Search

Electroconvulsive therapy (ECT) is considered to be an effective and safe treatment for depression in pregnant women in that it avoids the risk of psychotropic pharmacotherapy. However, clinicians should be cautious about the adverse effects in the fetus, such as fetal cardiac arrhythmia. Most of the previous studies have demonstrated a reduction in fetal heart rate associated with ECT. However, we encountered a case of fetal tachycardia after maternal ECT-induced convulsions. The patient was a woman who was 30 weeks’ pregnant and had severe depression; fetal tachycardia (180–200 bpm) occurred immediately after the electrical stimulation and lasted for more than 30 minutes. The fetal tachycardia might have been caused by maternal hypoxia and uterine contractions. To our knowledge, this is the first report of fetal tachycardia as an adverse effect of ECT. Prolonged fetal tachycardia may cause fetal heart failure. Therefore, oxygenation during convulsions and careful fetal cardiac monitoring are essential when administering ECT in pregnancy.

This was a multicenter study of rituximab, a chimeric monoclonal immunoglobulin G antibody directed against CD20, for the treatment of refractory autoimmune bullous diseases (pemphigus and pemphigoid). Ten patients (three with pemphigus vulgaris, six with pemphigus foliaceus and one with bullous pemphigoid) were treated with a single cycle of rituximab (four weekly infusions at a dose of 375 mg/m2 of body surface area). The primary end-points were the number of serious adverse events and rate of complete remission at 40 weeks. Five patients (50%) achieved complete remission with minimal therapy (defined as no active lesions with lower doses of systemic corticosteroids compared to that with prednisolone 10 mg/day). Improvements in clinical scores (Pemphigus Disease Area Index) and decreases in autoantibody titers in the sera were observed in the four pemphigus patients who failed to achieve complete remission. This suggests that rituximab was effective in nine of 10 cases. Two serious adverse events (Pneumocystis carinii pneumonia and septic shock due to infectious arthritis) were observed and adequately treated with hospitalization. CD19-positive B lymphocytes in the peripheral blood decreased on day 29 following rituximab treatment, and remained at low levels throughout the observation period (280 days). Our results confirmed the efficacy of rituximab therapy for refractory autoimmune bullous diseases in Japan.

BackgroundThere is currently no subjective, definitive evaluation method for therapeutic indication other than symptoms in aortic regurgitation. Energy loss, a novel parameter of cardiac workload, can be visualized and quantified using echocardiography vector flow mapping. The purpose of the present study was to evaluate whether energy loss in patients with chronic aortic regurgitation can quantify their subjective symptoms more clearly than other conventional metrics.MethodsWe studied 15 patients undergoing elective aortic valve surgery for aortic regurgitation. We divided the patients into symptomatic and asymptomatic groups using their admission records. We analyzed the mean energy loss in one cardiac cycle using transesophageal echocardiography during the preoperative period. The relationships between symptoms, energy loss, and other conventional metrics were statistically analyzed.ResultsThere were seven and eight patients in the symptomatic and asymptomatic groups, respectively. The mean energy loss of one cardiac cycle was higher in the symptomatic group (121 mW/m [96–184]) than in the asymptomatic group (87 mW/m [80–103]) (p = 0.040), whereas the diastolic diameter was higher in the asymptomatic group (65 mm [59–78]) than in the symptomatic group (57 mm [51–57]) (p = 0.040). There was no significant difference between the symptomatic and asymptomatic groups in terms of other conventional metrics.ConclusionsAn energy loss can quantify patients' subjective symptoms more clearly than other conventional metrics. The small sample size is the primary limitation of our study, further studies assessing larger cohort of patients are warranted to validate our findings.

Background: It is well known that SARS-CoV-2 genome accumulates point mutations constantly. However, whether non-synonymous mutations affect COVID-19 severity through altering viral protein function remains unknown. Methods: We performed SARS-CoV-2 genome sequencing in 90 patients with COVID-19 admitted at Keio University Hospital in Tokyo Metropolitan area between March and August 2020. Viral haplotypes were examined by counting the number of non-synonymous mutations to analyse phylogenic trees and comparative amino acid sequence. Statistically relevant non-synonymous mutations were functionally evaluated with structural analyses. Findings: The number of non-synonymous mutations correlated inversely with the COVID-19 severity. Phylogenic tree analyses identified two predominant groups which were differentiated by a set of six point mutations (four non-synonymous amino acid mutations). Among them, Pro108Ser in 3 chymotrypsin-like protease (3CLpro) and Pro151Leu in nucleocapsid protein occurred at conserved locations among β-coronaviruses. Patients with these mutations indicated significantly lower odds ratio for developing hypoxia which required supplemental oxygen (adjusted odds ratio 0·24 [95% CI 0·07-0·88, p-value = 0·032]) after adjustments for age and sex, versus those lacking this haplotype in the canonical Clade 20B spread in Japan. The Pro108Ser 3CLpro enzyme decreases in the activity by 58%, and the hydrogen/deuterium exchange mass spectrometry reveals that mechanisms for decline-of-function involve structural perturbation at the substrate-binding region which is positioned behind and distant from the 108th amino acid residue of the enzyme. Interpretation: Viral genome sequencing in Tokyo showed that the specific mutant strain containing Pro108Ser mutation in 3CLpro, ameliorates the COVID-19 severity. This Pro108Ser mutant in 3CLpro reduces the catalytic activity of the protein by 50%. The mutant strain rapidly outcompeted pre-existing variants to become the dominant one in Japan. Our results may benefit the efforts under way to design small molecular compounds or antibodies targeting 3CLpro. Funding: Keio Gijuku Academic Development Funds and AMED (Grant Number JP20he0622043) Declaration of Interests: Authors have no conflicts of interests. Ethics Approval Statement: The study protocol was approved by the Ethics Committee of Keio University School of Medicine (approval number: 20200062).

Extramammary Paget's disease (EMPD) is a rare cutaneous adenocarcinoma that clinicopathologically resembles breast cancer. The prognosis of metastatic EMPD is poor. Although several chemotherapies have been tried, the effects are temporary; better drugs and combinations are required. In the present study, we retrospectively analyze the efficacy and safety of combination of cisplatin, epirubicin, and paclitaxel in five metastatic EMPD cases. The efficacy was better than that for previously reported regimens: 80% partial responses, including two patients who were refractory to taxane- and/or platinum-based regimens. In terms of safety, four patients who were able to continue treatment exhibited acceptable tolerability. This is the first regimen to combine taxane and anthracycline. When treating breast cancer, anthracycline is regarded as the key cytotoxic agent, and anthracycline in combination with taxane constitutes a key chemotherapeutic regimen. Given our results, we speculate both drugs are critical chemotherapeutic agents for the treatment of metastatic EMPD.

Introduction: Malignant cutaneous epithelial tumors comprise various skin malignancies originating from the cutaneous epithelium, including cutaneous squamous cell carcinoma, basal cell carcinoma, and malignant cutaneous adnexal tumors. Treatment options are limited, as the rarity of these tumors, especially among Asians, renders well-controlled clinical trials extremely challenging to conduct. Thus, we designed a clinical trial to evaluate the efficacy and safety of the anti-programmed cell death-1 (PD-1) monoclonal antibody nivolumab in patients with metastatic cutaneous squamous cell carcinomas and other rare metastatic cutaneous epithelial tumors. Methods and analysis: This is an open-label, single-arm, multicenter, phase 2 clinical trial involving patients with metastatic malignant cutaneous epithelial tumors. Nivolumab (480 mg) will be administered intravenously every 4 weeks for a maximum of 26 doses. The primary outcome of the study will be the response rate based on response evaluation criteria in solid tumors, version 1.1. Assuming a null hypothesis of a response rate ≤5% and an alternative hypothesis of a 25% response rate, a minimum of 26 patients are required to achieve a 5% two-sided type I error and 80% power based on the exact binomial distribution. Finally, a target cohort size of 30 patients was determined as some patient dropout will be expected. Discussion: This is the first phase 2 clinical trial evaluating the efficacy and safety of the PD-1 inhibitor nivolumab in Asian patients with metastatic malignant cutaneous epithelial tumors. The findings of the study will contribute to the development of novel treatment approaches for patients with rare cutaneous malignancies, which remains an unmet clinical need. Trial registration: Registry number: jRCT 2031190048

Background Vector flow mapping, a novel flow visualization echocardiographic technology, is increasing in popularity. Energy loss reference values for children have been established using vector flow mapping, but those for adults have not yet been provided. We aimed to establish reference values in healthy adults for energy loss, kinetic energy in the left ventricular outflow tract, and the energetic performance index (defined as the ratio of kinetic energy to energy loss over one cardiac cycle). Methods Transthoracic echocardiography was performed in fifty healthy volunteers, and the stored images were analyzed to calculate energy loss, kinetic energy, and energetic performance index and obtain ranges of reference values for these. Results Mean energy loss over one cardiac cycle ranged from 10.1 to 59.1 mW/m (mean ± SD, 27.53 ± 13.46 mW/m), with a reference range of 10.32 ~ 58.63 mW/m. Mean systolic energy loss ranged from 8.5 to 80.1 (23.52 ± 14.53) mW/m, with a reference range of 8.86 ~ 77.30 mW/m. Mean diastolic energy loss ranged from 7.9 to 86 (30.41 ± 16.93) mW/m, with a reference range of 8.31 ~ 80.36 mW/m. Mean kinetic energy in the left ventricular outflow tract over one cardiac cycle ranged from 200 to 851.6 (449.74 ± 177.51) mW/m with a reference range of 203.16 ~ 833.15 mW/m. The energetic performance index ranged from 5.3 to 37.6 (18.48 ± 7.74), with a reference range of 5.80 ~ 36.67. Conclusions Energy loss, kinetic energy, and energetic performance index reference values were defined using vector flow mapping. These reference values enable the assessment of various cardiac conditions in any clinical situation.

Background Staged palliative surgery markedly shifts the balance of volume load on a single ventricle and pulmonary vascular bed. Blalock-Taussig shunt necessitates a single ventricle eject blood to both the systemic and pulmonary circulation. On the contrary, bidirectional cavopulmonary shunt release the single ventricle from pulmonary circulation. Case presentation We report a case of tricuspid atresia patient who underwent first palliative surgery and second palliative surgery. Volume loading condition was assessed by energetic parameters (energy loss, kinetic energy) intraoperatively using vector flow mapping. These energetic parameters can simply indicate the volume loading condition. Conclusion Vector flow mapping was useful tool for monitoring volume loading condition in congenital heart disease surgery. Electronic supplementary material The online version of this article (10.1186/s12947-017-0118-3) contains supplementary material, which is available to authorized users.

Extreme ultraviolet lithography is one of the promising high volume manufacturing processes for devices below 7 nm node and beyond. However, resist pattern collapse is one of the hurdles in achieving a comfortable process margin/window for resist patterning. To suppress this resist pattern collapse and to improve manufacturing process margin, undoubtedly it is a well-known fact that the rinse materials can mitigate a pattern collapse at development process. In this study, we focus on the relationship between resist swelling and pattern collapse, based on the assumption of resist pattern softening induced by resist swelling and further resist pattern bending or deformation which could be classified as pattern collapse. To verify this model, two samples of Rinse-A and Rinse-B which showed similar capillary force but different resist swelling behavior expected, respectively, were prepared. And the hardness of the resist films treated using those rinse materials together with de-ionized water as reference rinse, were measured using nano-indentation measurement technique. From the result, it was found that Rinse-A had the resist film hardness reduced by 7.9 % as compared to that on de-ionized water. On the other hand, Rinse-B which was designed to suppress the penetration of additives into the resist film, exhibited comparable hardness property to that on reference rinse, de-ionized water. Finally, the evaluation of lithographic performance of the photoresist in different rinse process conditions using Rinse-A and Rinse-B was carried out, and Rinse- B achieved 12.7 nm minimum pattern size of resist feature at 16 nm half pitch printing by interference projection system on Extreme Ultraviolet lithography, whereas Rinse-A showed 16.5 nm.

Vector flow mapping, a novel flow visualization echocardiographic technology, is increasing in popularity. Energy loss reference values for children have been established using vector flow mapping, but those for adults have not yet been provided. We aimed to establish reference values in healthy adults for energy loss, kinetic energy in the left ventricular outflow tract, and the energetic performance index (defined as the ratio of kinetic energy to energy loss over one cardiac cycle). Transthoracic echocardiography was performed in fifty healthy volunteers, and the stored images were analyzed to calculate energy loss, kinetic energy, and energetic performance index and obtain ranges of reference values for these. Mean energy loss over one cardiac cycle ranged from 10.1 to 59.1 mW/m (mean ± SD, 27.53 ± 13.46 mW/m), with a reference range of 10.32 ~ 58.63 mW/m. Mean systolic energy loss ranged from 8.5 to 80.1 (23.52 ± 14.53) mW/m, with a reference range of 8.86 ~ 77.30 mW/m. Mean diastolic energy loss ranged from 7.9 to 86 (30.41 ± 16.93) mW/m, with a reference range of 8.31 ~ 80.36 mW/m. Mean kinetic energy in the left ventricular outflow tract over one cardiac cycle ranged from 200 to 851.6 (449.74 ± 177.51) mW/m with a reference range of 203.16 ~ 833.15 mW/m. The energetic performance index ranged from 5.3 to 37.6 (18.48 ± 7.74), with a reference range of 5.80 ~ 36.67. Energy loss, kinetic energy, and energetic performance index reference values were defined using vector flow mapping. These reference values enable the assessment of various cardiac conditions in any clinical situation.

Spin-on-carbon hard mask (SOC HM) has been used in semiconductor manufacturing since 45nm node as an alternative carbon hard mask process to chemical vapor deposition (CVD). As advancement of semiconductor to 2X nm nodes and beyond, multiple patterning technology is used and planarization of topography become more important and challenging ever before. In order to develop next generation SOC, one of focuses is planarization of topography. SOC with different concepts for improved planarization and the influence of thermal flow temperature, crosslink, film shrinkage, baking conditions on planarization and filling performance are described in this paper.

Global ischemia selectively induces CA1 neuronal death in the hippocampus. Pretreatment with non-lethal ischemia (i.e. ischemic preconditioning) prevents CA1 neuronal death induced by lethal ischemia. While ischemic tolerance is a well-known phenomenon, the underlying molecular mechanisms are not fully understood. Cytoskeletal proteins including α-spectrin, tau, and microtubule-associated protein 2 (MAP-2) are indispensable for the maintenance of neuronal homeostasis. Here, we report the effects of ischemic preconditioning on the ischemia-induced degradation of cytoskeletal proteins α-spectrin, tau, and MAP-2 in the rat CA1 region. We found that most neurons of the CA1 region had died after 5 min of ischemia. However, exposing the brain to 3 min of ischemic preconditioning 3 days earlier significantly reduced the number of neuronal death. A significant degradation of α-spectrin and tau, but not of MAP-2, was found in the CA1 region after 5 min of ischemia. Ischemic preconditioning attenuated the ischemia-induced massive degradation of α-spectrin and tau. Our results suggest that the attenuation of ischemia-induced degradation of α-spectrin and tau by ischemic preconditioning may be associated with the neuroprotective mechanism of the ischemic tolerance.

The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V–FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V–FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells.

Hinesol is a unique sesquiterpenoid isolated from the Chinese traditional medicine, Atractylodes lancea rhizome. In a previous study, we screened various natural products in human leukemia HL-60 cells and identified an essential oil fraction from A. lancea rhizome that exhibited apoptosis-inducing activity in these cells; hinesol was subsequently shown to be the compound responsible for this apoptosis-inducing activity. In this study, we describe the cytotoxic effects and molecular mechanisms of hinesol in HL-60 cells. The antitumor effect of hinesol was associated with apoptosis. When HL-60 cells were treated with hinesol, characteristic features of apoptosis, such as nuclear fragmentation and DNA fragmentation, were observed. These growth-inhibitory and apoptosis-inducing activities of hinesol in leukemia cells were much stronger than those of β-eudesmol, another compound isolated from the essential oil fraction. Furthermore, hinesol induced activation of c-Jun N-terminal kinase (JNK), but not p38, prior to the onset of apoptosis. These results suggested that hinesol induced apoptosis through the JNK signaling pathway in HL-60 cells. Therefore, hinesol may represent a novel medicinal drug having indications in the treatment of various cancers, including leukemia.

The object of this study was to determine the details of morphological dynamics of spermatogenesis in Syrian hamsters exposed to both short photoperiod and low ambient temperature. Eight-week-old male hamsters, kept in a long photoperiod (14 h L, 10 h D), were transferred to a short photoperiod (6 h L, 18 h D) and kept there for 13 weeks to induce testicular regression. Some hamsters were then transferred from the room at 23 degrees C to that at 5 degrees C (5 degrees C group). Remaining hamsters were continuously kept at 23 degrees C (23 degrees C group). Thereafter, the morphology was examined. As a result, it took only 8 weeks until spermatogenesis recovered in the 23 degrees C group. However, it was not until 20 weeks that spermatogenesis was recognized in the 5 degrees C group. As the regulation of seasonal testicular activity is characterized by coordinated shifts in the relationships among mitosis, meiosis, and apoptosis, the changes in the proliferative and apoptotic activities were examined. Although no significant difference in proliferative activity of spermatogonia between the 5 degrees C and the 23 degrees C groups was confirmed, a notable increase in the rate of apoptosis was observed in the 5 degrees C group. Furthermore, this increase was more salient during the hibernation period. These findings suggest that both cold ambient temperature and hibernation caused the delay of testicular recrudescence and this delay arose from the increase of apoptotic activity but not the change in proliferative activity in spermatogonia in the 5 degrees C group.

Problem Chemokine receptors of placental trophoblasts possibly act as co-receptors or alternative receptors of maternal fetal infection by HIV. To clarify their possible expression and the physiological roles of chemokines on human placentae, we studied chemokine chemokine receptor expression and the effects of exogenous chemokines on choriocarcinoma cell lines. Materials and methods Placental samples were obtained from 13 placentae of various gestational ages. Villous tissue was mechanically dissected from samples. Trophoblasts were enriched by anti-human chorionic gonadotropin (hCG)-coated magnetic beads. Human choriocarcinoma cell lines (JAR, BeWo, JEG-3) were maintained in RPMI 1640 media supplemented with 10% FCS. Expression of chemokine receptors was studied by RT-PCR. The effects of MIP-1alpha, RANTES, MCP-1 on hCG production were estimated by EIA. Effects of chemokines on proliferation of choriocarcinoma cell lines were examined by MTT assay. Results We observed mRNA expression of CCR-1, 2, 3, 4, 5 and CXCR-1, 2, 4 in 1st trimester placental villi, CCR-I, 2, 4 and CXCR-1, 2. 4 in 2nd trimester placental villi, CCR-1, 2, 4 and CXCR-4 in 3rd trimester placental villi. Using MACS enriched trophoblasts, we observed identical results. A choriocarcinoma cell line BeWo expressed CCR-1, 3, 4 and CXCR-1, 2, 4 while JEG-3 and JAR expressed CCR-1, 3, 4, 5 and CXCR-1, 2, 4. Expression of the CCR-5 and CXCR-4 protein in choriocarcinoma cell lines and MACS-enriched trophoblats were confirmed by flow cytometry. Chemokine MCP-3, MIP-1alpha, RANTES mRNA were expressed by the 1st, 2nd and 3rd trimester placental samples and the three choriocarcinoma cell lines examined. MCP-1 was expressed by 1st and 2nd trimester placental villi. Administration of chemokines up-regulated proliferation (10(-1) - 10 ng/mL) and hCG production (10(-1) - 10(-2)ng/ mL) of the three choriocarcinoma cell lines examined. Conclusions Our results suggest possible roles of chemokines/chemokine receptors on placental physiology and their involvement in HIV transmission as alternative receptors.

PROBLEM: The mechanism of immunotherapy for patients with recurrent spontaneous abortions is not well understood. In order to investigate the suppressor mechanism of paternal lymphocyte immunization, we examined peripheral blood lymphocyte subpopulations and the repertoire of T-cell receptor (TCR) gene segments. METHOD OF STUDY: Twelve patients with recurrent miscarriage were treated with immunization with paternal lymphocyte vaccinations three times during 12-14 weeks. Before and 2 weeks after the final inoculation, lymphocyte subsets and intra-cellular interferon (IFN)-gamma and/or interleukin (IL)-4 production were examined by flow cytometry. TCR V beta and V gamma repertoires were examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: We found no significant difference in CD4/CD8 ratios, prevalence of CD56+CD3+ or CD57+CD3+ cells (possible extrathymic T cells), gamma(delta)T cells, and CD5+ CD19+ (B-1) cells. However, by in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, peripheral CD4 cells demonstrated a significant decrease of IFN-gamma-producing T helper 1 (Th1) cells and an increase of IL-4-producing T helper 2 (Th2) cells after immunotherapy. Seven of nine patients who exhibited remarkable decreases in Th1/Th2 ratios became pregnant within 6 months after three courses of immunotherapy, and four women delivered healthy babies, while none of the three patients who exhibited an increased or unchanged Th1/Th2 ratio had full-term pregnancies (chi2 < 0.0001). Further, changes in usage of TCR V beta and V gamma gene segments were observed after immunotherapy in six patients examined. CONCLUSION: Our findings suggest a shift of Th1-dominant to Th2-dominant status by vaccination might play important roles in maintaining successful pregnancies. Induction of some T cells that utilize different TCR repertoires possibly suppresses maternal rejection reactions.

Thrombin activates human platelets and other cells in part by cleaving an unusual G protein-coupled receptor. Thrombin cleavage of this receptor's amino-terminal exodomain unmasks a new amino terminus. This then binds intramolecularly to the body of the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying the domains responsible for thrombin receptor-G protein interactions, we examined the signaling properties of chimeric receptors in which thrombin receptor cytoplasmic sequences replaced the cognate sequences in the Gs-coupled β2-adrenergic receptor (β2AR) or the Gi-coupled dopamine D2 receptor (D2R). In Xenopus oocytes, a chimeric β2AR bearing the thrombin receptor second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic agonist, whereas a β2AR bearing the cognate C2 loop from the D2R did not. Similarly, in COS-7 cells, a chimeric D2R bearing the thrombin receptor C2 loop gained the ability to trigger phosphoinositide hydrolysis in response to dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in several different receptor contexts. These observations suggest that the thrombin receptor C2 loop specifies Gq coupling by directly contacting Gq or by contributing to a structure required for Gq coupling. The ability of the thrombin receptor C2 loop to function in the context of the D2R and β2AR strongly suggests that the transmembrane switching and G protein activation strategies used by the thrombin receptor must be very similar to those used by the D2R and β2AR despite the thrombin receptor's strikingly different liganding mechanism.

The negative tone development (NTD) process has proven benefits for superior imaging performance in 193nm lithography. Shrink materials, such as AZ® RELACS® have found widespread use as a resolution enhancement technology in conventional 248nm (DUV), 193 nm dry (ArF) and 193 nm immersion (ArFi) lithography. Surfactant rinses, such as AZ® FIRM® are employed as yield enhancement materials to improve the lithographic performance by avoiding pattern collapse, eliminating defects, and improving CDU. This paper describes the development and recent achievements obtained with new shrink and rinse materials for application in NTD patterning processes.

Constitutively active thrombin receptors were generated while constructing chimeric receptors to identify the structural basis for thrombin receptor agonist specificity. Substitution of eight amino acids from the Xenopus receptor's second extracellular loop (XECL2B) for the cognate sequence in the human thrombin receptor was sufficient to confer robust constitutive activity. Smaller substitutions within the XECL2B site yielded less constitutive activation, and substitution of several unrelated sequences at this site caused no activation. Expression of the XECL2B receptor caused high basal 45Ca efflux in Xenopus oocytes and high basal phosphoinositide hydrolysis and reporter gene induction in COS cells. Of note, a mutant receptor in which all four of the Xenopus thrombin receptor's extracellular segments replaced the cognate human sequences showed much less constitutive activity than XECL2B and preserved responsiveness to agonist. This partial complementation of the XECL2B phenotype by addition of other Xenopus extracellular structures suggests that the XECL2B mutation causes constitutive activation by altering interactions among the human receptor's extracellular domains. Thus, a change in an extracellular loop of a G protein-coupled receptor can transmit information across the cell membrane to cause signaling, perhaps via a conformational change similar to that caused by agonist binding. Indeed, the site of the activating mutation in XECL2B coincides with a putative agonist-docking site, supporting the hypothesis that agonist interactions with the thrombin receptor's extracellular loops contribute to receptor activation.

Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn in the Xenopus receptor's amino-terminal exodomain and Glu for Leu in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg in the human agonist peptide might normally interact with Glu in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu was converted to Arg. An “arginine scan” of the receptor's extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu region and to a lesser degree Phe as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the “correct” agonist and inhibit responsiveness to an “incorrect” agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.

Cleavage of the thrombin receptor's amino-terminal exodomain at the Arg41/Ser42 peptide bond within the sequence ... LDPR41/S42FLLRN ... is necessary and sufficient for receptor activation by proteases. The synthetic peptide SFLLRN activates the receptor independent of proteolysis. We proposed that the SFLLRN sequence is a tethered peptide ligand; receptor cleavage unmasks this agonist which then binds intramolecularly to effect receptor activation. The alternative hypothesis that receptor cleavage or exogenous SFLLRN effect receptor activation by disrupting tonic inhibitory interactions exerted by the receptor's amino-terminal exodomain has not been excluded. We report that delta AMINO, a mutant thrombin receptor lacking the amino-terminal exodomain, was not constitutively active and responded to SFLLRN but not thrombin when expressed in Xenopus oocytes or mammalian cells. Thrombin signaling was restored when delta AMINO was co-expressed with ATE-CD8 which encoded the receptor's amino-terminal exodomain fused to the transmembrane domain of CD8. Co-expression of a thrombin receptor lacking a functional tethered ligand domain ("F43A") with a non-signaling receptor mutant bearing an intact tethered ligand domain ("YYY") also reconstituted thrombin signaling. However, the EC50 for thrombin activation of cells co-expressing F43A and YYY was > 1000-fold that for cells expressing comparable levels of wild type receptor, while EC50s for activation by SFLLRN were similar. These and other data refute the release from inhibition hypothesis and suggest that while intermolecular liganding between two thrombin receptor molecules can occur, the intramolecular tethered liganding mechanism is the predominant mode of thrombin receptor activation.

Eph receptors and ephrin ligands are membrane-bound cell–cell communication molecules with well-defined roles in development. However, their expression and functions in the gastric epithelium are virtually unknown. We detected several EphB receptors and ephrin-Bs in the gastric corpus mucosa of the adult rodent stomach by RT-PCR amplification. Immunostaining showed complementary expression patterns, with EphB receptors preferentially expressed in the deeper regions and ephrin-Bs in the superficial regions of the gastric units. EphB1, EphB2 and EphB3 are expressed in mucous neck, chief and parietal cells, respectively. In contrast, ephrin-B1 is in pit cells and proliferating cells of the isthmus. In a mouse ulcer model, EphB2 expression was upregulated in the regenerating epithelium and expanded into the isthmus. Thus, EphB/ephrin-B signaling likely occurs preferentially in the isthmus, where receptor-ligand overlap is highest. We show that EphB signaling in primary gastric epithelial cells promotes cell retraction and repulsion at least in part through RhoA activation. Based on these findings, we propose that the EphB-positive progeny of gastric stem cells migrates from the isthmus toward the bottom of the gastric glands due to repulsive signals arising from contact with ephrin-Bs, which are preferentially expressed in the more superficial regions of the isthmus and gastric pits.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules that regulate the spatial organisation of cells in various tissues by repulsive or adhesive signals arising from contact between EphB- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the gastric epithelium and Brunner's glands are virtually unknown. We detected several EphB receptors and ephrin-B ligands in the pyloric and duodenal mucosa of the adult mouse by RT-PCR amplification. Immunostaining showed complementary expression patterns, with ephrin-B1 being preferentially expressed in the superficial part and EphB receptors in the deeper part of both epithelia. In the gastric pylorus, ephrin-B1 was expressed in pit cells and proliferating cells of the isthmus. In contrast, EphB2, EphB3, and EphB4 were expressed in pyloric glandular cells and proliferating cells of the isthmus. In the duodenum, ephrin-B1 was expressed in cells lining the ducts of Brunner's glands as well as those covering villi and the upper portion of the crypts of Lieberkuhn. In contrast, EphB2 and EphB3 were expressed in the gland segment of Brunner's glands and the lower portion of the crypts and EphB4, in the crypts. In both mucosae, EphB2, EphB3, and EphB4 were found to be tyrosine phosphorylated, suggesting that EphB/ephrin-B signalling might occur preferentially in the isthmus, crypts, and duct-gland transition of Brunner's glands, where the receptor and ligand expression overlaps. Based on these findings, we propose that EphB/ephrin-B signalling may regulate cell positioning within the pyloric and duodenal epithelium.

Eph receptors and ephrin ligands are membrane-bound cell–cell communication molecules with important roles not only in development but also in the physiology of many adult organs. However, their cellular localization and functions in the myocardium are virtually unknown and therefore, we have investigated the expression of EphB receptors and ephrin-B ligands in the rodent heart ventricles and their functions in the rodent cardiomyocytes of primary culture. Examinations by RT-PCR, immunohistochemistry and in situ hybridization revealed that the EphB receptors are preferentially expressed in cardiomyocytes and ephrin-B ligands in the vasculature in adult mouse heart ventricles. Interestingly, we found that inducing high levels of EphB receptor activation in primary cultures of rodent cardiomyocytes by stimulation with ephrin-B1-Fc desynchronized the contraction of adjacent clusters of cardiomyocytes that had contracted synchronously before the treatment. Co-immunoprecipitation experiments revealed that EphB4 physically associates with connexin43, a major component of gap junctions in the myocardium, and that EphB activation inhibits gap junctional intracellular communication between cardiomyocytes. The present findings suggest that ephrin-B-EphB signaling can modulate the electrical coupling of cardiomyocytes through effects on gap junctions.

A 67-year-old man presented with progressive quadriplegia. Magnetic resonance imaging (MRI) revealed spinal cord compression by a thickened dura ventral to the cord from C3 to C7. The lesion was isointense on both T1- and T2-weighted images, and showed contrast enhancement on T1-weighted gadorinium-enhanced images. A diagnosis of idiopathic hypertrophic spinal pachymeningitis was confirmed histologically after anterior decompression and fusion. Only partial excision was achieved. Marked improvement of the quadriplegia was attained only after steroid therapy. At the 3-month follow-up, the patient was able to walk with a cane. The affected site showed no remission on MRI despite continuous steroid therapy. At the 2-year follow-up, the patient could walk independently.

The P19CL6 cell line is a useful model to study cardiac differentiation in vitro. However, large variations were noticed in the differentiation rates among previous reports as well as our individual experiments. To overcome the unstable differentiation, we established P19CL6-A1, a new clonal derivative of P19CL6 that could differentiate into cardiomyocytes more efficiently and stably than the parent using the double stimulation with 5-Aza and DMSO based on the previous report. We also introduced a new software, Visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms. Using P19CL6-A1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. These indicate that a combination of P19CL6-A1 and Visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.

Epidural analgesia is available for postoperative pain relief except for the patients with bleeding tendency or under anticoagulation. Intravenous fentanyl analgesia can be applied for such patients but its effect has not been evaluated enough. We compared these two methods after abdominal surgery.In the intravenous fentanyl analgesia group (group iv, n = 15), 0.7 microg x kg(-1) x hr(-1) fentanyl infusion was started during operation and decreased to 0.5 microg x kg(-1) x hr(-1) on the next morning. In the epidural analgesia group (group e, n = 15), 0.4 microg x kg(-1) x hr(-1) fentanyl and 5 ml x hr(-1) 1% mepivacaine infusion was started during operation. The VAS pain score (at rest and at coughing), the level of consciousness, respiratory and cardiovascular depression, nausea and vomiting were evaluated for 3 days.The VAS scores at rest were similar in two groups but the VAS scores at coughing were similar or lower in the group iv. In the group iv, five patients suffered from nausea and one patient had somnolence. In the group e, only one patient had nausea but two patients had hypotension.Intravenous fentanyl analgesia is safe and possibly more effective than epidural analgesia.

In Japan, the multiplication table is learned predominantly by rote learning of reciting multiplication table (which is named Kuku) with a standardized mnemonic rhymes. The Kuku is memorized intensively by oral repetitions. Therefore, it is not clear whether the neuropsychological features of the deficit of the Kuku and that of the multiplication table in Western countries after brain injury are the same or not. Here we report the case of a 58-year-old right-handed man who suffered from an inability to perform calculations following right putaminal hemorrhage. His acalculia was caused, for the most part, by his failure to retrieve the Kuku, as well as his confusion over the positions of "0" when he wrote multiple-digit numbers. An analysis of his errors of the Kuku showed strong problem-size effect, which has been found not only in the studies of multiplication deficits both in patients and healthy subjects in Western countries, but also in the Japanese studies on children. Our patient applied the commutative law of multiplication and ordinal number line etc. to compensate for his deficit. Such compensation with preserved conceptual knowledge has also been previously reported in Western patients. Moreover, our patient did not make any errors when the first operand of the Kuku was 5. Sparing of multiplication errors when the first operand is 5 has been reported in another Japanese case study on acalculia as well as in studies of healthy Japanese children; however, this has never been reported in Western studies. Therefore, it was suggested that there were similarities as well as differences between the deficits of multiplication table in Western and Japanese patients after brain injury.

Chondroitin sulfate (CS) proteoglycans bind with various proteins through CS chains in a CS structure-dependent manner, in which oversulfated structures, such as iB (IdoA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate)), D (GlcA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate)), and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the critical functional module. In this study, we examined the expression and function of three CS sulfotransferases in the developing neocortex: uronyl 2-O-sulfotransferase (UST), N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST) and dermatan 4-O-sulfotransferase-1 (D4-ST), which are responsible for the synthesis of oversulfated structures. The CS chains of the neocortex of mouse embryos contained significant amounts of D and E units that are generated by UST and 4,6-ST, respectively. UST and 4,6-ST mRNAs were expressed in the ventricular and subventricular zones, and their expression increased during late embryonic development. In utero electroporation experiments indicated that knockdown of UST and 4,6-ST resulted in the disturbed migration of cortical neurons. The neurons electroporated with the short hairpin RNA constructs of UST and 4,6-ST accumulated in the lower intermediate zone and in the subventricular zone, showing a multipolar morphology. The cDNA constructs of UST and 4,6-ST rescued the defects caused by the RNA interference, and the neurons were able to migrate radially. On the other hand, knockdown of D4-ST, which is involved in the biosynthesis of the iB unit, caused no migratory defects. These results revealed that specific oversulfated structures in CS chains play critical roles in the migration of neuronal precursors during cortical development.

Chondroitin sulfate (CS) proteoglycans are major components of the cell surface and the extracellular matrix in the developing brain and bind to various proteins via CS chains in a CS structure-dependent manner. This study demonstrated the expression pattern of three CS sulfotransferase genes, dermatan 4-O-sulfotransferase (D4ST), uronyl 2-O-sulfotransferase (UST), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), in the mouse postnatal cerebellum. These sulfotransferases are responsible for the biosynthesis of oversulfated structures in CS chains such as B, D, and E units, which constitute the binding sites for various heparin-binding proteins. Real-time reverse transcription-polymerase chain reaction analysis indicated that the expression of UST increased remarkably during cerebellar development. The amounts of B and D units, which are generated by UST activity, in the cerebellar CS chains also increased during development. In contrast, the expression of GalNAc4S-6ST and its biosynthetic product, E unit, decreased during postnatal development. In situ hybridization experiments revealed the levels of UST and GalNAc4S-6ST mRNAs to correlate inversely in many cells including Purkinje cells, granule cells in the external granular layer, and inhibitory interneurons. In these neurons, the expression of UST increased and that of GalNAc4S-6ST decreased during development and/or maturation. D4ST was also expressed by many neurons, but its expression was not simply correlated with development, which might contribute to the diversification of CS structures expressed by distinct neurons. These results suggest that the CS structures of various cerebellar neurons change during development and such changes of CS are involved in the regulation of various signaling pathways.

Mono(2-ethylhexyl) phthalate (MEHP) is a well-characterized testicular toxicant. In this study, morphological alterations of mice testes caused by repeated administrations of MEHP were examined by light and transmission electron microscopy. Prepubertal male mice were given a range of MEHP doses (600-900 mg/kg/day) for 3 consecutive days in corn oil by oral gavage. Control animals were given only corn oil. Thereafter, the testes were excised, fixed in 4% paraformaldehyde for light microscopy and/or 5% glutaraldehyde for transmission electron microscopy. Then, they were embedded, and sectioned. TUNEL analysis was done to quantify the occurrence of apoptosis in the testis. Cellular damages were also observed. Results showed that administration of 700 mg/kg of MEHP caused a significant increase in TUNEL-positive cells. At the same time, mice treated with higher doses of MEHP showed presence of degenerating (apoptotic and necrotic) spermatogenic cells. Appearance of small vacuoles in the Sertoli cell cytoplasm and displacement of spermatogenic cells were also observed. Sloughed and shed spermatogenic cells found in the tubular lumen were identified to be necrotic and apoptotic in appearance, respectively.

The role of phagocytosis in eliminating apoptotic spermatogenic cells caused by mono(2-ethylhexyl) phthalate (MEHP) was studied. Twenty-one-day-old C57Bl/6N male mice were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage and sacrificed at 1, 3, 5, 7 and 9 days after initial exposure. At the same time, the role of phagocytosis in MEHP related apoptosis was examined using microinjection of annexin V into the seminiferous tubules of living mice. Results showed that mice treated with MEHP had a lower rate of testis weight gain (lower regression line) and a significant TUNEL-positive spermatogenic cell number compared to control. However, this incident was reversible, and the number of TUNEL-positive cells returned to normal after 9 days. Mice microinjected with annexin V and later treated with MEHP showed a large amount of TUNEL-positive cells compared to mice treated with MEHP only. This clearly proves that phagocytosis plays an efficient and highly important role in eliminating dead cells in the injured testis of mice treated with MEHP.

Chondroitin sulfate is a long sulfated polysaccharide with enormous structural heterogeneity that binds with various proteins, such as growth factors, in a structure-dependent manner. In this study, we analyzed the expression of chondroitin sulfate in the postnatally developing cerebellar cortex by using three monoclonal antibodies against chondroitin sulfate, MO-225, 2H6, and CS-56, which recognize different structural domains in this polysaccharide. During the first postnatal week, the patterns of immunohistochemical staining made by these antibodies were quite similar, and the molecular layer, the granule cell layer, and Bergmann glial fibers in the external granular layer were densely stained. After postnatal day 12 (P12), the expression of 2H6 epitopes was down-regulated in the molecular layer, and the expression of CS-56 epitopes in this layer was also reduced after P16. On the other hand, the strong expression of MO-225 epitopes, GlcA(2S)β1–3GalNAc(6S) (D unit)-containing structures, remained until adulthood. These chondroitin sulfate epitopes were observed around Purkinje cells, including cell soma and dendrites. Detailed immunohistochemical analysis suggested that chondroitin sulfate was deposited between Purkinje cell surfaces and the processes of Bergmann glia. Furthermore, the amount of pleiotrophin, a heparin-binding growth factor, in the cultured cerebellar slices was remarkably diminished after treatment with chondroitinase ABC or D unit-rich chondroitin sulfate. With the previous findings that pleiotrophin binds to D unit-rich chondroitin sulfate, we suggest that the D-type structure is important for the signaling of pleiotrophin, which plays roles in Purkinje cell–Bergmann glia interaction, and that the structural changes of chondroitin sulfate regulate this signaling pathway. © 2005 Wiley-Liss, Inc.

Usp9x, an X-linked deubiquitylating enzyme, is stage dependently expressed in the supporting cells (i.e. Sertoli cells and granulosa cells) and germ cells during mouse gametogenesis. Af-6, a cell junction protein, has been identified as a substrate of Usp9x, suggesting a possible association between Usp9x and Af-6 in spermatogenesis and oogenesis. In this study, we examined the expression pattern of Af-6 and Usp9x and their intracellular localization in testes and ovaries of mice treated with or without pregnant mare serum gonadotropin (PMSG), an FSH-like hormone. In both testes and ovaries, Af-6 expression was predominantly observed in supporting cells, as well as in steroidogenic cells, but not in any germ cells. In Sertoli cells, Af-6 was continuously expressed throughout postnatal and adult stages, where both Af-6 and Usp9x were enriched at the sites of Sertoli–Sertoli and Sertoli–spermatid junctions especially at stages XI–VI. In the granulosa cells, Af-6, as well as Usp9x, was highly expressed in primordial and primary follicles, but its expression rapidly decreased after the late-secondary follicle stage. Interestingly, in PMSG-treated mice, the expression levels of Af-6 and Usp9x were synchronously enhanced, slightly in Sertoli cells and strongly in granulosa cells of the late-secondary and Graafian follicles. Such closely correlated expression patterns between Af-6 and Usp9x clearly suggest that Af-6 may be deubiquitylated by Usp9x in both Sertoli and granulosa cells. It further suggests that the post-translational regulation of Af-6 by Usp9x may be one potential pathway to control the cell adhesion dynamics in mammalian gametogenesis.

The phthalate esters have been used as plasticizers for various plastic products, and their testicular toxicity has been reported. In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of the phthalate esters, on prepubertal rat testes in vitro were examined. The testes of 20-day-old Sprague Dawley (SD) rats were cut into smaller pieces and seeded in medium, and then the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, TUNEL-positive spermatogenic cells were identified, and they gradually increased in number in time- and dose-dependent manners. Ultrastructurally, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still-functioning cell organelles, and packed cell contents in membrane-bounded bodies), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), apoptotic Sertoli cells (highly condensed nuclei and nuclear membrane lysis) and necrotic Sertoli cells (marginated chromatins along the nuclear membrane, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, based on transmission electron microscopic observations, MEHP treatment may affect spermatogenic cells, and lead them to necrosis. Thus, testicular tissue cultures and cell cultures are of advantageous for screening testicular toxicity of chemicals.

The Sertoli cell of the lesser mouse deer, Tragulus javanicus, was examined using light and transmission electron microscopy. Similar to other ruminants, a multivesicular nuclear body (MNB) and laminated smooth endoplasmic reticulum (sER) were observed in the lesser mouse deer Sertoli cell. The MNB was present within the Sertoli cell nucleus, and consisted of vesicles, irregular tubules and ribosome-like structures. It was infrequent in the lesser mouse deer, which differs from domestic ruminants. Vesicles and irregular tubules seem to contain some materials with low and/or middle electron density, and be surrounded by electron dense materials. The diameter of vesicles was between 30 nm and 180 nm. Since the MNB, though less developed compared to that of bulls and goats, was present even in the Sertoli cell nucleus of the primitive ruminant-lesser mouse deer, it should be a common structure of ruminant Sertoli cells.

In mouse gonadal differentiation, the center-to-pole Sry expression pattern suggests the regionally distinct potencies of the genital ridge, which induce testis differentiation. In this study, we examined the anteroposterior axis-dependent differences in testis-differentiation potencies by using cultures of anterior, middle, and posterior segments of the XY genital ridge. The inducible pattern of Sertoli cell differentiation showed a center-to-pole wave similar to the initial Sry expression pattern. In contrast, the ability to induce Leydig cell differentiation emanated from the anterior segment and then spread to the posterior side. These findings suggest the presence of two distinct dynamic waves in the capacity of the genital ridge to induce Sertoli or Leydig cell differentiation at early phases of testis differentiation.

During the Drosophila oogenic processes, Fat facets (Faf), an ubiquitin-specific protease essential for normal development of oocyte and eye, becomes localized at the posterior pole and is incorporated into the pole cells. This is dependent on Oskar, a key factor for pole cell determination, and suggests a role for Faf in germ cell differentiation and development. Here we show that Usp9x, an X-linked ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during mouse gonadal development in stage- and sex-dependent manners. Usp9x was first detected in PGCs at 10.5 days post coitum (dpc), and thereafter its expression both at mRNA and protein levels was enhanced in PGCs of both sexes at 11.5-13.5 dpc. In testis, Usp9x expression rapidly decreased to an undetectable level by 15.5 dpc and after birth to adult, no expression was found in any spermatogenic cells, except for weak expression in Sertoli cells. In the ovary, Usp9x expression in embryonic oocytes was also reduced at the newborn stage, its expression reappeared in oocytes at secondary follicle stage, and its products were highly accumulated in the cytoplasm of Graaffian follicles in adults. Although follicular epithelial cells also expressed Usp9x at a moderate level during postnatal development, its expression was downregulated from early secondary follicle stage. Thus, the present study is not only the first to demonstrate a conserved expression of fat facets in PGCs between mouse and fly, but also sex- and stage-dependent changes of a specific component of the deubiquitylation system during mammalian gonadal development.

The distribution of glycoconjugate in the feline hair follicle and hair was studied by light and electron microscopic histochemical methods. The hair apparatus was found to contain considerable amounts of complex carbohydrates with different saccharide residues (alpha-D-mannose, beta-D-glucose, alpha-L-fucose, beta-N-acetyl-D-glucosamine). Variations of those were detected in the plasma membrane of the hair follicle cells during the course of their differentiation and keratinization, namely, alph-D-glucose, alpha-L-fucose and beta-N-acetyl-D-glucosamine in the suprabulbar and bulbar regions. The reaction level of sialic acid residues in the plasma membrane decreased in some cell layers during the course of differentiation. The results obtained from the present study indicated that interaction between saccharide residues of neutral carbohydrates and sialyl groups during the anagen phase might contribute to cell keratinization in hair follicles and hairs. It is discussed whether the existence of glycogen in outer root sheath cells might enable these cells to provide other hair apparatus cells with energy when necessary. Moreover, it became obvious from variations in sialyl residue distribution that cell differentiation processes terminate first of all in Huxley's and Henle's layers within the suprabulbar region of the hair follicle, as followed by the hair cortex.

We exploited the mechanism underlying thrombin receptor activation to develop a novel screening method to identify peptide agonists. The thrombin receptor is activated by limited proteolysis of its amino-terminal exodomain. Thrombin cleaves this domain to unmask a new amino terminus, which then functions as a tethered peptide agonist, binding intramolecularly to the body of the receptor to trigger signaling. The thrombin receptor's amino-terminal exodomain can also donate the tethered agonist intermolecularly to activate nearby thrombin receptors. We utilized this ability by co-expressing a "tethered ligand library," which displayed the thrombin receptor's amino-terminal exodomain bearing random pentapeptides in place of the native tethered ligand together with target receptors in Xenopus oocytes. Clones that conferred thrombin-dependent signaling by intermolecular ligation of the target receptor were isolated by sib selection. Agonists for the thrombin receptor itself (GFIYF) and for the formyl peptide receptor (MMWLL) were identified. Surprisingly, the latter agonist was quite active at the formyl peptide receptor even without N-formylation, and its formylated form, fMMWLL, was more potent than the classical formyl peptide receptor agonist fMLF. In addition to identifying novel peptide agonists for targets of pharmacological interest, this method might be used to discover agonists for orphan receptors. It also suggests a possible evolutionary path from peptide to protease-activated receptors.