Search

Biosimilars are less expensive than their originators, and Japanese government policies call for their development and promotion. However, the adoption and prescription of some biosimilars, especially antibody/its-related ones, have been delayed for use in Japan, possibly due to concerns on the differences in quality attributes such as glycan structures between the originators and their biosimilars, and that clinical efficacy/safety studies are conducted for usually one disease and its results extrapolated to other indications. We conducted a questionnaire survey among physicians in four disease areas (hematology, medical oncology, rheumatoid arthritis, and inflammatory bowel disease), where biosimilars of antibody/its-related drugs have been approved, regarding their thoughts on the adoption and prescription of biosimilars in Japan from January to April 2020. We received totally 1024 responses. When adopting biosimilars and explaining them to patients, physicians requested specific information including the comparative results of phase III clinical trials and quality characteristics between biosimilars and their originators; the results of clinical studies on switching from originators to their biosimilars; and a comparison of the estimated cost on patients in consideration of the high medical cost payment system. Priority differed depending on the studied disease areas. In terms of post-marketing information, physicians requested a variety of information. When explaining biosimilars to the patients, physicians would like to use general material from government describing the comparability between originators and their biosimilars. These results suggest that physicians sought more comparative information on the quality, efficacy, and patients' cost between originators and their biosimilars when adopting or prescribing biosimilars.

Previous cross-sectional studies have reported that adolescent patients with anorexia nervosa (AN) showed global gray matter volume (GMV) reductions at the acute phase which were restored at the weight-recovered phase, compared with healthy controls (HC). However, few studies have investigated white matter volume (WMV) or cortical thickness in the context of AN, and results have been inconsistent. Voxel-based morphometry analyses for GM and WM, and cortical thickness analyses for GM were conducted in 31 adolescent patients with AN (vs. 18 HC) in the acute phase, and 16 patients with AN (vs. 13 HC) in the follow-up weight-recovered phase, over an approximately 1-year follow-up interval. At the acute phase, the AN patients showed significant reductions of GMVs and cortical thickness in widespread brain regions, compared with HC. Significant WMV reductions were identified in the bilateral superior longitudinal fascicle, superior thalamic radiation, corona radiata, and fornix, pons, and medulla in the patients. At the weight-recovered phase, the AN patients showed a significant GMV reduction in the left hippocampus, and a WMV reduction in the pons, compared with the HC. There was no difference in cortical thickness between two groups at the weight-recovered phase. In conclusion, the widespread volumetric reductions in GM and WM, and reduced cortical thickness observed in AN patients in the acute phase were not evident in the follow-up weight-recovered phase. The volume reductions observed in the hippocampus and pons in the weight-recovered phase could potentially reflect delayed neurogenesis or recovery from starvation in the AN patients.

Water and energy are essential for the human body. The doubly labeled water (DLW) method measures water turnover (WT) and total energy expenditure (TEE), which serves as a benchmark for the adequate intake (AI) of water and estimated energy requirements (EER). The objective of the current study was to examine the association of WT and TEE with physical activity and body composition in Japanese preschool children. We included 41 preschool children (22 girls, 19 boys) aged 3–6 in this study. WT, TEE, and fat-free mass (FFM) were obtained using DLW. Physical activity was measured using a triaxial accelerometer and categorized as light (LPA; 1.5–2.9 Metabolic equivalents, METs) and of moderate-to-vigorous intensity (MVPA; ≥3.0 METs). Exercise duration (Ex) was defined as ≥4.0 METs of physical activity. WT and TEE moderately positively correlated with Ex, but not with LPA. WT moderately positively correlated with BW and FFM while TEE strongly. We established predictive equations for WT and TEE using body weight (BW), FFM, step count, and Ex to guide the AI of water and EER in Japanese preschool children. We found that FFM and step count are the determinants of TEE, and that BW and Ex are the determinants of WT in preschool children.

Problematic Internet use (PIU) is common and likely to coexist with mental health problems among adolescents with school refusal behavior. To date, no study has revealed to what extent PIU relates to the daily burden compared with other mental health problems. This study has examined the association between daily difficulties and PIU among adolescents with school refusal behaviors.This cross-sectional study involved all first-visit patients, regardless of diagnosis, aged 10 to 18 years at 2 child/adolescent psychiatric outpatient clinics in Yokohama City, Japan, from April 2016 to March 2018. The Questionnaire-Children with Difficulties (QCD) were obtained from parents. Simultaneously, the severity of PIU was evaluated using the Internet Addiction Test and depressive and anxiety symptoms were evaluated using the Patient Health Questionnaire-9 and General Anxiety Disorder-7 scale in the 2 weeks before the first-visit. From 684 first-visit patients, 227 with school refusal behaviors were enrolled in the study.PIU was observed in 40% of adolescents with school refusal behaviors. The QCD scores among patients with PIU were significantly lower than those in patients without PIU. Linear regression analysis revealed relationships between PIU and lower QCD scores throughout the day (except at night) and the total score of the day, after controlling for confounders such as depressive and anxiety symptoms.In conclusion, among adolescents with school refusal behaviors, PIU may affect their parent-assessed daily difficulties particularly experienced throughout the day.

幼児期における身体活動量の実態および身体活動量と体格や体力との関連を明らかにし、この年代の体力向上施策に活かすため、2009年からH幼稚園の観察調査研究を継続している。本稿では、2012年度調査の結果について、幼稚園児の身体活動量・体格・体力の実態と相互の関連性について検討した。対象はT市H幼稚園の園児83名(男児47名、女児36名)である。身体活動量は、1日あたりの総エネルギー消費量(TEE)、歩数、身体活動レベル(PAL)、運動強度別(低強度、中等度、高強度)活動時間を測定した。体力は、25m走、立ち幅跳び、テニスボール投げ、両足連続跳び越し、体支持持続時間、握力、捕球の7種目で評価した。身体活動量指標には、学年差は少ないが、男女差、平日休日間差が認められる指標が多かった。体力には、明らかな学年差があり、5歳児では3種目に男女差がみられた。身体活動量は、体格と有意な関連を示さなかったが、体力には有意な関連が認められ、歩数および4METs以上活動時間の低値群は、中値群、高値群に比べ、いずれの体力測定値とも低値を示した。本研究で対象とした都市部の幼稚園児において、身体活動量は体力に関連し、高い身体活動量レベルの者の体力が高値であった。

知的障害児者の肥満対策の基礎資料を得ることを目的として、知的障害のある小学校特別支援学級在籍児童6名と成人4名を対象に、身体活動量、食生活を調査し、成人4名に対しては成人の骨格筋量(細胞内量)を測定した。栄養摂取量では、摂取エネルギー量は同年代値に近い値が多く、児童で炭水化物、成人で脂質が多い傾向があった。身体活動量は、同年代値と比較すると、児童では高く、成人では女性は低く、男性は高い値であった。平日休日の活動量は、児童では休日に減少する傾向があり、成人では半数は休日に多かった。成人の体重あたりの筋細胞量は、肥満度II・IIIである女性各1名は、同世代値より少なく、肥満度Iの男性では平均的であった。これらのことから、児童と成人では食生活、活動量に異なる特徴が認められ、成人においては身体活動量と筋細胞量の関連が示唆された。

幼稚園に在籍する園児を対象に、1週間の身体活動量と体力(立ち幅跳び・テニスボール投げ・25m走・両足連続跳び越し・体支持持続時間・握力)を、3年間に渡って測定した。活動量は、歩数、総エネルギー消費量、活動指数、運動強度別活動時間で評価した。1日の歩数は男児16502歩、女児14685歩で、男児の歩数が有意に高い値を示し、ほとんどの活動量指数で平日が休日より上回った。体力における男女差は、全学年のテニスボール投げ、5歳児の25m走と握力で男児が有意に高い値を示したものの、立ち幅跳びと両足連続跳び越しでは男女差は認められなかった。

聴覚に障害のある子どもにとって早期発見・早期対応の必要性は高く、多くは専門的な支援機能をもつ機関において早期教育を受ける。一方で、聴覚障害児が幼稚園に在籍するケースもあり、幼稚園における支援のあり方が問題となる。本研究では、聴覚支援学校幼稚部から一般の幼稚園の5歳児クラスに転園してきた聴覚障害児の保育実践事例を通して、幼稚園における聴覚障害児への効果的な園内支援のあり方を明らかにすることを目的とする。幼稚園教員や保護者へのインタビューから、保育における支援の成果と課題を明らかにしたうえで、幼稚園での活用に効果的で、聴覚障害幼児のニーズに対応する「個別の指導計画」のフォームを考案し作成した。幼稚園における聴覚障害児の支援には、保護者との連携による支援、健聴児との育ち合い、園内支援の活性化の視点をもって取り組むことが重要であることが分かった。

The aim of the present study was to clarify the knowledge of and perspectives on pharmacovigilance among pharmacists in the Miyagi and Hokkaido regions of Japan. In this cross-sectional, self-administered questionnairebased study, we contacted 3,164 pharmacists who belonged to the Miyagi Prefecture Hospital Pharmacists Association or the Hokkaido Society of Hospital Pharmacists during the 3-month period between January and March 2013. Of the 1,851 respondents (

Rationale and objectives The purpose of this study was to evaluate the utility of magnetic resonance (MR) imaging for indicating the extent of true tissue necrosis of the liver after radiofrequency (RF) ablation in comparison with histopathologic findings in dog models and an autopsy case. Materials and methods RF ablation of the liver parenchyma was performed on three dogs under general anesthesia. MR appearances of the RF-ablated regions on T1-weighted fast-low angle shot (FLASH; repetition time/echo time [TR/TE]/flip angle: 120/3.8/70),T2-weighted turbo spin echo (3000/80/echo train = 25) and contrast-enhanced T1-weighted images were compared with histopathologic findings. An autopsy case with hepatocellular carcinoma was also enrolled in this study. Results All ablated regions showed three zones on T1-weighted FLASH images: a central zone with low intensity, a broad hyperintense middle zone, and a surrounding hypointense band. The central and middle zones corresponded to the degrees of coagulation necrosis observed during histopathologic examination, whereas no viable cells were seen in these zones during the microscopic examination using nicotinamide adenine dinucleotide diaphorase stain. The surrounding hypointense band corresponded to sinusoidal congestion in the acute phase and to fibrotic change in the subacute phase. Conclusion MR imaging using the FLASH sequence can accurately determine the extent of the necrotic area after RF ablation.

Purpose To investigate extraocular muscle (EOM) path shift and prolapse of posterior eyeball from muscle cone in acquired esotropia with high myopia (AEHM), using magnetic resonance imaging. Design A case-control study. Methods There were 16 eyes with AEHM, 11 with high myopia (HM), 12 with moderate myopia (MM), and 11 control eyes. Extraocular muscle shift was evaluated by measuring angles formed by the line connecting orbital centroids and the line connecting each orbital centroid and each EOM centroid. The ratio of the prolapse in the posterior eyeball from the muscle cone was also measured. Results Both inferior shift of lateral rectus (LR) and nasal shift of superior rectus (SR) muscle were observed in the AEHM group, compared with HM, MM, and control groups. Neither shifted significantly in the HM group compared with control group. The prolapse ratio in AEHM group was higher than in the HM, MM, and control groups. Greater EOM shifts and eyeball prolapse were observed when the AEHM was more severe, as in esotropia fixus. Conclusion In AEHM, a prolapsing eyeball shifts LR inferiorly and SR nasally; these findings were not observed in high myopia with neither ocular deviation nor restriction. These shifts reduce abduction and supraduction and increase infraduction and adduction in AEHM. The shifts would be predicted to create a hypoesodeviation, which is a common finding in AEHM. Both EOM shifts and superotemporal eyeball prolapse tend to be greater in esotropia fixus.

The purpose of this report is to investigate the actual working condition of food preparers in both small and medium-size day nurseries and their consciousness regarding day nursery feeding. In spite of the importance of day nursery feeding, it has become clear that food preparers working condition as well as their status has been quite insufficient, and the importance of their work has not been highly esteemed. On the other hand, it has been recognized that food preparers enthusiasm regarding the education of dietary food for infants is very strong.

We have identified a stimulatory monoclonal antibody (mAb) from autoimmune mice that selectively stimulates granulocyte colony-stimulating factor (G-CSF) gene expression in a mouse macrophage cell line. The induction was observed not only in the cell line, but also in normal peritoneal macrophages. This mAb bound to the monocyte/macrophage cell lines and pre-B leukemia cell lines, but also in normal peritoneal macrophages, whereas it did not bind to normal T and B cells in the spleen or fibroblastic cell lines. It could even bind to a human promyelocytic leukemia cell line, when they were differentiated into monocytic cells. On Western blotting, this mAb mainly recognized an approximately 30-kDa band and it was unique because there have been no reports of membrane-associated proteins with a similar molecular mass found in macrophages. These results suggest that there could be a specific gateway molecule to induce G-CSF in macrophages.

The relation between the structure of haptens and the esterolytic activities of antibodies was investigated. We synthesized two phenylalanine analogs, the negatively charged phosphonate derivative 1 and the neutral phosphonamidate derivative 2. Seventeen out of 41 monoclonal antibodies generated against the hapten 1 hydrolyzed the relevant phenylalanine ester R-12. On the contrary, none of 27 monoclonal antibodies generated against the hapten 2 had catalytic activity. An ab initio study of the structural and electronic properties of the modeled haptens showed that the value of the negative electrostatic potential around the phosphonyl oxygen was an important factor affecting the induction of esterolytic antibodies.

In order to scrutinize the adherence-dependent interactions for induction of granulocyte colony-stimulating factor (G-CSF) in peripheral monocytes/macrophages, a sensitive reporter gene assay was constructed using the mouse macrophage cell line transfected with the mouse G-CSF promoter region in conjunction with the luciferase gene as a reporter. With this system, lipopolysaccharide (LPS) showed a markedly positive response. Among the extracellular matrix (ECM) proteins, both fibronectin (FN) and vitronectin (VN) markedly induced luciferase activity, but others did so but much lesser extent. Among the synthetic peptides having Arg-Gly-Asp (RGD) sequences, only FLEPP with multiple RGD significantly induced luciferase activity. Pretreatment of the cells with anti-integrin alpha 6, alpha M, beta 1 and beta 2 monoclonal antibodies (mAbs) significantly reduced the LPS-induced responses and anti-alpha 1, alpha 2 and beta 3 mAbs to lesser extent, and anti-alpha 5, alpha 6, alpha M, beta 1 and beta 2 mAbs blocked the FN-induced response. In the cell-to-cell interactions, significantly positive increase was observed by direct contacting this cell line with a G-CSF-dependent promyelocytic leukaemia cell line, known to stimulate the induction of G-CSF to the stromal cells. Its effect was mostly blocked by pretreatment with anti-integrin alpha 5, alpha L, beta 1 and beta 2 and anti-ICAM-1 mAbs. These results indicate that there are several pathways via the cell-to-ECM and cell-to-cell interactions triggering the induction of G-CSF in the macrophages.

Cellular interactions between hematopoietic cells and stromal cells play crucial roles in the proliferation and differentiation of the hematopoietic cells. Interleukin-6 (IL-6)-dependent 7TD1 cells markedly proliferated without IL-6 when they were co-cultured with hematopoietic-supportive bone marrow stromal cells, HESS-5 cells and HESS-1 CL.3 cells, which can support long-term hematopoiesis in vitro with but not without direct cell contact, cell contact being prevented with a microporous membrane. The production of IL-6 and the amount of IL-6 mRNA in hematopoietic-supportive stromal cells but not 7TD1 cells significantly increased only when the stromal cells were co-cultured in direct contact with 7TD1 cells. Furthermore, the amount of IL-6 mRNA increased according to the number of 7TD1 cells co-cultured. These inductions were not observed on co-culture with a murine myeloid cell line, M1 cells, or on the addition of the co-culture supernatant. These results suggest that 7TD1 cells transmit the signal to stromal cells that enhances IL-6 production by stromal cells via direct cell contact. A certain specific molecule for transduction of the signals may exist on the surface membrane of stromal cells and hematopoietic cells.

Two cDNA clones homologous with human neuropeptide (NP) Y-Y1 receptor have been isolated from a mouse bone marrow cDNA library. One was thought to be the cognate of the human NPY-Y1 receptor, termed Y1α receptor, and the other form, termed Y1β receptor, differed from the Y1α receptor in the seventh transmembrane domain and C-terminal tail. Analysis of the mouse genomic DNA showed that both receptors originated from a single gene. The different peptide sequences of the Y1β receptor were encoded by separate exons, hence, these receptors were generated by differential RNA splicing. High affinity binding of [125I]NPY to each receptor expressed in Chinese hamster ovary (CHO) cells and sequestration of [125I]NPY after binding to each receptor were observed. In the CHO cells expressing the Y1α receptor, intracellular Ca2+ increase, inhibition of forskolin-induced cAMP accumulation, and mitogen-activated protein kinase (MAPK) activation were observed by stimulation of NPY, and these responses were abolished by pretreatment with pertussis toxin. Since wortmannin completely inhibited NPY-elicited MAPK activation, we speculate that wortmannin-sensitive signaling molecule(s) such as phosphoinositide 3-kinase may lie between pertussis toxin-sensitive G-protein and MAPK. In contrast, these intracellular signals were not detected in CHO cells expressing the Y1β receptor. Northern blots and reverse transcriptase-polymerase chain reaction analyses indicated that the Y1α receptor was highly expressed in the brain, heart, kidney, spleen, skeletal muscle, and lung, whereas the Y1β receptor mRNA was not detected in these tissues. However, the Y1β receptor was expressed in mouse embryonic developmental stage (7 and 11 days), bone marrow cells and several hematopoietic cell lines. These results suggest that the Y1β receptor is an embryonic and a bone marrow form of the NPY-Y1 receptor, which decreases in the expression during development and differentiation.

The authors analyzed the 62 patients who underwent hepatic arterial infusion (HAI) chemotherapy using W-spiral (WS) catheter. The catheter was successfully inserted into the hepatic artery without a coil fixation in 57 cases. After cessation of chemotherapy, the catheter was removed in 32 cases without difficulty, which resulted in improved patients' quality of life as well as preservation of patency of hepatic arteries. This catheter has a special shape-memory alloy in its tip, which allows preferable fixation without coils and removal of the catheter if desired. HAI using this catheter and subsequent removal of it is a reasonable strategy in the era with potent systemic chemotherapy.

Measurement of released granule components, popularly used to quantify mast cell exocytosis, does not deliver real-time information about degranulation at the single-cell level nor the ratio of responding/non-responding cells. Rather it provides, only end-point, bulk-population data. Here we studied degranulation of rat peritoneal mast cells dispersed in a narrow horizontal channel between a silicon substrate and a glass plate. Upon exposure to a concentration gradient of a soluble stimulus, degranulation started from those cells facing towards the highest concentration of stimulus. We captured images of exocytosing cells without the need for phase-contrast or differential interference-contrast microscopy. This was achieved using the reflection caused by the silicon substrate. The time-lapse images of cells in the channel were segmented into multiple concentration belts to identify the proportion of degranulated cells in each belt region. Maximum ratios of degranulated cells in the belt regions determined by time-course curve fitting calculations were then plotted against the distance from the stimulus injection site, resulting in a sigmoidal response curve. This method provides a powerful means for real-time analysis of concentration- and stimulus-dependent degranulation of mast cells and allows comparison of cell responses under different conditions. To show its effectiveness, we evaluated the effect of a protein kinase C (PKC) inhibitor, Go6976, on degranulation induced by various stimuli. In contrast to stimulation with concanavalin A+lysophosphatidylserine (lysoPS) or nerve growth factor+lysoPS (completely inhibited by Go6976 over the whole range of stimulus concentrations used) or compound 48/80 and mastoparan (no inhibition by Go6976), stimulation with ionomycin, a known Ca(2+) ionophore, showed a concentration-dependent inhibition by Go6976, with a major inhibition at low stimulus concentrations and a diminished one at higher ionomycin concentrations. The results indicate that ionomycin-induced degranulation is mainly induced via a PKC-independent signal cascade at high stimulus concentrations, whereas below a certain concentration, degranulation is completely dependent on PKC.

A rare case of deep penetrating burn injury caused by misuse of a high-pressure acetylene burner is reported. A 35 year old man was admitted with second and third degree burns involving the right arm cubital area and a subcutaneous burn on his right arm caused by a high-pressure acetylene gas flame. Early surgical debridement and secondary skin grafting using a preserved subcutaneous vascular network skin graft (PSVNSG) proved effective in this patient. Skin contracture was prevented and function was recovered. The basis of PSVNSG is that the vascular system existing in the graft is used as a permanent vascular system without degeneration. This case shows that, in this kind of burn injury, subcutaneous tissue damage should be suspected and that it is important to perform surgical debridement early after admission.

Purpose To determine the usefulness of nonenhanced T1-weighted spoiled gradient-recalled acquisition in the steady-state (SPGR) MRI in the early assessment of the efficacy of radio frequency (RF) therapy for hepatocellular carcinoma (HCC). Materials and Methods A total of 23 patients with 28 HCC nodules treated with percutaneous RF ablation underwent nonenhanced MRI within two days after the RF procedure and contrast-enhanced computed tomography (CT) one week after. MR assessment of ablation efficacy according to the concentric zonal pattern on T1-weighted SPGR imaging was compared with the one-week CT and presence of local recurrence by means of follow-up study for 12 months or more. Results In 18 of 28 ablated nodules, SPGR images revealed a central hyperintense zone covering the entire tumor, CT showed a nonenhanced area covering the entire tumor, and no local recurrence was demonstrated in the follow-up studies. In nine of 28 nodules, the central hyperintense zone did not cover the entire tumor; and local recurrence was demonstrated in three nodules. In the remaining one nodule, no signal change was seen in the treated area on SPGR images and CT showed the presence of residual viable tumor. Conclusion Nonenhanced T1-weighted MRI was considered useful for early assessment of the efficacy of RF therapy for HCC. J. Magn. Reson. Imaging 2007. © 2007 Wiley-Liss, Inc.

The purpose of this study was to evaluate the utility of magnetic resonance (MR) imaging for indicating the extent of true tissue necrosis of the liver after radiofrequency (RF) ablation in comparison with histopathologic findings in dog models and an autopsy case.RF ablation of the liver parenchyma was performed on three dogs under general anesthesia. MR appearances of the RF-ablated regions on T1-weighted fast-low angle shot (FLASH; repetition time/echo time [TR/TE]/flip angle: 120/3.8/70),T2-weighted turbo spin echo (3000/80/echo train = 25) and contrast-enhanced T1-weighted images were compared with histopathologic findings. An autopsy case with hepatocellular carcinoma was also enrolled in this study.All ablated regions showed three zones on T1-weighted FLASH images: a central zone with low intensity, a broad hyperintense middle zone, and a surrounding hypointense band. The central and middle zones corresponded to the degrees of coagulation necrosis observed during histopathologic examination, whereas no viable cells were seen in these zones during the microscopic examination using nicotinamide adenine dinucleotide diaphorase stain. The surrounding hypointense band corresponded to sinusoidal congestion in the acute phase and to fibrotic change in the subacute phase.MR imaging using the FLASH sequence can accurately determine the extent of the necrotic area after RF ablation.

Purpose: To review the results of a muscle transposition procedure in which the halves of the vertical rectus muscle bellies are sutured onto the sclera, without tenotomy of vertical recti as in Hummelsheim's procedure or surgical treatment of the lateral rectus (LR) as in Jensen's procedure. Methods: Ten patients with abducens palsy received the procedure. We measured the ocular deviation and the field of single binocular vision, and observed the LR using magnetic resonance imaging (MRI). Results: Preoperative or postoperative deviation was distributed from +27 to +58 prism diopters (PD) or orthophoria to +12 PD, respectively, in 7 patients with unilateral paresis, and +75 to +120 PD or +2 to +37 PD in 3 patients with bilateral paresis. The average correction was 42.4 PD per eye. Seven patients were able to regain the field of single binocular vision at least in the primary position. No postoperative complications were observed. MRI showed that the LR was atrophic and floppy, lacking muscle tension. Conclusions: Our procedure enabled the patients to obtain satisfactory postoperative results without treatment of the LR or tenotomy of the transposed muscles. This procedure can reduce operative damage to the eye more than Hummelsheim's or Jensen's procedure.

One of the major obstacles to acquiring catalytic antibodies is that it requires labor-intensive procedures to select catalytic antibodies from huge repertories of antibodies. Here, we selected potential catalytic Abs by utilizing their affinity towards a short transition-state analog which contained only the transition-state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited against a phosphonate derivative, the TSA1, were screened by a three-step screening process: step 1, ELISA for TSA1-BSA; step 2, ELISA for the short TSA4; and step 3, competitive-inhibition by the short TSA2. Only 22. 8% of positive mAbs from step 1 were found to be catalytic. The rate of catalytic Abs increased to 45.7% using screening steps 1 plus 2, and reached 83.3% using all three screening steps. This clearly suggests that our screening protocol is an efficient method to select potential catalytic Abs. Furthermore, we characterized the properties of both the catalytic Abs and the noncatalytic Abs in detail. The catalytic Abs tended to have lower Kd for TSA1 and the short TSA2 than noncatalytic Abs. It was also observed that catalytic Abs showed clear enantiospecificity toward substrate 6 containing d-phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and binding parameters for these antibodies gives us further insight into catalytic antibodies.

Platelet-activating factor (PAF) is a mediator with physiological and inflammatory activities. Numerous actions of PAF are mediated through specific membrane receptors.1 Accurate and rapid determination of PAF in biological samples is essential for a comprehensive understanding of its role and mode of action. To date, various biological,2 massspectrometrical3 and immunological4 assays for PAF have been developed, but all have problems such as interference by endogenous PAF inhibitors, lack of specificity, data standardization or reproduction, tedious procedures for derivatization of PAF, or contamination with other membrane phospholipids which interfer with the determination5. Ra-dioreceptor binding assays for PAF were developed to measure PAF produced by cells or homogenates of tissues.6,7 However, these methods also have drawbacks in being tedious procedures involving the preparation of rabbit platelet membranes or unreliable due to significant deviations in binding capacity depending on the membrane batches. We have now overcome these difficulties by using membranes from CHO cells carrying a cloned human PAF receptor and [3H]WEB 2086, a PAF antagonist. We describe in this paper the characteristics of this novel assay system and its application in quantifying PAF in biological samples.

A cDNA clone homologous with the human neuropeptide Y (NPY)-Y2 receptor has been isolated from a mouse brain cDNA library. Analysis of the predicted amino-acid sequence indicates that the polypeptide encoded by this cDNA is 94% homologous to the human NPY-Y2 receptor. In Chinese hamster ovary (CHO) cells expressing the mouse NPY-Y2 receptor, an increase in intracellular Ca2+ and inhibition of forskolin-induced cAMP accumulation were observed due to stimulation with NPY, NPY-(13–36) and peptide YY, but not with pancreatic polypeptide or [Leu31,Pro34]NPY. The fact that the NPY-induced increase in intracellular Ca2+ and inhibition of forskolin-induced cAMP accumulation were eliminated by pretreatment with pertussis toxin suggests that the NPY-Y2 receptor couples to PTX-sensitive G-protein(s), probably Gi/Go, in CHO cells.

Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5´-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0±5.4 nM, Bmax = 707±11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca2+-independent/ wortmannin-sensitive pathway in CHO cells.

A simple and reproducible radioreceptor assay (RRA) has been developed using membranes from CHO cells which can stably express human platelet-activating factor (PAF) receptor. The CHO cells expressing the PAF receptor, termed CHO · 1F8, showed a significant intracellular Ca 2+ response to PAF, and the same binding properties to [ 3 H]WEB 2086, a PAF antagonist, as reported ( K d , 13.6 ± 1.9 nM; B max , 2.5 ± 0.4 pmol/mg protein ( n = 6)). A competitive binding assay was done using the CHO · 1F8 cell membranes and [ 3 H]WEB 2086. The minimum detectable dose of PAF was 0.3 nM (≈ 30 pg per well) and the assay was highly specific for PAF. This method makes it possible to handle large numbers of samples rapidly and simultaneously, since the receptor membrane is prepared in advance and the binding assay can be completed within 3 h. Using this method, we have determined the production and cell association of PAF in human neutrophils.

Introduction Magnetic resonance imaging (MRI) gives a much more detailed picture of the soft tissue than computerized tomography (CT). In blowout fracture cases, it is very easy to observe the incarcerated orbital tissue. Subjects We performed MRI in 19 blowout fracture cases. Results After evaluating the images, we found three advantages of MRI. The first is that even small herniation of the orbital contents can easily be detected because the orbital fatty tissue contrasts well around the other tissues in MRI. The second is that the incarcerated tissues can be clearly differentiated because a clear contrast between the orbital fatty tissue and the extraocular muscle can be seen in MRI. The third is that the running images of the incarcerated muscle belly can be observed because any necessary directional slies can be taken in MRI. Conclusion These advantages are very important in the diagnosis of blowout fractures. MRI should be employed in blowout fracture cases in addition to CT.

The reactions of methyl-, ethyl-, n-propyl-, n-butyl-, allyl-, and benzyl-magnesium halides with ethyl N-trichloroethylidenecarbamate (1) gave 1-substituted ethyl 2,2,2-trichloroethylcarbamates, which afforded α-amino-acids on hydrolysis and decarboxylation. By this method DL-alanine and DL-norleucine were obtained in 53 and 60% overall yields, respectively. When branched alkyl Grignard reagents such as isopropyl-, isobutyl- and s-butyl-magnesium bromide were used, attack on the imino-group was hindered by the trichloromethyl group and only reduction occurred to give ethyl 3,3-dichloro-1-ethoxycarbonyl-4-trichloromethylazetidin-2-ylcarbamate (4) and ethyl [2,2,2-trichloro-1-(N-ethoxycarbonyl-2,2,2-trichloroethylamino)ethyl]carbamate (5).

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum which recognizes strictly the structure of phosphatidylethanolamine (PE) and forms an equimolar complex with the phospholipid on biological membranes. To use the peptide as a probe for analyzing the transbilayer movement of PE, we labeled the amino-terminal amino acid of the peptide with biotin without changing either the reactivity or specificity of the peptide. The amount of the peptide bound to the membrane was measured by enzyme linked immunosorbent assay (ELISA) after extraction of the peptide from the membrane. The peptide showed a strict temperature-dependent binding to human erythrocytes and the binding increased with increasing temperature. Since the peptide bound to PE in a temperature-independent manner and the binding to membrane PE is not affected by membrane proteins, the present temperature-dependent binding of the peptide to the cell membranes was likely to reflect temperature-dependent translocation of PE. The binding of the peptide to erythrocytes differed greatly among animal species. The peptide also showed temperature-dependent binding to a human histocytic lymphoma cell line, U937, suggesting that the peptide will provide a novel and convenient probe for analyzing the transbilayer movement of PE in eukaryotic cells.