A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC₅₀ at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.     

Guinea pig bone marrow cells treated with platelet-activating factor generate factor(s) which affects their DNA synthesis and microbicidal activity

We have previously reported that platelet-activating factor (PAF) induces proliferation and microbicidal activity of guinea pig bone marrow cells. In the present study, we have found that the conditioned medium of PAF- or nonmetabolizable PAF agonist-treated guinea pig bone marrow cells augmented DNA synthesis and induced microbicial activity of bone marrow cells. A PAF specific antagonist, CV-6209, inhibited generation of the active conditioned medium by PAF. Addition of the PAF antagonist only partially suppressed the augmentative effect of the active conditioned medium on DNA synthesis; this is consistent with the fact that, because of the rapid breakdown, no appreciable amount of PAF remained in the conditioned medium of PAF-treated cells. Although mouse bone marrow cells did not respond to PAF unlike guinea pig cells, their DNA synthesis was significantly enhanced by the conditioned medium of PAF-treated guinea pig bone marrow cells. Thus, some newly generated factor(s) distinct from the originally inoculated PAF seemed to modulate the bioactions of PAF on bone marrow cells. An appreciable amount of PAF was produced by calcium ionophore-treated guinea pig bone marrow cells. These findings indicate that PAF synthesized in guinea pig bone marrow cells induces generation in the cells of some factor(s) which affects proliferation or microbicidal activity. Presented at The Third International Conference on Platelet-Activating Factor and Structurally Related Ether Lipids, Tokyo, Japan, May 1989.     

日本著名时装设计师小筱弘子访谈

本文是在国际服饰文化与教育研讨会期间,本栏目主持人、我校服装艺术与工程学院讲师孙雪飞对日本著名时装设计师小筱弘子进行访谈的整理.     

Doing Business with Theory: Communities of Practice in Knowledge Management

We explore how the notion of communities of practice (CoPs) was translated and popularized from its original inception by Lave and Wenger in 1991. We argue that the Institute for Research on Learning (IRL), a spin-off of Xerox PARC, proved instrumental in enrolling CoPs into the knowledge management (KM) discipline. IRL objectified, packaged, and made a business out of CoPs. CoPs in KM are now a formalized process coupled with technological artifacts to build groups of people who effectively share knowledge across boundaries. Drawing from participant observations, archival documents, and interviews with KM practitioners in the aerospace industry as well as key players of IRL, our research seeks to unveil the invisible history that the popularization of a theory can often obscure. We argue that CoPs provide a case study for understanding how abstract concepts in science are strategically and subconsciously reified, or made objects of inquiry, and appropriated by actors. This reification of a “soft” science blurs the line between theory and technology.     

Colorimetric microdetermination of sulphites in foods by use of the modified rankine apparatus. IV.

Summary Detection and determination of traces of sulphites in foods was attempted by use of the modified Rankine apparatus and pararosaniline colorimetry. Replacement of alkaline titration reported previously by pararosaniline colorimetry lowered the absolute detection limit from 30 g (titration method) to 2 g. In view of clean analysis, in the color developing system, 0.1 N-sodium hydroxide was used in place of mercuric chloride solution commonly used as an absorbant of sulphites. In order to prevent oxidative decomposition of sulphites during operation, nitrogen gas was used as carrier instead of air. Dimedone and sodium azide were used for the elimination of aldehydes and nitrites, respecitvely, in the sample, which will disturb the color development of sulphites with pararosaniline-formaldehyde reagents. With this improved method, it was possible to determine the residual sulphites in frozen peeled shrimps, sugared beans and other foods with low sulphite contents accurately.

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Colorimetrische Mikrobestimmung von Sulfiten in Lebensmitteln bei Anwendung der modifizierten IV. Rankine Apparatur

Zusammenfassung Geringe Sulfitmengen in Lebensmitteln (geschälte Garnelen, gezuckerte Bohnen) können colorimetrisch bestimmt werden. Die neuentwickelte Methode beruht auf einer Kombination von colorimetrischer Bestimmung mittels p-Rosanilin und der Bestimmungsmethode nach Rankine. Auf diese Weise lassen sich Gehalte von 2 g noch genau bestimmen. Bei der Farbentwicklung wurde das giftige Quecksilbertetrachlorid durch 0.1 n-NaOH ersetzt, anstelle von Luft Stickstoff als Trägergas verwendet und somit eine Oxydation des Sulfits während der Bestimmung vermieden. Da Nitrit und Aldehyde die Farbentwicklung stören, wurde ihr Einfluß durch Dimedon und Natriumazid ausgeschaltet.

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Studies on the Analyses of Sulphites in Foods (IV)     

Bile Acid–Drug Interaction via Organic Anion-Transporting Polypeptide 4C1 Is a Potential Mechanism of Altered Pharmacokinetics of Renally Excreted Drugs

Patients with liver diseases not only experience the adverse effects of liver-metabolized drugs, but also the unexpected adverse effects of renally excreted drugs. Bile acids alter the expression of renal drug transporters, however, the direct effects of bile acids on drug transport remain unknown. Renal drug transporter organic anion-transporting polypeptide 4C1 (OATP4C1) was reported to be inhibited by chenodeoxycholic acid. Therefore, we predicted that the inhibition of OATP4C1-mediated transport by bile acids might be a potential mechanism for the altered pharmacokinetics of renally excreted drugs. We screened 45 types of bile acids and calculated the IC₅₀, K_i values, and bile acid–drug interaction (BDI) indices of bile acids whose inhibitory effect on OATP4C1 was >50%. From the screening results, lithocholic acid (LCA), glycine-conjugated lithocholic acid (GLCA), and taurine-conjugated lithocholic acid (TLCA) were newly identified as inhibitors of OATP4C1. Since the BDI index of LCA was 0.278, LCA is likely to inhibit OATP4C1-mediated transport in clinical settings. Our findings suggest that dose adjustment of renally excreted drugs may be required in patients with renal failure as well as in patients with hepatic failure. We believe that our findings provide essential information for drug development and safe drug treatment in clinics.     

Simple Method for Measuring the Peroxide Value in a Colored Lipid

We developed a simple method for quantification of the peroxide value (PV) in colored lipids on the basis of the reaction between triphenylphosphine (TPP) and oxidized oil to afford triphenylphosphineoxide (TPPO). Diphenylphosphineoxide (DPPO) was employed as internal standard. The formed TPPO was analyzed by high-pressure liquid chromatography–UV spectroscopy with absorption at 260 nm. The conditions that gave the highest correlative calibration curve between the peak area on the chromatogram and peroxide value were identified: the optimum TPP–oxidized oil mix ratio, reaction temperature, and reaction time were found to be 2:1, 40 °C, and 30 min, respectively. Linear calibration curves, passing through the origin, were obtained for PV versus TPPO and TPPO versus DPPO. The quantification limit for this method was 2.01 pmol hydroperoxyl group, which corresponds to a PV value of 0.2 meq/kg in a 10-mg oil sample. This method was used to measure the PV in colored fats and oils or lipids extracted from dark meat and processed food containing a coloring agent. Though the official method could not measure the PVs in the colored lipids, the method proposed here, which uses an inexpensive chemical reagent and machine, could. The developed method could play an important role for food quality control.     

Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.