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This study aims to explore the structural characteristics of the inhomogeneous top layer of thin-film composite membranes when pretreated by different methods: room temperature–oven, ethanol–hexane in a solvent exchange process, and freeze-drying. An evaluation of the nano-order free-volume pore size of the polyamide samples was carried out by nanopermporometry (NPP) and was quantitatively compared with the free-volume pore estimated from normalized Knudsen-based permeance (NKP) and with positron annihilation characterization (PALS). NPP results denoted a bimodal polyamide membrane structure described by a dense matrix and highly permeable regions. The application of different condensable vapors (water, hexane, and isopropanol) resulted in a free-volume pore size smaller than dp = 0.6 nm for dense regions, which was confirmed after NKP and PALS. In addition, the influence of highly permeable regions on permeance decreased in the following order: ethanol–hexane > freeze-drying > room temperature–oven sampl...

Three new saponarin analogs, 6'''-sinapoylsaponarin, 6'''-feruloysaponarin and 4'-glucosyl-6'''-sinapoyl-saponarin were isolated together with four known compounds from young green barley (Hordeum vulgare var. nudum) leaves. Their anti-oxidative effects towards superoxide, photo-oxidation of vitamins and the 1, 1-diphenyl-2-picrylhydrazyl radical were tested. The superoxide-scavenging activity depended on the number of free hydroxy groups. In contrast, the inhibition of vitamin oxidation seemed to be due to the flavone skeleton. In addition, the sinapoyl moiety seemed to be important for radical-scavenging activity.

We have previously reported the isolation of a vitamin E analog ( α -tocopherol succinate) from a green barley leaf extract, which stimulates the release of prolactin and growth hormone from rat anterior pituitary cells in vitro. In the present study, we tested the commercially available forms of a-tocopherol and also succinic acid. Normal anterior pituitary cells were treated with different commercially available forms of tocopherol (100 μ g/ml) and succinic acid (50 to 100 μ g/ml). Only α -tocopherol succinate caused a significant increase ( P α -tocopherol, δ-tocopherol, α -tocopherol acetate, α -tocopherol nicotinate, succinic acid disodium salt, and succinic acid butenedoic acid did not affect the prolactin release in vitro. The data demonstrate that of all the biologically active forms of vitamin E, only α -tocopherol succinate possesses the ability to enhance anterior pituitary hormone release in vitro.

A chemically synthesized gene encoding human CuZn superoxide dismutase (hSOD) was cloned into the shuttle vector pBAX18R and expressed in Anacystis nidulans 6301 (Synechococcus sp. strain PCC 6301) under the control of a ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbc) promoter derived from A. nidulans 6301. The sequences immediately upstream from the hSOD coding region and the distances between the ribosomal binding site and ATG initiation codon strongly affected the expression of the hSOD gene in A. nidulans cells. Optimal expression of hSOD was obtained with the expression vector pBAXSOD8-I, which contained a GGAGAG sequence. In defined conditions, irradiation with light increased hSOD enzyme activity in the transformants > 18-fold and the level of the hSOD protein reached a value of about 3% of the total soluble protein. The transformants that expressed hSOD acquired the ability to extenuate photooxidative damage induced by methyl viologen.

Many aldehydes formed from lipid peroxidation are reportedly implicated in various diseases, including arteriosclerosis. Lecithin, a blood plasma lipid, and cod liver oil (known to have a beneficial effect on coronary artery disease) were oxidized by Fenton's reagent. The oxidation products, malonaldehyde (MA) and glyoxal, were analyzed in the presence of α-tocopherol or 2″-O-glycosylisovitexin (2″-O-GdM) by gas chromatography after derivatization to 1-methylpyrazol and quinoxaline, respectively. α-Tocopherol inhibited MA and glyoxal formation effectively from 5 mg of lecithin at low doses (1 μmol), whereas 2″-O-GdM required 6 μmol to produce the same level of inhibition

We have previously reported that green barley leaf extract, a dried extract of young green barley leaves that is widely used in Japan and other countries as a nutritional supplement, contains a molecule(s) that enhances the release of growth hormone and/or prolactin from rat anterior pituitary cells in vitro. Using reverse-phase high performance liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS) we have now isolated and chemically characterized the molecule possessing this neuroendocrine activity. The molecule is α-tocopherol succinate, an analog of α-tocopherol or vitamin E. This is the first report documenting a role for this vitamin E analog on pituitary endocrine release.

An ethanol extract obtained from freeze-dried young green barley leaves was fractionated with column chromatography. The fraction exhibiting strongest antioxidative activity was further purified by HPLC. The active component was identified as 2''(3'')-O-glycosylisoviterin by gas chromatography, W, FABMS, 1 H NMR, and 13 C NMR

Pritumumab is a human IgG1 kappa antibody that has been derived from a B-cell isolated from a regional draining lymph node of a patient with cervical carcinoma. Specificity analysis of the antibody with human tissues showed the antigen, altered tumor-associated vimentin, to be highly restricted to various cancers and not normal cells and tissues. In various clinical trials in Japan 249 patients with brain cancer were treated with pritumumab. The overall response rate was between 25-30% with several survivors beyond 5-years post-treatment. The patients were on a low dose regimen of 1mg given twice a week for a course of 24 weeks for a total dose of 48 mgs per course. Pritumumab appears to be a safe and effective therapy in patients with malignant gliomas.

The gene encoding modified human superoxide dismutase (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD. Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl beta-D-thiogalactoside by inserting the gene into the expression vector pKK223-3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codon showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing. The main isomer was purified to homogeneity and characterized. The h-SOD-Ala6 showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum, but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10 degrees C. Thus, the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.

By pre-stimulating the lymphocytes of cancer patients with BCGF plus anti-mu chain specific antibody, we have generated human X human hybridomas. The resulting hybridomas preferentially produced IgG class antibody compared with those obtained from non-stimulated lymphocytes. With non-stimulated lymph-node lymphocytes, from stomach cancer patients we selected a hybrid (TOS/H8) producing IgM with broad reactivity against human tumor cells. In contrast, with pre-stimulated lymph-node lymphocytes from hepatoma patients we selected a hybrid (TOH/D5) producing IgG having a specific reactivity with HuTu80 (duodenum carcinoma).

Ever since the development of human monoclonal antibody CLN-IgG in 1982, we anticipated the identification of the antigen that is recognized by this antibody. Despite its scarce expression on the cell surface, susceptibility to proteolytic enzymes and adherence to experimental equipment, we finally succeeded in determining the antigen moiety that is recognized by this antibody by means of CLN-IgG conjugated column affinity chromatography, two-dimensional electrophoresis, MALDI-TOF/MS and use of glioblastoma cell line U-251MG. The antigen was found to be vimentin, a cytoskeletal protein, and we succeeded in determining a 79 amino acids sequence of the epitope which turned out to comprise a part of the c2 (coil 2 of the central rod) domain of vimentin.

The clinical value of a human monoclonal antibody, CLN-IgG, as an antitumor antibody has been studied at the molecular level. By use of computer-aided graphic design, an anti-idiotypic antibody to CLN-IgG termed idio-No. 3 was evaluated as having an epitope image of a tumor-associated antigen TA226; on the other hand, the combining site of CLN-IgG was seen to be in conformity with a counterimage of the epitope of TA226 and presumed to be part of the ligand image to TA226. An anti-paratactic antibody, idio-No. 3 behaves as a mimic antigen on TA226 and binds to p34 antigen that is expressed on glioma cells. Idio-No. 3 was used for monitoring anti anti-idiotypic antibody intensification in the sera of glioma patients to whom CLN-IgG had been administered.

CLN-IgG is a monoclonal antibody (Ab) produced from a human-human hybridoma recognizing the tumor common antigen (Ag) TA226. In a clinical trial of CLN-IgG treatment conducted in 16 cases of glioma, 1 mg of the Ab was administered to patients twice a week for more than 8 weeks. The induction of antianti-idiotypic Ab (Ab3) in the serum of patient was measured using anti-CLN-IgG idiotype Ab (idiono.33). In 2 cases of complete remission and 3 cases of partial remission treated with CLN-IgG, high levels of Ab3 were detected. The induction of Ab3 was correlated to the clinical effect of the treatment. CLN-IgG is beneficial for the long-term maintenance therapy of glioma without an adverse side effect.

A cyanobacterial expression vector was constructed using ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter and terminator sequences derived from Synechococcus PCC 6301. recombinant plasmid, designated pARUB19, has an ampicillin-resistant (ApR) gene as a selectable marker and four unique restriction sites to allow the insertion of foreign genes. Using this vector, the luciferase gene from the firefly, Photinus pyralis, was introduced into Synechococcus PCC 6301 cells. The luciferase expression vector could be maintained stably in the host cells. Light production of luciferin/luciferase was detected in the transformants. Luciferase amounted to 1.2% of the total soluble protein. This plasmid may facilitate higher levels of foreign gene expression in Synechococcus PCC 6301.

A novel cell line called Trioma, which can proliferate and secrete a human monoclonal antibody in the basal medium, was established. Trioma was generated by somatic cell hybridization with human x human hybridoma and A431c, which is a cell line able to grow autonomously in the basal medium. A Trioma called TriH8 has been kept growing in the DMEM/F-12 medium for over 1 year and stably producing stomach cancer-reactive human IgM into culture medium at 10 micrograms/ml. TriH8 has a characteristic cytological phenotype, that is, to diminish cell growth in the presence of epidermal growth factor.

Human anaplastic gliomas still have a poor prognosis despite current aggressive therapies such as microsurgery, advanced radiation therapy, combined chemotherapy and/or immuno-therapy. It has been widely reported that a major limitation of these treatments is the lack of tumor specificity. Hybridoma technology has made it possible to generate large quantities of monoclonal antibodies (MAB) which are highly specific to human malignant tumors. Such tumor-associated MABs are potentially powerful tools in the diagnosis and treatment of human malignant tumors. A large number of monoclonal antibodies has been produced by hybridomas from mouse × mouse or mouse × human cells, and those antibodies were used in the diagnosis and the treatment of human cancers [1–3].

Alpha-Tocopherol Succinate, But Not Alpha-Tocopherol Or Other Vitamin E Analogs Stimulates Prolactin And Growth Hormone Release From Rat Anterior Pituitary Cells in vitro

Two kinds of human monoclonal antibodies, CLN-IgG and SLN-IgG, were obtained from human x human hybridomas, termed CLNH11 and SLNF10, respectively. These antibodies recognized different molecules which were expressed simultaneously on the cell surface of tumor cells. When both antibodies were added into culture medium in the presence of complement, a remarkable synergistic cytotoxic-effect against glioblastoma cells was observed.

A protease capable of hydrolyzing casein with optimum pH 10 (alkaline protease), perhaps functional in hydrolysis of non-histone proteins and Hl histone, was found to exist at the state bound with chromatins of various normal and tumorous tissues of rats, in addition to the protease capable of hydrolyzing histone with optimum pH 8 (neutral protease). Alkaline protease was not observed in other subcellular fractions than nuclear fraction. It had approximately 18,000 daltons, and was chymotrypsin-like as inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor and Chymostatin. Its contents were significantly high in rapidly proliferating cells; Yoshida sarcoma⪢ Rhodamine sarcoma≥ AH 130≥ thymus> spleen⪢ kidney≥ liver⪢ brain.

A human X human hybridoma, CLNH11, derived from a lymphocyte of a patient with cervical carcinoma, produces a human monoclonal antibody of gamma 1 and kappa isotypes (CLN-IgG). Immunoperoxidase staining showed that CLN-IgG reacted with frozen tissue sections of human malignant tumors (cervical carcinoma, gall bladder carcinoma, glioblastoma), but not with their normal counterparts. Enzyme-linked immunosorbant assay also demonstrated that CLN-IgG reacted with various human tumor cell lines, but not with non-tumorigenic cells such as some fibroblasts, peripheral blood lymphocytes and red blood cells. Indirect and direct immunofluorescence staining indicated that the tumor antigens recognized by CLN-IgG were located in restricted areas close to the cell surface and exposed on the outer surface of the cell membrane. A protein antigen of Mr 226,000 was purified to homogeneity by affinity chromatography with CLN-IgG from the plasma membrane fraction of A549 lung tumor cell line. The antigen consisted of alpha (Mr 60,000) and beta subunit (Mr 53,000) which were linked by disulfide bond(s) (TA60K/53K). The TA60K/53k antigens were expressed commonly in other tumor cell lines originated from histologically different tissues.

It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 00-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.

UC 729-6, a 6-thioguanine-resistant human lymphoblastoid B-cell line, was fused with normal and malignant human lymphocytes. Parent UC 729-6 cells were diploid with a 21p+ marker chromosome, expressed surface and cytoplasmic IgM kappa, and doubled every 17 hr. The resulting human-human hybridomas were pseudotetraploid containing the 21p+ marker, doubled every 20-30 hr, and secreted 3-9 micrograms of human Ig per ml per 10(6) hybrid cells for greater than 9 months in continuous culture. Human Ig-secreting hybridomas were generated in 88% (14/16) of the fusions carried out and were cloned by limiting dilution (one cell per three wells) without the use of feeder layers. The mean fusion frequencies (number of wells, plated at 10(5) cells per well, showing hybrid growth per 10(6) lymphocytes fused) of UC 729-6 with normal lymphocytes ranged from 0.45 to 2.9 and with malignant B lymphocytes, from 3 to 10. Analysis of the human-human hybridomas derived from lymphocytes isolated from regional draining lymph nodes of cancer patients revealed several that secreted human monoclonal antibody that reacted by an enzyme immunoassay with some carcinoma cell lines but not with normal fibroblast cell lines. These data suggest that (i) UC 729-6 can be fused with human lymphocytes to generate stable human-human hybridomas, some of which secrete antibody reactive to human cell surface antigens, and (ii) UC 729-6 can be used to rescue Ig from nonsecretory malignant B cells and thereby allow for the production of anti-idiotype antibodies.