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Fish consumption has both the risk of methylmercury (MeHg) poisoning and the benefit of obtaining n-3 polyunsaturated fatty acids (n-3 PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). However, the cellular interaction between MeHg and PUFAs remains unknown. Therefore, the aim of this study was to investigate the effects of MeHg and n-3 PUFA exposure on mouse embryonic fibroblasts (MEFs). The results showed that EPA had a negligible effect on MeHg-induced cell death, whereas DHA promoted it. Thiobarbituric acid reactive substance (TBARS) concentrations in cells exposed to DHA and MeHg were higher than in those exposed to EPA and MeHg. Treatment with DHA and MeHg markedly induced the expression of endoplasmic reticulum (ER) stress (CHOP and DNAJB9) and Nrf2 target gene (p62 and HMOX-1) mRNA levels. Unexpectedly, EPA supplementation in addition to DHA and MeHg attenuated DHA- and MeHg-induced cell death and suppressed ER stress and expression of Nrf2 target genes. Our results revealed a differential impact of DHA and EPA on MeHg-induced cell death, and combined treatment with DHA and EPA along with MeHg attenuated MeHg-induced toxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Methylmercury (MeHg) is a highly toxic pollutant, and is considered hazardous to human health. In our previous study, we found that MeHg induces autophagy and that Atg5-dependent autophagy plays a protective role against MeHg toxicity. To further characterize the role of autophagy in MeHg-induced toxicity, we examined the impact of autophagy on microtubules and nuclei under MeHg exposure using Atg5KO mouse embryonic fibroblasts (MEFs). Low concentrations of MeHg induced a decrease in α-tubulin and acetylated-tubulin in both wild-type and Atg5KO cells. While α-tubulin acetylation was promoted by treatment with tubacin, a selective inhibitor of histone deacetylase 6, MeHg treatment inhibits the increase of tubacin-induced acetylated-tubulin. However, similar effects were observed for treatment with either tubacin or tubacin + MeHg in wild-type and Atg5KO cells. We also found a significant increase in the number of multinuclear cells upon MeHg exposure in Atg5KO MEFs compared to wild-type MEFs. In addition, DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), markedly increased in Atg5KO MEFs compared to wild-type MEFs. Our results therefore suggest that autophagy is not a simple elimination pathway of MeHg-induced damaged proteins, but that it also plays a protective role in the context of MeHg-associated DSBs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Umbilical cord blood-derived platelet-rich plasma (UCB-PRP) containing growth factors has attracted attention as a biomaterial useful for regenerative medicine. The osteoblastic differentiation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) can be induced by UCB-PRP., Materials and Methods: Nine samples of UC and UCB were used to conduct an in vitro study that determined the contents of three growth factors (i.e., platelet-derived growth factor, transforming growth factor β-1, and vascular endothelial growth factor) and that examined, by staining with Alizarin red, their ability to induce the osteoblastic differentiation of UC-MSCs at the baseline, 3 months, and 3 years of cryopreservation., Results: The contents of growth factors in cryopreserved UCB-PRP were markedly elevated compared to those found in UCB at baseline. The samples of UCB that were added with cryopreserved UCB-PRP and those with bone morphogenetic protein-2 were stained granularly with Alizarin red, thus indicating the presence of calcium. The samples of UCB that were not added with UCB-PRP were not stained with Alizarin red. The above-mentioned contents and ability were maintained at 3 years of cryopreservation. Cryopreserved UCB-PRP possibly and advantageously induced the osteoblastic differentiation of UC-MSCs., Conclusion: The potential clinical application of cryopreserved UCB-PRP to regenerative medicine was suggested., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Methylmercury (MeHg) is one of the most toxic environmental pollutants, presenting a serious health hazard worldwide. In this study, we examined the potential of derivatives of oleanolic acid (OA), such as OA 3-glucoside, OA 28-glucoside, and OA 3,28-diglucoside, to mitigate MeHg toxicity in vitro and in vivo. We found that OA 3-glucoside suppressed the cellular MeHg uptake by 63.4% compared with that of the control and improved the cell viability from 75.4% to 107.9% upon exposure to cytotoxic MeHg in Caco-2 cells. To verify the anti-MeHg activity of OA 3-glucoside, mice were orally administered MeHg (0, 1.0, or 5.0 mg kg -1 ·d -1 ), with or without OA 3-glucoside, and then mercury accumulation was measured in various organs of the mice. The mice co-treated with MeHg and OA 3-glucoside showed significantly lower mercury content in organs such as the cerebrum, cerebellum, liver, kidney, and spleen, with 83.1%, 68.7%, 71.7%, 82.1%, and 18.2% of those in the OA 3-glucoside-untreated group, respectively. This suggested OA 3-glucoside had the potential as an anti-MeHg compound, owing to its ability to suppress the distribution of MeHg into organs. Supporting this hypothesis, the mice treated with MeHg and OA 3-glucoside showed a tendency to survive one day longer than the control mice. Our findings suggest OA 3-glucoside administration alleviates the toxicity of MeHg by suppressing MeHg accumulation in organs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cancer cells enhance autophagic activity as a survival measure against metabolic and therapeutic stresses. The inhibition of autophagy may represent a valuable sensitizing target for cancer treatment. Recently, we examined the ability of various cytochalasins to inhibit autophagy and demonstrated the potent inhibitory effect of cytochalasin E (CE) on autophagic flux. The present study was conducted to investigate whether CE inhibited autophagosome-lysosome fusion, and to determine whether CE enhanced chemotherapy-induced cell death. Cell exposure to CE led to the accumulation of microtubule-associated protein light chain 3-II (LC3-II) and sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62) in a dose- and time-dependent manner. Cells treated with CE exhibited distinct formation of p62-positive structures on lysosome-associated membrane protein 2 (LAMP2)-positive lysosomal vesicles. CE treatment following serum starvation robustly reduced cell viability and increased expression levels of LC3-II and p62, in comparison to those of cells treated with CE alone. Furthermore, combination treatment with CE and bortezomib, an inhibitor of the 26S proteasome, showed a synergistic effect in targeting human lung cancer A549 cells. Altogether, our results demonstrated that CE treatment inhibited autophagosome-lysosome fusion, and this activity, in part, augmented bortezomib-induced cell death. Therefore, we concluded that CE may be a potentially effective therapeutic agent against lung cancer, especially in a combination therapy with proteasome inhibitors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Autophagy is a cell survival process that represents a therapeutic target in cancer treatment. Many types of cytochalasins have been identified and some of them have been reported to interfere with the formation of the autophagosome, although only limited data are available to assess their potential effects. Therefore, in this study, we examined the effects of cytochalasins and structurally related compounds on cell survival and the regulation of autophagy in human lung A549 adenocarcinoma cells. Cytochalasin D (CD) and cytochalasin E (CE) prominently inhibited the growth of A549 cells in a dose-dependent manner. Following treatment with CE, F-actin filaments were disrupted, and the proportion of binucleated cells increased, whereas no such effects were observed with the seven other cytochalasins tested. We found that cytochalasin H (CH), CD, and especially CE could induce the up-regulation of autophagy-related protein (LC3-II) and SQSTM1/p62. Using bafilomycin A 1 , we demonstrated that CD, CE, and CH inhibited autophagosome turnover, resulting in a dysfunctional autophagic process. The results of this study reveal that CE is the most potent cytochalasin in terms of its ability to induce cell death and inhibit autophagy. CE may therefore be an effective therapeutic agent against lung cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Methylmercury (MeHg) is a widespread environmental pollutant and causes a serious hazard to health worldwide. However, molecular mechanisms underlying MeHg toxicity remain elusive. We show that MeHg reduced mouse embryonic fibroblast (MEF) viability in a dose-dependent manner. Furthermore, MeHg treatment increased levels of autophagy markers LC3-II and p62, possibly by acting on the MAPKs signaling pathway in several cell types. MeHg exposure elevated the number of LC3 puncta in stable GFP-LC3 MEFs and the number of autophagic vacuoles. The accumulation of LC3-II and p62 increased further when complementing MeHg with autophagy inhibitor, chloroquine. Moreover, we found that autophagy-related gene 5-deficient (Atg5 -/- ) MEFs exhibited higher sensitivity and higher levels of p62 compared to their wild-type counterparts following MeHg exposure. This suggested that p62 was upregulated at the transcription level by MeHg and degraded by Atg5-dependent autophagy. Our data demonstrate that MeHg exposure promotes autophagy, and Atg5-dependent autophagy serves to protect cells from MeHg cytotoxicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Management of the protruding/deviated premaxilla in patients with complete bilateral cleft lip and palate is a challenging problem for surgeons and orthodontists. Various passive and active methods have been developed for the presurgical orthopaedic treatment. However, most of these treatments are complicated and laborious for the patient's parents and clinicians. Here, we describe our original active intraoral appliance comprising two components, that is, the premaxillary and palatine process plates, connected with two elastic chains, and we assess its therapeutic efficacy., Patients and Methods: We retrospectively evaluated 15 patients treated using this appliance during 2006-2012, followed up for an average of 60.3 months (range, 18-97 months). We analysed the cleft widths and maxillary size, obtained pretreatment, post-treatment and pre-palatoplasty., Results: Cleft widths and premaxillary protrusion were significantly decreased post treatment; however, the transverse dimensions were not significantly altered. In all cases, the protruding/deviated premaxilla was set into a suitable position within 1 month, and we could perform one-stage cheiloplasty using the modified Mulliken method with low tension., Conclusion: Our appliance is technically simple to use, less invasive to the skin and bone and cost-effective, with reliable and predictable outcomes. In the follow-up period, we observed no detrimental growth of the maxilla or dentition. Therefore, we consider our appliance to be useful for application in presurgical orthopaedic treatments of complete bilateral cleft lip and palate., (Copyright © 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background and Aim: For patients with a wide, complete, unilateral cleft lip and palate, pre-surgical maxillary orthodontic treatments have been used to reduce the alveolar gap before cheiloplasty. However, most of these treatments are complicated and laborious for patients and for medical professionals. Thus, we developed an original pre-surgical orthodontic device made with two separate acrylic resin plates connected with a spring-shaped β-titanium wire (β-TW). When the device was applied on the palate, each segment of the maxilla was automatically aligned for our target formation with the elastic force of β-titanium alloy. This study aimed to evaluate the efficacy of the new device and the size of the maxilla in comparison with the conventional Hotz procedures., Patients and Methods: A total of 47 patients with a wide unilateral cleft lip and palate were retrospectively evaluated; 33 patients were treated with our new device (β-TW plate group) and 14 were treated with a Hotz plate (HP group). We evaluated the alveolar gap reduction and the size of the maxilla between the two groups, obtaining intraoral maxillary impressions at birth, at 3 months and 1 year., Results: The width of the alveolar gap in the β-TW plate group was significantly reduced compared with that in the HP group 1 month after the treatment (p < 0.001). The alveolar gap reduction continued until the age of 1 year (p = 0.02). By contrast, no significant difference in the maxillary size was observed between the two groups at any examination period., Conclusion: Our treatment protocol using the β-TW plate was not only easy and simple to apply but it was also cost-effective, with highly predictable outcomes. Moreover, it provided the ideal alveolar cleft reduction without detrimental collapse of the alveolar segments. Therefore, we consider our β-TW plate device to be useful for application in pre-surgical orthodontic treatments., (Copyright © 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Listeria monocytogenes is a foodborne pathogen that causes the potentially life-threatening illness listeriosis. Previously, a few clones of L. monocytogenes persisting in a cold-smoked fish processing plant were isolated from the plant's products continuously. To evaluate the role of biofilms in the persistence of L. monocytogenes strains specific to this plant, the abilities of the persistent strain (PS) and transient strain (TS) of L. monocytogenes found in this plant to form biofilms were compared, as was resistance to the sanitizing effects of benzalkonium chloride (BC). The PS produced more biofilm than the TS in 48 h. The half-maximal effective concentration (EC50), the BC concentration at which the ATP bioluminescence of each bacterial strain decreased by 50 % relative to its maximum activity, was about 150-fold higher in the PS than in the TS. In contrast, when these values were measured in organisms in a planktonic state, the EC50 of the PS was only 2.2-fold higher than that of the TS. Extracellular polymeric substances (EPS) were extracted from biofilms, and the glucose content of these biofilms was determined with the phenol-sulfuric acid method to estimate the quantity of EPS. The total amount of EPS in the PS biofilm was higher than that in the TS biofilm. These findings suggest that the PS produces greater amounts of biofilm and EPS than the TS, which results in greater resistance of the PS to disinfectants. The persistence of the strain in the fish processing plant might be attributable to these properties.

Background & Aims: The gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) often depicts non-hypervascular hypointense hepatic nodules during the hepatobiliary phase in patients with hepatocellular carcinoma (HCC). It is unclear whether the presence of these nodules is associated with HCC recurrence after hepatectomy. We conducted a prospective observational study to investigate the impact of the presence of non-hypervascular hypointense hepatic nodules on the hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI on the recurrence of HCC after hepatectomy., Methods: A total of 77 patients who underwent hepatectomy for primary, non-recurrent, hypervascular HCC were prospectively followed up after hepatectomy. Post-operative recurrence rates were compared according to the presence of non-hypervascular hypointense nodules on preoperative Gd-EOB-DTPA-enhanced MRI., Results: Recurrence rates after hepatectomy were higher in patients with non-hypervascular hypointense nodules (risk ratio 1.9396 [1.3615-2.7222]) and the presence of non-hypervascular hypointense nodules was an independent factor associated with postoperative recurrence (risk ratio 2.1767 [1.5089-3.1105]) along with HCC differentiation and portal vein invasion. While no differences were found in the rate of intrahepatic metastasis recurrence based on the preoperative presence of non-hypervascular hypointense hepatic nodules, the rate of multicentric recurrence was significantly higher in patients with preoperative non-hypervascular hypointense hepatic nodules., Conclusions: Patients with preoperative non-hypervascular hypointense hepatic nodules detected during the hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI are at higher risk of HCC recurrence after hepatectomy, mainly due to multicentric recurrence., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Gentle ovarian stimulation protocols, such as 'mini-IVF', have several potential advantages over conventional IVF protocols, including less medication and fewer injections, producing fewer eggs, but eggs of higher quality. The particular 'mild' stimulation protocol called 'mini-IVF' is described. This protocol requires a reliable and cheap method for embryo cryopreservation such as vitrification, because of the negative impact of clomiphene citrate on the endometrium and since cryopreserved embryo transfers with this protocol have yielded much higher pregnancy rates than fresh transfers. In this series, patients were not denied treatment based on their day-3 FSH value or ovarian reserve. Yet very acceptable pregnancy rates were achieved (20% for fresh embryo transfers and 41% for cryopreserved embryo transfers). These results strengthen the argument for a mini-IVF protocol and vitrification as an alternative to standard conventional IVF stimulation protocols. Now a randomized control trial with cryopreserved single-embryo transfer is required., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background/aims: This study sought to identify the independent risk factors involved in the development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV) infection who have normal alanine aminotransferase (ALT) levels., Methods: A total of 519 patients with average ALT integration values less than or equal to 40 IU/L over 10 years were included. Baseline ultrasound was done in all patients and 68 patients underwent liver biopsy at the start of this study. Factors associated with the cumulative incidence of HCC were determined., Results: HCC occurred in 48 of 519 patients (9.2%). The following factors were significantly associated with the incidence of HCC: age>65 years (adjusted hazard ratio: 2.006 [95% confidence interval: 1.078-3.733]), ALT>20 IU/L (6.242 [1.499-25.987]), platelet count<15.0x10(4)/m(3) (2.675 [1.407-5.085]), total bilirubin>1.2mg/dL (2.798 [1.257-6.228]), ALP>338 IU/L (2.486 [1.327-4.657]), and total albumin<3.5g/dl (2.707 [1.177-6.223]). The 5- and 10-year cumulative incidences of HCC were 4.4% and 26.5% in patients with ALT>20 IU/L and platelet count<15.0x10(4)/m(3), respectively., Conclusions: High ALT level and low platelet count are closely associated with the development of hepatocarcinogenesis. Therefore, individuals within this group are candidates for antiviral therapy.

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.

Background/aims: Blood concentration levels of alpha-fetoprotein like the Lens culinaris agglutinin-reactive fraction (AFP-L3) are a useful marker for predicting the long-term prognosis of hepatocellular carcinoma. This study investigated the relationship between serum AFP-L3 and various imaging modalities., Methods: Sixty-three patients with small hepatocellular carcinomas < or = 2 cm in diameter were studied. Serum AFP-L3 concentrations were measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting and expressed as % AFP-L3 (the percent of AFP-L3 as total AFP). A clinical "cutoff level" of 10% was used in this study to indicate the presence of hepatocellular carcinoma. Selective hepatic intraarterial digital subtraction angiography (DSA), ultrasonographic angiography with carbon dioxide microbubbles (USAG), and computed tomography during arterial portography (CTAP) were performed to evaluate the hemodynamics of hepatic nodules., Results: Fourteen (22.2%) of the 63 patients were positive for % AFP-L3. The % AFP-L3 levels (n=45, 4.4%) of patients with hypervascular tumors were significantly higher than those (n=15, 0.0%) of patients with isovascular or hypovascular tumors as determined by USAG (p=0.0061). The % AFP-L3 levels (n=53, 4.4%) of patients with a negative portal blood supply were significantly higher than the % AFP-L3 levels (n=7, 0.0%) of patients with a positive portal blood supply as determined by CTAP (p=0.0140). The % AFP-L3 levels of patients with tumors with a long doubling time (DT) were significantly lower than for patients with tumors with a short DT (p=0.0176)., Conclusion: AFP-L3 is a positive indicator which may be more specific for small advanced hepatocellular carcinoma.

Background/aims: We estimated the significance of the vascularity of small hepatocellular carcinoma (HCC) as a predictor of long-term prognosis in patients treated with percutaneous ethanol injection therapy (PEIT/PEI)., Methods: Fifty-four patients who have been followed-up in our hospital and who had HCC less than 20 mm in diameter were observed for 199 to 2074 days. Hepatic angiography (digital subtraction angiography; DSA and ultrasound angiography with intraarterial CO2 microbubbles; USAG) was performed before treatment in all cases, and the vascularity of the tumor was clinically evaluated. The survival rate was analyzed according to this vascularity., Results: Of the 54 tumors, 24 had tumor stain on DSA, while 30 did not, and 38 showed enhancement on USAG, while 16 did not. The 3- and 5-year survival rates were 48.7 and 34.1% and 89.7 and 69.7% of patients with negative staining HCC (p = 0.0723). The rates were 48.6 and 36.7%, respectively, of patients with positive enhancement HCC on USAG, and both rates were 85.7% of patients with negative enhancement HCC (p = 0.0231)., Conclusions: Tumor vascularity will play a role in the long-term prognosis of these patients with small HCC when they are treated with PEIT/PEI.

Background/aims: We evaluated the significance of the quasispecies nature of HCV as a predictor of the response to alpha interferon therapy in patients with chronic hepatitis C., Methods: Natural alpha interferon was administered in 62 patients for 24 weeks (daily for 2 weeks, then three times weekly for 22 weeks) and factors were analyzed that could affect the response. HCV subtype, HCV RNA concentrations and the number of HCV quasispecies were evaluated before treatment. HCV RNA concentrations were measured by branched DNA probe assay. The number of HCV quasispecies was measured by fluorescence single-strand conformation polymorphism analysis., Results: The HCV RNA concentration (p < 0.0001), HCV subtype (p = 0.0076), and the number of HCV quasispecies (p = 0.0024) were significantly associated with a complete response. Multivariate analyses showed that the number of HCV quasispecies was an independent predictor of the disappearance of HCV RNA during the administration of alpha interferon, but did not predict a relapse after its completion. Pretreatment concentration of HCV RNA was the only factor that was related to a long-term disappearance of HCV RNA., Conclusions: The number of HCV quasispecies was significantly related to the response to alpha interferon early in its administration. The pretreatment concentration of HCV RNA was mainly related to a relapse following completion of treatment.

Two P-450s from adult pig testis were purified to specific contents of 11.2 and 12.0 nmol P-450/mg protein and shown to have minimum molecular weights of 45,000 and 46,000, respectively. The absorption spectra were typical of P-450s. The P-450s were separated from the two fractions by CM-C50 Sephadex column chromatography. One P-450 (M(r) = 45000) exhibited 17,20-lyase activity of 6.78 nmol of androstenedione/min/nmol P-450, on incubation with 17 alpha-hydroxyprogesterone as a substrate. The other P-450 (M(r) = 46,000) exhibited no 17,20-lyase activity. Both P-450s exhibited 17 alpha-hydroxylase activity that amounted to 10 nmol of steroid products. Accordingly, the two molecular species of P-450 are thus markedly different in 17,20-lyase activity toward 17 alpha-hydroxyprogesterone.

Lactobacillus helveticus strain TN-4, a spontaneous mutant strain of Lactobacillus helveticus TY1-2, produced an exocellular polysaccharide from reconstituted skim milk. On the basis of the results of methylation analysis, enzymatic digestion, mild Smith degradation, mild acid hydrolysis, acetolysis, and 1D and 2D 1H-NMR spectroscopy, it was concluded that the polysaccharide has a D-galactofuranose containing hexasaccharide repeating unit with the following structure: [formula see text]

Lactobacillus helveticus TY1-2 produced an exocellular polysaccharide when it was cultured in reconstituted skim milk. This polysaccharide is a high molecular weight heteropolymer of D-glucopyranosyl, D-galactopyranosyl, and 2-acetamido-2-deoxy-D-glucopyranosyl residues in the molar ratio 3.0:2.8:0.9. The primary structure of the polysaccharide was shown by glycose analysis, methylation analysis, Smith degradation, and NMR spectroscopy to be composed of branched heptasaccharide repeating units having the following structure: [formula: see text]

A cytochrome P-450 from adult pig testicular microsomes was purified to a specific content of 12 nmol P-450/mg protein. P-450 has a minimum molecular weight of 46000 +/- 1000, as judged on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Adult testicular P-450 is prepared in the low-spin form with an absorbance maximum at 417 nm. The substrate-induced difference spectrum with progesterone is a typical I difference spectrum. P-450 was incorporated into liposomal membranes composed of phosphatidylcholine, and 17 alpha-hydroxylase activity was shown to amount to 15.5 nmol product/min/nmol of P-450. Furthermore, testicular cytochrome b 5 did not increase the 17 alpha-hydroxylase activity, and the activity was largely inhibited by the addition of sodium cholate, Emulgen 913 and testicular lipid.

Branched polysaccharides having D-mannopyranose, D-arabinofuranose, or oligo-D-arabinofuranose side-chains were synthesized by the reaction of 3,4,6-tri-O-acetyl-(1,2-O-ethylorthoacetyl)-beta-D-mannopyranose, 3,5-di-O-benzoyl-(1,2-O-ethylorthobenzoyl)-beta-D-arabinofuranose, or 3-O-benzoyl-(1,2,5-O-orthobenzoyl)-beta-D-arabinofuranose with cellulose acetate or curdlan acetate, followed by desterification. The structure and antitumor activity of the water-soluble portion of the polysaccharides thus obtained were investigated. Polysaccharides synthesized from (1----3)-beta-D-glucan as the main chain with oligo-D-arabinofuranose side-chains exhibited high antitumor activity.

The p-aminophenyl beta-glycoside of 6-O-alpha-D-xylopyranosyl-D-glucopyranose (isoprimeverose), the disaccharide unit of plant xyloglucan, was coupled to bovine serum albumin, and the resulting glycoconjugate was used as an immunogen for the immunization of a rabbit. The immunochemical specificities of the rabbit antiserum raised against the glycoconjugate were characterized by immunodiffusion, quantitative precipitation, and hapten inhibition. After removal of anti-bovine serum albumin antibodies, the antiserum exhibited a specificity for the introduced disaccharide unit of the artificial antigen. The antibody-combining site was also shown to recognize the aglycon portion of the introduced hapten. The antiserum interacted with some xyloglucans, such as those from tamarind seed and the cell wall of pea stem. beta-Isoprimeverose and alpha-D-xylopyranosides were good inhibitors of the xyloglucan-antibody precipitation system, indicating that the antibodies recognize the beta-isoprimeverose unit of the xyloglucan.

p-Nitrophenyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside was condensed with 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide, the product deprotected, and the disaccharide glycoside converted into p-trifluoroacetamidophenyl 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta- D-glucopyranoside. p-Nitrophenyl 3-O-benzoyl-4,6-di-O-benzylidene-alpha-D-mannopyranoside was condensed with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl bromide, and the product was deprotected, to yield p-nitrophenyl 2-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D-mannopyranoside. p-Nitrophenyl 2-acetamido-3,4-di-O-benzoyl-2-deoxy-beta-D-glucopyranoside was condensed with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide, and, after reduction, trifluoroacetylation, and deprotection, p-trifluoroacetamidophenyl 2-acetamido-2-deoxy-6-O-alpha-L-fucopyranosyl-beta-D-glucopyranoside was obtained.

A (1--3)-beta-D-glucan branched by O-6 substitution (FCAP), obtained from the cold-alkali extract of the fruiting body of V. volvacea, exhibited potent growth-inhibitory activity against implanted tumors in mice. It contained protein and appeared to be heterogeneous. Fractionation by DEAE-Toyopearl column chromatography yielded an unbound, protein-free glucan fraction ([alpha]D - 30 degrees in M NaOH, mol. wt. 1.5-2 x 10(6)), which showed the highest antitumor activity. The polysaccharide had a moderately branched structure, consisting of a backbone chain of beta-(1--3)-linked-D-glucose residues, one out of five or six being substituted at O-6 with single glucosyl of beta-(1--6)-linked diglucosyl groups. Digestion of the glucan with exo-(1--3)-beta-D-glucanase yielded glucose and ++gentiobiose (molar ratio, 8:2:1.0), and a highly branched (d.b. 1/3), degraded glucan. Digestion with endo-(1--3)-beta-D-glucanase gave D-glucose, laminarabiose, a trisaccharide beta-D-Glcp-(1--6)-beta-D-Glcp-(1--3)-D-Glc, a tetrasaccharide beta-D-Glcp-(1--3)-beta-D-Glcp-(1--3)-[beta-D-Glcp-(1--6)]-D-Glc, and a highly branched (d.b. 1/2), enzyme-resistant glucan. The results suggest that the Volvariella glucan is structurally heterogeneous with regard to the distribution of branches, having less branched, moderately branched, and highly branched segments.