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Inherited thrombophilia has been suggested as a cause of micro-thrombosis resulting in femoral head necrosis in Legg-Calve' Perthes Disease (LCPD). Protein C, hepatic zymogens, acts as a thrombotic agent and regulates the coagulation cascade. Genotype variations in the promoter region of the protein C gene (T-1654C; A- 1641G & A-1476T) have been reported to be associated with plasma protein C levels and thrombotic risk. In order to clarify any association of these polymorphic variations to LCPD, we analyzed the promoter region of gene for protein C (from –1704 to –1198 nt) in 70 Japanese LCPD cases (8 early-onset & 62 lateonset type) and 93 controls. Results of direct sequencing of the protein C gene promoter region showed a significantly high incidence of T-1654C change in early-onset LCPD cases than among the late-onset LCPD and control groups. Our results, therefore, suggest a significant association between early onset LCPD and the T-1654C mutation in the protein C promoter region. Key Words: Protein C gene promoter region polymorphism, micro thrombosis, early-onset LCPD.

Purpose: The purpose of this study was to investigate the utility of automated focal plane merging with the collection of gonio-photographs with different depths of field (DOF) using an established focus-stacking algorithm., Methods: A cross-sectional study was conducted at Shimane University Hospital, Izumo, Japan. Sixteen eyes from 16 subjects from the glaucoma clinic were included in this study. Image processing was performed for the images of 16 eyes from 16 angle sector following the successful gonio-photography. The 256 sets of focus-stacked and best-focused images were prepared in random order and were compared for the DOF and informativeness to diagnose angle pathology by masked observers in each set as the subjective assessments. Moreover, the energy of the Laplacian (average |ΔI|), which is an indicator of image sharpness between the photographs with and without the focus-stacking processing was also analyzed with the Laplacian filter as the objective assessment., Results: The automated image processing was successfully performed in all stacks of images. The significant deepening of DOF and improvement of informativeness achieved in 255 (99.6%) and 216 (84.4%) images (P < 0.0001 for both, sign test) and the energy of the Laplacian also significantly increased in 243 (94.9%) images (P < 0.0001, sign test)., Conclusions: Focal plane merging by the automated algorithm can make the gonio-images deeper focus compared with the paired best-focused images subjectively and objectively, which would be useful for angle pathological assessment in clinical practice., Translational Relevance: Focal plane merging algorithm for the automated gonio-photography can facilitate the angle assessment by providing informative deep-focus image, which would be useful for glaucoma care.

Purpose: To develop a novel evaluation system for retinal vessel alterations caused by hypertension using a deep learning algorithm., Design: Retrospective study., Participants: Fundus photographs (n = 10 571) of health-check participants (n = 5598)., Methods: The participants were analyzed using a fully automatic architecture assisted by a deep learning system, and the total area of retinal arterioles and venules was assessed separately. The retinal vessels were extracted automatically from each photograph and categorized as arterioles or venules. Subsequently, the total arteriolar area (AA) and total venular area (VA) were measured. The correlations among AA, VA, age, systolic blood pressure (SBP), and diastolic blood pressure were analyzed. Six ophthalmologists manually evaluated the arteriovenous ratio (AVR) in fundus images (n = 102), and the correlation between the SBP and AVR was evaluated manually., Main Outcome Measures: Total arteriolar area and VA., Results: The deep learning algorithm demonstrated favorable properties of vessel segmentation and arteriovenous classification, comparable with pre-existing techniques. Using the algorithm, a significant positive correlation was found between AA and VA. Both AA and VA demonstrated negative correlations with age and blood pressure. Furthermore, the SBP showed a higher negative correlation with AA measured by the algorithm than with AVR., Conclusions: The current data demonstrated that the retinal vascular area measured with the deep learning system could be a novel index of hypertension-related vascular changes., (© 2021 by the American Academy of Ophthalmology.)

Background: Luteal phase support (LPS) is essential for hormone replacement therapy (HRT) for frozen-thawed embryo transfer (FET). However, the optimal dose and serum progesterone (P4) levels required for pregnancy are controversial. We attempted to determine the association between pregnancy outcomes and serum P4 levels administered via vaginal suppository for HRT-FET cycles on embryo transfer day., Materials and Methods: This was a secondary analysis of the dataset from the EXCULL trial, which prospectively investigated pregnancy outcomes of four different P4 vaginal suppositories (Lutinus, Utrogestan, Luteum, and Crinone) for HRT-FET. It was conducted at a private fertility clinic between December 2016 to December 2017. During this trial, 235 cycles were divided into four groups based on serum P4 values (quartile [Q] 1 group: <7.8 ng/mL; Q2 group: 7.8-10.8 ng/mL; Q3 group: 10.8-13.7 ng/mL; Q4 group: >13.7 ng/mL). We investigated clinical pregnancy rate (CPR), positive fetal heart rate (FHR), live birth rate (LBR), and miscarriage rate (MR) for each group. A logistic regression analysis was performed using age, body mass index (BMI), and transferred embryos as covariates., Results: Serum P4 values (ng/mL) of each drug were as follows: Lutinus, 13.3 ± 4.9; Utrogestan, 9.3 ± 3.3; Luteum, 13.6 ± 4.2; and Crinone, 8.7 ± 3.2 (mean ± standard deviation, P<0.001).The percentages of Utrogestan and Crinone were higher in the Q1 group, while the percentages of Lutinus and Luteum were higher in the Q4 group. Nonetheless, there were no statistical differences between the Q1 and Q4 groups in CPR, FHR, LBR, and MR., Conclusion: When vaginal P4 was used for FET, although serum P4 levels on transfer day differed based on the drug that was administered, no relationship was observed between serum progesteronelevels and pregnancy outcomes (Registration number: UMIN000032997)., Competing Interests: The authors declare no conflicts of interest., (Copyright© by Royan Institute. All rights reserved.)

Purpose: The time-lapse system is a device that allows continuous monitoring without removing embryos from the incubator. Using a time-lapse system, we retrospectively investigated cleavage speed time points as potential indicators for selecting high-quality viable blastocysts., Methods: This study included 963 zygotes of two pronuclei retrieved from 196 patients between January 2015 and December 2016. All embryos in culture were monitored by time-lapse after intracytoplasmic sperm injection. Of 492 blastocysts developed in vitro, 128 vitrified-warmed single blastocyst transfers were classified into pregnancy and non-pregnancy groups, and the parameters were compared., Results: In the pregnancy group, timing of both morula compaction and regular blastocyst formation was significantly faster than in the non-pregnancy group. Furthermore, the optimal cutoff values for compacted morula (94.9 hours) and regular blastocyst (113.9 hours) were determined using the receiver operator characteristic curve analysis. Embryos that formed compacted morulae within 94.9 hours and developed into regular blastocysts within 113.9 hours were associated with a significantly higher pregnancy rate than those that did not (44.4% vs 16.0%)., Conclusion: The timing of morula compaction and regular blastocyst formation is important as an indicator of high-quality blastocysts to increase odds for pregnancy after embryo transfer., Competing Interests: Conflict of interest: The authors declare no conflict of interest. Human rights statements and informed consent: All procedures were followed in accordance with ethical standards of the institutional ethical committee and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients in this study, and the study design was approved by the ethics committee of Kinutani Women's Clinic, Hiroshima, Japan. Animal studies: This article does not contain any study with animal participants that was performed by any the authors., (© 2019 The Authors. Reproductive Medicine and Biology published by John Wiley & Sons Australia, Ltd on behalf of Japan Society for Reproductive Medicine.)

Purpose: To investigate the efficacy of four vaginal progesterones, Lutinus, Utrogestan, Luteum, and Crinone, as luteal phase support (LPS) in frozen-thawed embryo transfer (Frozen-ET) cycles., Methods: Patients undergoing autologous Frozen-ET of one cleavage-stage embryo or one blastocyst. Two hundred fifty-nine Frozen-ET cycles were randomized to four groups for LPS: Lutinus, Utrogestan, Luteum, and Crinone. The clinical pregnancy rate (CPR), fetal heartbeat rate (FHR), and miscarriage rate (MR) were analyzed using the Mann-Whitney or Kruskal-Wallis test and Fisher exact test., Results: Two hundred thirty-five Frozen-ET cycles were analyzed: 63 cycles in the Lutinus group, 60 in the Utrogestan group, 56 in the Luteum group, and 56 in the Crinone group. No significant differences were observed between the four groups in CPR (Lutinus, Utrogestan, Luteum, and Crinone: 34.9%, 33.3%, 37.5%, and 35.7%, respectively; P  = .976), FHR (26.9%, 31.6%, 30.3%, and 25.0%, respectively; P  = .857), and MR (31.8%, 10.0%, 19.0%, and 30.0%, respectively; P  = .306). Multivariate logistic regression analysis also revealed that there were no statistically significant differences between the four groups with regard to CPR, FHR, and MR., Conclusion: There was no clinically significant difference in pregnancy outcomes between the four vaginal progesterone groups for LPS in Frozen-ET cycles., Competing Interests: None. Human rights statement and informed consent: This study was approved by the Ethical Committee of Kinutani Women's Clinic (ethical review number 2016‐1110‐1) and registered in the UMIN Clinical Trials Registry (name of the trial register: exploratory test to investigate the CPR between four vaginal progesterones: Lutinus vaginal tablet (300 mg/d), Utrogestan vaginal capsule (600 mg/d), Luteum vaginal suppository (800 mg/d), and Crinone vaginal gel (90 mg/d), registration number: UMIN000032997). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients included in the study. Animal studies: This article does not contain any study with animal participants that have been performed by any of the authors., (© 2019 The Authors. Reproductive Medicine and Biology published by John Wiley & Sons Australia, Ltd on behalf of Japan Society for Reproductive Medicine.)

We report a case of rocuronium-induced anaphylaxis in a previously healthy 7-year-old boy. The first presenting sign of anaphylaxis was bronchospasm, appearing 11 min after he received intravenous doses of rocuronium (1 mg/kg) (Eslax ® , MSD Co. Ltd., Tokyo, Japan), propofol (2 mg/kg), and cefazolin sodium (25 mg/kg). After the administration of adrenalin and ephedrine hydrochloride, bronchospasm resolved, and the vital signs became stable. Percutaneous pinning of his left humeral supracondylar fracture was performed without problems. The next day, he was successfully liberated from the ventilator support and discharged on the fifth hospital day. On the 76th postoperative day, we performed intradermal tests of rocuronium, propofol, and cefazolin. It showed that diluted rocuronium alone induced 14 mm of flare and 8 mm of wheal within 5 min, both of which disappeared within 15 min after the intradermal injection. The reaction was too quick to mention the possible contribution of rocuronium-specific IgE. His rapid reaction at the rocuronium skin test and anaphylactic reaction upon the first exposure to this drug may highlight the association of rocuronium anaphylaxis with IgE independent mast cell stimulation through mas-related G-protein coupled receptor X2 (MRGPRX2 receptor).

Decoding sequence information is equivalent to elucidating the design principles of proteins. For this purpose, we conducted systematic alanine insertion analysis to reveal the regions in the primary structure where the sequence continuity cannot be disrupted. We applied this method to dihydrofolate reductase (DHFR), and examined the effects of alanine insertion on structure and the enzymatic activity by solubility assay and trimethoprim resistance, respectively. We revealed that DHFR is composed of "Structure Elements", "Function Elements" and linkers connecting these elements. The "Elements" are defined as regions where the alanine insertion caused DHFR to become unstructured or inactive. Some "Structure Elements" overlap with "Function Elements", indicating that loss of structure leads to loss of function. However, other "Structure Elements" are not "Function Elements", in that alanine insertion mutants of these regions exhibit substrate- or inhibitor-induced folding. There are also some "Function Elements" which are not "Structure Elements"; alanine insertion into these elements deforms the catalytic site topology without the loss of tertiary structure. We hypothesize that these elements are involved essential interactions for structure formation and functional expression. The "Elements" are closely related to the module structure of DHFR. An "Element" belongs to a single module, and a single module is composed of some number of "Elements." We propose that properties of a module are determined by the "Elements" it contains. Systematic alanine insertion analysis is an effective and unique method for deriving the regions of a sequence that are essential for structure formation and functional expression.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB&#95;4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

Background: Automatic recognition of relations between a specific disease term and its relevant genes or protein terms is an important practice of bioinformatics. Considering the utility of the results of this approach, we identified prostate cancer and gene terms with the ID tags of public biomedical databases. Moreover, considering that genetics experts will use our results, we classified them based on six topics that can be used to analyze the type of prostate cancers, genes, and their relations., Methods: We developed a maximum entropy-based named entity recognizer and a relation recognizer and applied them to a corpus-based approach. We collected prostate cancer-related abstracts from MEDLINE, and constructed an annotated corpus of gene and prostate cancer relations based on six topics by biologists. We used it to train the maximum entropy-based named entity recognizer and relation recognizer., Results: Topic-classified relation recognition achieved 92.1% precision for the relation (an increase of 11.0% from that obtained in a baseline experiment). For all topics, the precision was between 67.6 and 88.1%., Conclusion: A series of experimental results revealed two important findings: a carefully designed relation recognition system using named entity recognition can improve the performance of relation recognition, and topic-classified relation recognition can be effectively addressed through a corpus-based approach using manual annotation and machine learning techniques.

We describe a system that extracts disease-gene relations from Medline. We constructed a dictionary for disease and gene names from six public databases and extracted relation candidates by dictionary matching. Since dictionary matching produces a large number of false positives, we developed a method of machine learning-based named entity recognition (NER) to filter out false recognitions of disease/gene names. We found that the performance of relation extraction is heavily dependent upon the performance of NER filtering and that the filtering improves the precision of relation extraction by 26.7% at the cost of a small reduction in recall.

Linearly polarized light in the near-infrared portion of the spectrum has recently been associated with a variety of musculoskeletal disorders including temporomandibular disorders. The purpose of this study was to determine whether short-term linearly polarized near-infrared light radiation in the trigeminal region affects sensory and pain perception thresholds in the trigeminally mediated region and in the cervically mediated region of normal subjects. Thirty-five normal female volunteers participated in this study. Each subject received an 8-minute course of irradiation in the right cheek, and sensory/nociceptive perception thresholds were compared before and immediately after the irradiation in the right cheek and the right forearm. As a result, this study demonstrated a significant elevation of the heat-induced pain threshold in both regions and a tendency for the warm sensation threshold to elevate in the cervical region. In addition, a significant increase in vibratory sensitivity was observed in the trigeminal region. In conclusion, our results provided additional evidence that the warming sensation has a negative feedback influence on heat pain intensity in humans, and provides a theoretical basis for the application of linear polarized near-infrared light radiation to the trigeminal region.

We implanted 60 posterior stabilized total knee prostheses (P.F.C. Sigma, DePuy, Warsaw, USA). In 30 cases, we used a CT-free navigation system (Vector Vision, Brain LAB, Heimstetten, Germany), and in 30 matched-paired controls, we used a conventional manual implantation. We compared postoperative long-leg radiographs in the two groups. The results revealed a significant difference in favor of navigation. In addition, we compared the preoperative anteroposterior dimension of the femoral condyle with the postoperative value. While there were no significant differences in the preoperative anteroposterior dimension of the femoral condyle between the two groups, the postoperative value in the navigation group was significantly larger than that of the preoperative value. Therefore, surgeons using navigation systems should guard against the possibility of oversizing when determining the size of the femoral component.

The purpose of this study was to compare hardness characteristics of the masseter muscle to those of the biceps brachii muscle during repetitive muscle movements. Seventeen asymptomatic female subjects participated in this study. Each subject, on separate days, undertook a 5-minute unilateral chewing gum task on the right side and a 5-minute flexion-extension exercise on the right hand with a 2kg dumbbell. Using a handheld hardness meter, muscle hardness was measured in the right masseter and in the biceps brachii muscle at eight time points (before the task, immediately after the task, and at 1, 3, 5, 10, 30, and 60 minutes after the task), and the data obtained before and after the task on each muscle were compared. Comparisons of the normalized data were also performed between the two muscles at each time point. As a result, a significant increase in muscle hardness was seen at 1 minute after the task in the biceps brachii muscle (p=0.0093). In contrast, the masseter muscle showed a tendency to lower hardness, with the lowest point of hardness occurring at 10 minutes after the task (p = 0.0160). Between the two muscles, there was a difference in the normalized data immediately after the task, and at 1, 5, and 10 minutes after the task (0.01

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology., Competing Interests: The authors have declared that no conflicts of interest exist.

Today, physical therapy is recognized as an effective, reversible, and conservative treatment for temporomandibular disorders (TMD). The purpose of this investigation is to explore the feasibility of utilizing counter irritation with ischemic pain at a remote site outside of the head and neck region as a method for restoring muscle force in a course of physical therapy. Twenty healthy asymptomatic female subjects were recruited for this study. The experiments were performed over two days, with the two experimental days randomly assigned to the experiment performed with or without counter irritation. The counter irritation was applied to the subject's left hand using a submaximal effort tourniquet procedure. The maximal bite, finger-pinch, and handgrip forces were measured on the right side, and the results from the days with and without the counter irritation were compared. As a result, a significantly higher mean maximal bite force and a trend toward higher finger-pinch force were observed with the irritation than without the irritation, while there was no significant difference in the handgrip force. These findings indicate that counter irritation outside of the head and neck may be useful for increasing bite force, and may be applicable in the treatment of TMD for the restoration of masticatory muscle force.

This study was designed to evaluate the intra-examiner and inter-examiner reliability of measurements of masticatory muscle hardness and to confirm that the muscle hardness increases with contraction using a commercially available muscle meter. Twenty healthy asymptomatic female subjects participated in this study. Hardness was expressed as numerical relative values (0-100). First, muscle hardness was measured at a standardized point located in the masseter muscle and temporal muscle in a randomized order by two examiners, and again by one of the same examiners after ten minutes for the reproducibility study. Then the muscle hardness was measured at each point for 0 kgf, 3 kgf, 6 kgf, and 9 kgf levels of bite force. As a result, intraclass correlation coefficient (ICC) analysis revealed good intra-examiner reliability in the masseter muscle (ICC = 0.711), good intra-examiner reliability in the temporal muscle (ICC = 0.643), good inter-examiner reliability in the masseter muscle (ICC = 0.631), and unacceptable inter-examiner reliability in the temporal muscle (ICC = 0.008). Also, our results showed that muscle hardness increased with contraction, and relationships with a slope of 1.229, a y-intercept of 62.513, and a correlation coefficient of 0.448 were observed in the masseter muscle. However, no correlation was found between muscle hardness and bite force in the temporal muscle. The findings indicate that measurement of hardness provides reliable physiological information about the masseter muscle in this setting.

Electromyographic turns, or reversals of direction, per second (TS) and mean amplitude per turn (AT) are variables that have been widely used to assist and support an electrodiagnosis in neuromuscular disorders. The purpose of this study was to explore the feasibility of utilizing the TS and AT in conjunction with surface electrodes as a method for assessing masticatory muscle function. Ten normal subjects were instructed to exert brief isometric contractions at various levels, and the TS and AT were calculated from the surface electromyographic signals of the masseter muscle. Our results showed that the AT increased with contraction at all contraction levels, demonstrating a linear relationship, while the TS showed a nonlinear increase characterized by an initial steep rise followed by a plateau, which was simulated with a third polynomial function. It is considered that the TS and AT may provide valuable physiological information about the underlying mechanisms of recruitment and the firing of motor units.

The purpose of this study was to examine whether or not temporomandibular disorder (TMD) patients with chronic masticatory myalgia have increased pain sensitivity at remote sites outside of the head and neck region, and to evaluate whether the endogenous pain inhibitory systems triggered ischemic pain functions favorably in those patients. Twenty female TMD patients with chronic myalgia and 20 controls participated in this study. Ischemic pain was produced to activate endogenous opioids. The pain threshold time, pain tolerance time, pain intensity and pain unpleasantness were compared between the TMD patients and controls. The pressure pain thresholds in the hand were also compared before, between, and immediately after the ischemic pain. The TMD patients showed higher severe pain intensity and unpleasantness values and had lower pressure pain thresholds in the hand. Although both groups showed an increase in the pressure pain threshold, there was less of an increase in the pressure pain threshold in the TMD patients than in the controls. These findings indicate that TMD patients have increased pain sensitivity at remote sites, and also indicate additional evidence that the endogenous opioid systems may become impaired in TMD patients with chronic masticatory myalgia.

A high rate of induction (9 of 10 cases) of human autologous cytotoxic T lymphocytes (CTL) was achieved in vitro from peripheral blood mononuclear cells of renal carcinoma patients by applying an interleukin (IL)-cocktail consisting of IL-1, -2, -4, and -6. The CTL specifically lysed their own target carcinoma cells within 24 h but did not kill neighboring autologous normal kidney cells or allogeneic renal cancer cell lines. In the case of TUHR4TKB, for which autologous CTL were not induced, no expression of MHC class-I molecules was observed on the surface of these carcinoma cells, although they were sensitive to autologous natural killer cells. The results imply that adoptive immunotherapy for metastasized renal carcinoma will be feasible with autologous CTL in combination with natural killer cells.

Tumor-specific human cytotoxic T lymphocytes (CTL) were induced by co-culturing peripheral blood mononuclear cells with X-ray-irradiated human lung squamous carcinoma cells, SQ-5, in the medium supplemented with interleukin(IL)-1, IL-2, IL-4 and IL-6, and 5% autologous plasma for 3 or 5 days. The CTL grew in serum/plasma-free medium containing these four interleukins and 0.5% bovine serum albumin for over a month and maintained killing activity of target cells within 48 h at an effector/target ratio of 1.25. Their growth was essentially dependent on the target SQ-5 cells, which were renewed every 5 days. Under these conditions, IL-4 and IL-6 could be omitted. When anti-CD3 monoclonal antibody was added to the serum/plasma-free medium supplemented with IL-1 and IL-2, the target tumor cells were not required to maintain the specific killing activity of the CTL. A large number of CTL (10(11)) were obtained in 35 days.

Activin A stimulated DNA synthesis and transient c-fos expression in quiescent Swiss 3T3 fibroblasts. The activin A-induced DNA synthesis was dose-dependent with a half-maximal effect obtained at 0.3 nM. The maximal response obtained at 10 nM was comparable with that induced by 5 ng/ml basic fibroblast growth factor. Swiss 3T3 fibroblasts expressed abundant high affinity binding sites for 125I-labeled activin A with a Kd value of 0.63 nM and the number of binding sites at 24,000/cell. Northern blot analysis revealed that Swiss 3T3 fibroblasts express a high level of type II activin receptor mRNA. In an attempt to elucidate the mechanism of mitogenic action of activin A, we examined the effect of activin A on mitogen-activated protein kinase activation. Unexpectedly, however, activin A did not induce kinase activation under conditions in which basic fibroblast growth factor and endothelin-1 at similar or even less potent mitogenic concentrations did. Furthermore, activin A did not induce phosphorylation of the Erk2 species of mitogen-activated protein kinase. These observations strongly suggest that the activation of mitogen-activated protein kinase is not a necessary step for activin A-induced DNA synthesis in Swiss 3T3 fibroblasts.

1. The vascular effects of endothelin and localization of endothelin-like immunoreactivity were characterized in isolated cerebral arteries of dogs. 2. Endothelin-like immunoreactivity was detected in a few populations of endothelial cells of dog basilar artery. 3. Endothelin-1, endothelin-2 and endothelin-3 contracted isolated ring preparations of cerebral arteries in a dose-dependent manner independently of the presence of endothelium. The ED50 values (and 95% confidence intervals) for the contraction were 411 pM (242-697 pM) and 478 pM (295-776 pM) for endothelin-1 and endothelin-2, respectively. Endothelin-3 induced vascular contraction at a higher concentration (ED50 = 26.5 nM, 95% confidence interval = 15.7-45.7 nM). 4. The increases in tone induced by endothelin-1 and endothelin-2 did not return to the resting level after repeated washings, while a rinse with Krebs solution reversed the vasoconstrictor response to endothelin-3. The endothelins did not cause any vasodilator response in arteries precontracted with uridine 5'-triphosphate even in the presence of intact endothelial cells. 5. NiCl2 (1 mM) attenuated the contractions induced by endothelin-3 (10-300 nM) and those to relatively low doses (1 nM) but not higher doses (10-100 nM) of endothelin-1 and endothelin-2. The contractions in response to endothelin-1, endothelin-2 and endothelin-3 were greatly attenuated in Ca(2+)-free solutions although high concentrations of endothelin-1 and endothelin-2 still evoked contractions. 6. These results suggest that the vasoconstriction induced by endothelin-3 and lower doses of endothelin-1 and endothelin-2, largely depends on the influx of Ca2+ ions. The apparent insensitivity to Ni2+ shows that additional distinct mechanisms also operate in the vasconstrictor responses to high concentrations of endothelin-1 and endothelin-2. 7. The presence of endothelin-like immunoreactivity in endothelial cells suggests that endothelin is a potential endogenous spasmogen.

In a few populations of endothelial cells of dog basilar arteries, endothelin (ET)-like immunoreactivity was detected to be present. ET-1, ET-2, and ET-3 caused vasoconstrictor responses but not vasodilator responses in isolated ring preparations of dog cerebral arteries in vitro. The ED50 values for the contractions were 411 pM, 478 pM, and 26.5 nM for ET-1, ET-2, and ET-3, respectively. NiCl2 (10(-3) M) attenuated the contractions induced by ET-3 (10(-8)-3 x 10(-7) M) and those to relatively low doses (10(-9) M) but not higher doses (10(-8)-10(-7) M) of ET-1 and ET-2. The contractions in response to ET-1, ET-2, and ET-3 were greatly attenuated in Ca(2+)-free solutions, although high concentrations of ET-1 and ET-2 still evoked contractions. These results suggest that the vasoconstriction induced by ET-3 and lower doses of ET-1 and ET-2 largely depends on the influx of Ca2+ ions. Furthermore, additional distinct mechanisms may contribute to the vasoconstrictor effects of high concentrations of ET-1 and ET-2. The presence of endothelin-like immunoreactivity in endothelial cells suggests that endothelin is a potential endogenous spasmogen.

Since the discovery of endothelin-1 (ET-1), its involvement in cerebral vasospasm after subarachnoid hemorrhage (SAH) has been suspected. We performed various experiments, first to demonstrate the presence of ET in both patients and dogs with SAH, and second to examine the effects of ET synthesis inhibition in experimental vasospasm. Here we report that ET was present in both plasma and cerebrospinal fluid (CSF) in SAH, but did not correlate with vasospasm. However, ET was locally expressed in the vascular endothelium in vasospasm. Several therapeutic approaches causing the inhibition of ET synthesis were effective in preventing the development of vasospasm. Such approaches utilized drugs that inhibited RNA and DNA synthesis. Among them, actinomycin D treatment was most effective. We also utilized phosphoramidon, a recently found conversion inhibitor of big ET to ET. However, this product failed to ameliorate the development of vasospasm. Therefore, although we cannot yet conclude that ET is the main cause of cerebral vasospasm, it may, at least, act as one of the modifying factors in cerebral vasospasm.

Endothelin, a recently discovered endothelium-derived peptide, has been reported to produce potent vasoconstriction in various vessels of experimental animals. To study the involvement of endothelin in the regulation of vascular tonus in humans, isolated human mesenteric arteries were investigated by both pharmacological and immunohistochemical methods. The vasoconstrictor action of endothelin-1 was examined on ring segments of human mesenteric arteries. Endothelin-1 induced a slowly developing and sustained contraction, with an EC50 value (half-maximal effective concentration) of 2.9 x 10(-9) M, two orders of magnitude smaller than that of norepinephrine (EC50 of 3.9 x 10(-7) M), indicating that the vasoconstrictor action of endothelin-1 is about 100 times more potent than that of norepinephrine. The contractile effect of endothelin-1 was affected neither by adrenergic, cholinergic, histaminergic, nor serotonergic antagonists, nor by inhibitors of arachidonic acid metabolism. The vasoconstrictor response to endothelin-1 was effectively antagonized by nicardipine, a dihydropyridine Ca2+ channel blocker. Endothelin-1 profoundly augmented contractile response to Ca2+ in partially depolarized tissues. Immunohistochemical studies revealed for the first time that endothelin-like immunoreactivity was localized in endothelial cells of human mesenteric artery. The results of the present study indicate that endothelin-1 is one of the most potent vasoconstrictors in the human mesenteric artery and that it induces vasoconstriction via an ultimately accelerating Ca2+ influx through voltage-dependent Ca2+ channels. Since endothelin-1 can be located in human endothelial cells, it may play an important physiological or pathophysiological role.

The rate of elimination and the fate of endothelin-1 (ET-1) from the circulating blood was studied in urethane-anesthetized rats by intravenous injection of [125I]-labeled ET-1. The vasoconstrictor activities of the iodinated ET-1 were confirmed to be similar to those of native ET-1. Following i.v. bolus injection of 30 pmol/kg of [125I]-ET-1 into the femoral vein, the total radioactivity of the right atrial blood decayed rapidly, with a half-life of 7 min. At 5 min after the injection, the administered radioactivity distributed chiefly to the parenchyma of the lungs, kidneys, and liver. The analysis of the chemical form of labeled peptides from the plasma by reverse-phase high-performance liquid chromatography (HPLC) demonstrated no appreciable amount of degraded forms of [125I]-ET-1 in the blood for up to 60 min. [125I]-ET-1 was also stable for up to 60 min upon incubation in vitro with heparinized rat blood at 37 degrees C. Even when the same amount of labeled ET-1 was injected together with a pressor dose (1,500 pmol/kg) of cold ET-1, the half-life of the radioactivity in the bloodstream was exactly identical to that for [125I]-ET-1 alone. Nevertheless, the pressor response continued for more than 90 min after i.v. bolus injection of 1500 pmol/kg of ET-1 to the rat. These results clearly indicate that the elimination of ET-1 from circulating blood and the ET-1-induced pressor response are not in parallel, and the relatively rapid disappearance of ET-1 from the bloodstream is mostly due to the removal of the peptide by the parenchymal tissues, in the anesthetized rat. The long-lasting pressor action of ET-1 may be ascribed to our previous finding that the dissociation of ET-1 from its specific binding sites on vascular smooth muscle cells is extremely slow.