subsequent cooling process. These results suggest that the oil droplet aggregation induced by the thermal treatment at the “critical temperature” and the subsequent cooling occurred via further adsorption of proteins. Electron spin resonance measurement demonstrated the dramatic reduction of fluidity of triacylglycerol molecules at the oil droplet surface in cream B during the cooling process after thermal treatment at temperatures below “critical”. Based on these results, we speculated on the mechanism for the destabilization of thermally treated creams during the cooling process.

Soybean lipoxygenase-3 reaction in emulsions stabilized by proteins and low molecular weight surfactants was investigated. High lipoxygenase activity was observed in the emulsions stabilized by proteins, whereas the reaction rates were very low in the surfactant-stabilized emulsions. The specificity of hydroperoxide isomers produced by lipoxygenase reaction was independent of the type of emulsifier. The effects of neutral salts and phospholipids addition on lipoxygenase reaction in protein-stabilized emulsions were also studied. Lipoxygenase activity decreased with increasing concentration of neutral salts. The efficiency of inhibition of lipoxygenase activity by salts was related to the activity of salts to decrease ζ-potential at the surfaces of substrate oil droplets. With respect to the effect of addition of phospholipids, it was shown that the lowering of interfacial tension and the fall in ζ-potential at the oil droplet surfaces were responsible for the inhibition of lipoxygenase activity.

Polymer formation of α-lactalbumin (α-LA) caused by the incubation with transglutaminases from guinea pig liver (GTGase) and Streptoverticillium mobaraense (MTGase) in the presence of dithiothreitol (DTT) was analyzed by gel permeation chromatography combined with a multi-angle laser light scattering detector (GPC–MALLS) system using the elution buffer containing sodium dodecyl sulphate. Polymer formation by the two enzymes was carried out in the absence as well as in the presence of Ca 2+ , because we had found previously that not only Ca 2+ -independent MTGase but also Ca 2+ -dependent GTGase could cross-link α-LA in the absence of Ca 2+ . In the Ca 2+ -containing system, the incubation with TGases produced precipitates in 30 min, thereby inhibiting the growth of polymer. The maximum sizes of polymers were constant from 30 to 60 min, i.e. 3.0×10 6 and 7.0×10 6 , for GTGase-catalyzed and MTGase-catalyzed cases, respectively. On the contrary, the large polymers (>1×10 7 ) were formed by the incubation with the enzymes in the Ca 2+ -free system. These results suggest that TGases could not make full use of catalytic activity in the presence of Ca 2+ because of the difficult access of the enzymes to the targeting sites in aggregates formed via electrostatic interactions. Irrespective of the presence or absence of Ca 2+ in the reaction mixtures, GTGase showed the high cross-linking activity at the early stages of incubation, namely, the more rapid decrease of monomer α-LA and the more rapid growth of large polymers. In the late stages (prolonged incubation), however, reverse results were observed with respect to the efficiency in polymer formation, i.e. MTGase was capable of forming larger polymers than GTGase.