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After activation, some invariant natural killer T (iNKT) cells are differentiated into Klrg1 + long-lived effector NKT1 cells. However, the regulation from the effector phase to the memory phase has not been elucidated. Zeb2 is a zinc finger E homeobox-binding transcription factor and is expressed in a variety of immune cells, but its function in iNKT cell differentiation remains also unknown. Here, we show that Zeb2 is dispensable for development of iNKT cells in the thymus and their maintenance in steady state peripheral tissues. After ligand stimulation, Zeb2 plays essential roles in the differentiation to and maintenance of Klrg1 + Cx3cr1 + GzmA + iNKT cell population derived from the NKT1 subset. Our results including single-cell-RNA-seq analysis indicate that Zeb2 regulates Klrg1 + long-lived iNKT cell differentiation by preventing apoptosis. Collectively, this study reveals the crucial transcriptional regulation by Zeb2 in establishment of the memory iNKT phase through driving differentiation of Klrg1 + Cx3cr1 + GzmA + iNKT population., (© 2023. Springer Nature Limited.)

Bevacizumab, a monoclonal antibody against vascular endothelial growth factor, may be associated with arterial embolisms. We herein report a case of acute myocardial infarction caused by coronary embolism during combination chemotherapy with mFOLFOX-6 and bevacizumab in a patient with metastatic colon cancer. Thromboembolism occurred only in the distal right posterolateral branch without stenotic lesions or plaque rupture in the proximal branch of the right coronary artery. Sole thromboaspiration was successfully performed; the final angiogram demonstrated no stenosis in the right coronary artery. Bevacizumab may be associated with acute coronary syndrome in patients with coronary risk factors, despite no significant coronary narrowing.

Background: A key to success of cancer immunotherapy is the amplification and sustenance of various effector cells. The hallmark of prominent antitumor T cells is their long-term effector function. Although interleukin (IL)-2 is an attractive cytokine, several attempts have been made towards developing IL-2 modalities with improved effectiveness and safety that enhance natural killer (NK) cells or T cells in cancer models. However, whether such IL-2 modalities can simultaneously support long-term innate and adaptive immunity, particularly stem-like memory, has not been shown. To resolve this issue, we compared the antitumor cellular mechanism with two IL-2/anti-IL-2 complexes (IL-2Cxs) administered in combination with a therapeutic cancer vaccine, which we had previously established as an in vivo dendritic cell-targeting therapy., Methods: Two types of IL-2Cxs, CD25-biased IL-2Cx and CD122-biased IL-2Cx, together with a Wilms' tumor 1-expressing vaccine, were evaluated in a leukemic model. The immunological response and synergistic antitumor efficacy of these IL-2Cxs were then evaluated., Results: When CD25-biased or CD122-biased IL-2Cxs in combination with the vaccine were assessed in an advanced-leukemia model, the CD122-biased IL-2Cx combination showed 100% survival, but the CD25-biased IL-2Cx did not. We first showed that invariant natural killer T (NKT) 1 cells are predominantly activated by CD122-biased IL-2Cx. In addition, in-depth analysis of immune responses by CD122-biased IL-2Cx in lymphoid tissues and the tumor microenvironment revealed a dramatic increase in the distinct subsets of NK and CD8 + T cells with stem-like phenotype (CD27 + Sca-1 hi , CXCR3 hi , CD127 + TCF-1 + T-bet + Eomes + ). Moreover, CD122-biased IL-2Cx combination therapy maintained long-term memory CD8 + T cells capable of potent antitumor protection. After the high dimensional profiling analysis of NK and CD8 + T cells, principal component analysis revealed that the stem-like-NK cell and stem-like-CD8 + T cell state in the combination were integrated in the same group., Conclusions: CD122-biased IL-2Cx combined with the vaccine can induce a series of reactions in the immune cascade, including activation of not only NKT1 cells, but also NK and CD8 + T cells with a stem-like memory phenotype. Since it can also lead to a long-term, strong antitumor response, the combination of CD122-biased IL-2Cx with a vaccine may serve as a potential and competent strategy for patients with advanced cancer., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Safe and oncologically acceptable segmentectomy outcomes were reported for early-stage lung cancer. The high-resolution computed tomography allowed us to find detailed structures inside the lungs, such as the pulmonary ligaments (PLs). Hence, we have presented the relatively anatomically challenging thoracoscopic segmentectomy, for the resection of the lateral basal segment, the posterior basal segment, and both segments through the PL as a PL approach. This study aimed to retrospectively examine the lung lower lobe segmentectomy, excluding the superior and basal segments (from S7 to S10), using the PL approach as an option to treat the lower lobe tumors of the lung. We then compared the efficacy of the PL approach in terms of safety with the interlobar fissure (IF) approach. The characteristics of the patients, intra- and postoperative complications, and surgical outcomes were analyzed., Methods: Of the 510 patients who underwent segmentectomy for malignant lung tumors from February 2009 to December 2020, 85 were included in this study. Among them, 41 underwent a complete lung lower lobe thoracoscopic segmentectomy, excluding S6 and basal segments (from S7 to S10), using the PL approach, and the remaining 44 used the IF approach., Results: The median age in 41 patients in the PL group was 64.0 years (range, 22-82), and that in 44 patients in the IF group was 66.5 years (range, 44-88), with significant differences in gender between these groups. Video-assisted thoracoscopic surgery and robot-assisted thoracoscopic surgery were performed on 37 and 4 patients in the PL group and 43 and 1 patient in the IF group, respectively. Postoperative complication frequency was not significantly different between these groups. The most common complications were the air leaks that persisted for over 7 days in 1 and 5 patients in the PL and IF groups, respectively., Conclusions: Complete thoracoscopic segmentectomy of the lower lobe, excluding S6 and basal segments, using the PL approach is a reasonable option for lung lower lobe tumors compared with the IF approach., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-22-1535/coif). MK has received honoraria from Intuitive Surgical Japan. The other authors have no conflicts of interest to declare., (2023 Journal of Thoracic Disease. All rights reserved.)

Bioprosthetic aortic valves have limited durability. We herein report sudden progression of structural valve deterioration (SVD) and a successful case of emergency transcatheter aortic valve (TAV) implantation for acute decompensated heart failure (ADHF) caused by SVD. A 79-year-old man who had undergone a Bentall operation 11 years prior was diagnosed with ADHF due to suddenly progressive SVD. Emergency TAV implantation in the surgical bioprosthetic valve was selected based on the surgical risk. Ours and previous case reports suggest that SVD can progress suddenly, even after months of stability, and that emergency TAV implantation is effective.

Background: The high resolution of computed tomography has found the pulmonary ligaments that consists of a double serous layer of visceral pleura, forms the intersegmental septum, and enters the lung parenchyma. This study aimed to investigate the clinical feasibility of thoracoscopic segmentectomy (TS) of the lateral basal segment (S9), posterior basal segment (S10), and both through the pulmonary ligament (PL)., Methods: Between February 2009 and November 2021, 542 patients underwent segmentectomy for malignant lung tumors at Tokyo Women's Medical University Hospital (Tokyo, Japan). This study included 51 patients. Among them, 40 underwent a complete TS of the S9, S10, or both by the PL approach (PL group), and the remaining 11 by the interlobar fissure approach (IF group)., Results: Patients' characteristics did not significantly differ between the two groups. In the PL group, 34 underwent video-assisted thoracoscopic surgery (VATS), and 6 underwent robot-assisted thoracoscopic surgery. In the IF group, all 11 underwent VATS. Operation duration, estimated blood loss, and postoperative complication frequency were not significantly different between these groups, but the maximum tumor diameter showed a significant difference., Conclusions: Complete TS of the S9, S10, and both through the PL is a reasonable option for tumors located in such segments. This approach is a feasible option for performing TS., (© 2023. The Author(s).)

Ciliated muconodular papillary tumor (CMPT) is a rare type of tumor with both benign and malignant characteristics. Herein, we report the surgical case of a 65-year-old man with CMPT. Chest computed tomography revealed a solitary cavitary lesion with a maximum diameter of 11-mm in S 10 b of the right lower lung. A thoracoscopic lung wedge resection was subsequently performed. On microscopic examination, the tumor was composed of highly columnar cells with tubular-to-papillary and cystic growth patterns. On immunostaining, it was positive for the epidermal growth factor receptor. The tumor was diagnosed as a CMPT, exhibiting no recurrence after two years of follow-up., (© 2023. The Author(s).)

Background: This study aims to investigate the effects of robot- and videoassisted thoracoscopic lobectomy on the learning curve of lobectomy., Methods: Between September 2013 and February 2020, the first 68 consecutive patients (28 males, 40 females; median age: 71 years; range, 33 to 86 years) who were operated for lung malignancies and scheduled for robot-assisted thoracoscopic lobectomy were retrospectively analyzed. The characteristics of the patients and operative data were analyzed, and the operation times of the first 51 cases of video-assisted thoracoscopic lobectomy were compared with those of robot-assisted thoracoscopic lobectomy performed by a single surgeon., Results: Of the patients, 62 had primary lung cancer and six had metastatic lung tumors. The majority of primary lung cancer patients (87.1%) had an adenocarcinoma. The most common clinical stage was IA1 (30.9%). There was no emergent conversion to thoracotomy in any of the patients. The median operation time was 223.5 min, and console time was 151 min. The most common complication was an air leak. All patients were alive. Compared to video-assisted thoracoscopic lobectomy, the median operation time was significantly longer in the robot-assisted thoracoscopic lobectomy group (p=0.0002). Similar to the operation time learning curve of the video-assisted thoracoscopic surgery group, the operation time learning curve of the robotassisted thoracoscopic surgery group increased from the first to ninth case (Phase 1), plateaued from the 10th t o 14th c ase ( Phase 2 ), and decreased from the 15th case (Phase 3). There was a statistically significant decrease in the operation time between Phase 1 and Phase 3 (p=0.0063)., Conclusion: The results of robot-assisted thoracoscopic lobectomy by a single surgeon show that this surgery has a longer operation time, but the perioperative outcomes are satisfactory. The learning curve of this surgery may be gradual for experienced video-assisted thoracoscopic surgeons., Competing Interests: Conflict of Interest: Masato Kanzaki received honoraria from the Intuitive Surgical Inc., Japan. All other authors declared no conflicts of interest with respect to the authorship and/or publication of this article., (Copyright © 2021, Turkish Society of Cardiovascular Surgery.)

Acute type A aortic dissection is a potentially fatal disease, and emergency surgery should be considered when it is diagnosed. We herein report two cases of retrograde type A aortic dissection with intramural hematoma, followed by re-dissection, rupture, and cardiac tamponade. The diagnoses in these cases had to be made carefully, as the false lumen of the ascending aorta was sometimes unclear on contrast-enhanced computed tomography.

Background: Thoracoscopic segmentectomy of the lateral and posterior basal segments is extremely technically challenging. Appropriate segmentectomy requires exposure and recognition of the branches of the bronchi and pulmonary vessels deep in the lung parenchyma. Although various approaches for these segmentectomies have been reported, the use of a pulmonary ligament approach is rational because it does not require any interlobar separation. Here, we report a successful case of portal robotic segmentectomy of the lateral and posterior basal segments through the pulmonary ligament approach., Case Presentation: A 60-year-old Japanese man with a history of low anterior resection for rectal cancer was referred to our department because of a lung nodule. His chest computed tomography revealed a 15-mm tumor in the left posterior basal bronchus. Robotic left S9-10 segmentectomy through the pulmonary ligament was performed with five-port incisions., Conclusions: An extremely technically challenging thoracoscopic segmentectomy of the lateral and posterior basal segments was performed through the pulmonary ligament using a robotic surgical system.

The number of robot-assisted thoracoscopic surgery (RATS) procedures performed for thoracic disease has been increasing. Recently, a narrow-profile vascular stapler (Signia small-diameter reload, Covidien Japan, Tokyo, Japan) has been developed to provide superior access and precise staple placement in video-assisted thoracoscopic surgery (VATS) procedures. A 70-year-old man who was an exsmoker visited our hospital with a complaint of coughing. Chest computed tomography revealed a tumor with a cavity, ~55 mm in diameter, in the right-middle lobe. A 4-arm RATS right-middle lobectomy was performed with a 4-cm utility thoracotomy with da Vinci Xi (Intuitive Surgical, Sunnyvale, CA, USA). The pulmonary vessels were divided using a narrow-profile vascular stapler through an 8-mm port. No complications occurred in the postoperative course. With a narrow-profile vascular stapler, portal RATS can be performed after VATS lung biopsy using 8-mm ports., (Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2021.)

The number of robotic surgical procedures for thoracic disease is increasing. The automatic linear stapler has become a necessary device in endoscopic surgery. A 67-year-old man with a history of smoking was referred to our hospital for an abnormal finding on chest x-ray. Computer tomography of the chest revealed a ground glass nodule in the right middle lobe. We performed right middle wedge resection through the assistant port using a long-shaft stapler (LSS). Because the nodule was diagnosed as an adenocarcinoma on the intraoperative frozen section, the patient underwent robotic-assisted thoracoscopic surgery (RATS) right middle lobectomy using the da Vinci ® Xi system. The pulmonary arteries, veins, right middle bronchus and minor fissure were divided using the LSS through the utility thoracotomy or assistant port. The postoperative course was uncomplicated. This case suggests that the LSS can be used in RATS lung resection for cancer., (Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2020.)

Dantrolene capsule, an effective therapeutic agent for the treatment of spasticity, is administered to children who cannot swallow the capsule after reformulation into a powder. The powdered drug can alter the specified dosage and it is also difficult to dispense the powdered formulation because of its bulky and sticky nature. To resolve these problems, we reformulated dantrolene capsules into granules using a centrifugal planetary mixer in the pharmacy. The granules containing lactose-cornstarch, D-mannitol, or microcrystalline cellulose as a diluent were examined to determine particle size distribution, flowability, drug content uniformity, and disintegration time. The granules with microcrystalline cellulose were superior to the other forms, owing to their smaller size, good drug content uniformity, and rapid disintegration. We further investigated the usability of the granules in the dispensing procedure (dividing and packing) and in the dosing process (retrieval from package) using the powders as controls. The deviation of the divided amount and loss on dosing were reduced relative to the powders. In addition, drug dissolution properties and storage stability for 12 months were the same as those of the powders. Therefore, we concluded that dantrolene granules are excellent alternatives as an extemporaneous preparation in pharmacies.

Background: Ectopic mediastinal parathyroid tumor (EMPT) is a rare cause of primary hyperparathyroidism (PHPT); it is difficult to resect using the cervical approach. We describe a case of using video-assisted thoracic surgery (VATS) for EMPT resection., Case Presentation: A 67-year-old woman with a history of postoperative thyroid cancer had no symptoms. She was diagnosed with PHPT and underwent thyroid cancer surgery. She had serum calcium and intact parathyroid hormone (PTH) levels of 11.1 mg/dL and 206 pg/mL, respectively. Chest computed tomography showed a 10-mm nodule in the anterior mediastinum. Technetium-99 m methoxyisobutyl isonitrile scintigraphy showed an abnormal uptake lesion in the anterior mediastinum. She was diagnosed with PHPT caused by EMPT and underwent VATS. The pathological examination confirmed parathyroid adenoma. Her serum calcium and intact PTH levels were normal from 15 min after tumor resection. She has had no recurrence of EMPT., Conclusions: The VATS approach was effective for the resection of EMPT.

The therapeutic effects of fullerene derivatives on many models of inflammatory disease have been demonstrated. The anti-inflammatory mechanisms of these nanoparticles remain to be elucidated, though their beneficial roles in allergy and autoimmune diseases suggest their suppressive potential in acquired immunity. Here, we evaluated the effects of C 60 pyrrolidine tris-acid (C 60 -P) and polyhydroxylated fullerene (C 60 (OH) 36 ) on the acquired immune response in vitro and in vivo. In vitro, both C 60 derivatives had dose-dependent suppressive effects on T cell receptor-mediated activation of T cells and antibody production by B cells under anti-CD40/IL-4 stimulation, similar to the actions of the antioxidant N-acetylcysteine. In addition, C 60 -P suppressed ovalbumin-specific antibody production and ovalbumin-specific T cell responses in vivo, although T cell-independent antibodies responses were not affected by C 60 -P. Together, our data suggest that fullerene derivatives can suppress acquired immune responses that require T cells.

For large-area ion-implanted vertical-cavity surface-emitting lasers (VCSELs), side emission from the edges of a chip disturbs the laser emission of a VCSEL mode, and suppression of it is fundamental. In this paper, we present results of a numerical investigation of the side emission from large-area VCSELs. We have modeled a VCSEL structure by an infinitely broad layer structure with mirror loss at the edge surfaces. Estimated threshold gains indicate that laser emission occurs either in a VCSEL mode or in an edge-emitting Fabry-Perot (EEFP) mode. Calculated emitter length dependence of the threshold gain of these modes shows good agreement with experimental results, and the side emission is verified to be the laser emission of the EEFP mode. We have also discussed the way to suppress the side emission and confirmed that our recent achievement of over 200 W quasi-continuous-wave output from an ion-implanted VCSEL array is due to antireflection coatings of the edges and introduction of optical losses in ex-emitter regions.

Eight new caffeic acid oligomers, clinopodic acids J-Q (1-8), were isolated from whole plants of Clinopodium gracile, together with nine known caffeic acid oligomers. The caffeic acid oligomers with two to four dihydrobenzofuran rings were isolated as natural products for the first time. Clinopodic acid M (4) showed the strongest hyaluronidase inhibitory activity, IC(50) (19 µM) among the 22 compounds isolated from this plant.

Effects of squalane-dissolved fullerene-C60 (Sql-fullerene) on macrophage activation and adipose conversion with oxidative stress were studied using an inflammatory adipose-tissue equivalent (ATE) and OP9 mouse stromal preadipocyte-U937 lymphoma cell co-culture systems. Differentiation of OP9 cells was initiated by insulin-rich serum replacement (SR) as an adipogenic stimulant, and then followed by accumulation of intracellular lipid droplets and reactive oxygen species (ROS), both of which were significantly inhibited by Sql-fullerene. In the OP9-U937 cell co-culture system, U937 cells rapidly differentiated to macrophage-like cells during SR-induced adipogenesis in OP9 cells. The ROS accumulation was in the co-culture more marked than in OP9 cells alone, suggesting that the interaction between adipocytes and monocytes/macrophages promotes inflammatory responses. Sql-fullerene significantly inhibited macrophage activation and low-grade adipogenesis in the OP9-U937 co-culture system. We developed a three-dimensional inflammatory adipose-tissue model "ATE" consisting of, characteristically, U937 cells in the culture-wells, and, in addition, mounted a culture insert containing OP9 cells-populated collagen gel. ATE is enabled with suitable stimulation to represent the pathology of inflammatory disorders, such as macrophage infiltration in adipose tissue. Five-day culturing of ATE in SR medium occurred U937 macrophage migration and intracellular oil-droplet accumulation that were significantly inhibited by Sql-fullerene. Our results suggest that Sql-fullerene might be explored as a potential medicine for the treatment of metabolic syndrome or other obesity-related disorders., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We evaluated the safety of water-soluble polymer-enwrapped fullerenes (PVP/fullerenes) as antioxidants in cosmetic and pharmaceutical preparations by studying the genotoxicity, phototoxicity, and pro-oxidant effects of these fullerenes. These materials were not mutagenic to any of the tested bacterial strains and did not induce chromosomal aberrations in cultured mammalian cells. The PVP/fullerenes did not exhibit cytotoxicity under ultraviolet or sham irradiation in the alternative phototoxicity test. Moreover, they did not show any pro-oxidant effect in the presence of Fe(2+) or Cu(2+). Thus, we concluded that PVP/fullerenes are safe for use in cosmetic and pharmaceutical applications. This is the first study in which toxicity tests were performed on PVP/fullerenes.

The safety of an oil-degrading bacterium, C2 strain, was evaluated for utilization in an open system for bioremediation of oil-contaminated environments. The C2 strain was identified as Rhodococcus erythropolis by performing an alignment analysis of the whole 16S rRNA sequence. R. erythropolis was classified as a nonpathogenic (category 1) bacterium. Biological and biochemical properties of the C2 strain also confirmed its nonpathogenicity. The pathogenicity and basic ecotoxicity were studied in laboratory animals and in a variety of test species, respectively. General and inhalation toxicities were not detected; additionally, there was no evidence of skin irritation, mutagenic potential, eye irritation, skin sensitization, ecotoxicity or notable pathogenicity. The comparison of these results with human exposure levels and previously published data indicates that the C2 strain appears to be safe for utilization in bioremediation of polluted environments, requires no special occupational health precautions during the application process, and has a low environmental impact. This study suggests that the C2 strain could be suitable for bioremediation of oil-contaminated environments.

A simple and rapid method (slow-stirring method) for extracting environmental DNA (eDNA) from soils was constructed by physical mild stirring with chemical treatment. eDNA was extracted efficiently with minimal damage from various kinds of soil. The amount of eDNA and soil bacterial biomass showed a linear proportional relation [Y=(1.70x10(8))X, r2=0.96], indicating that bacterial biomass could be evaluated by quantifying levels of eDNA. Consequently, the average bacterial biomass in an agricultural field was calculated as 5.95x10(9) cells/g sample, approximately 10-100 times higher than that in non- and oil-polluted fields.

A cDNA, which has a high homology with teleost Platichthys flesus [Arg8] vasotocin (AVT) receptor (GenBank: AK033957), was found in mouse genome database. Analyses of the deduced amino acid sequence revealed that a cDNA has several features of AVT receptor. We tentatively named it as a mouse vasotocin receptor (MVTR). A two-electrodes voltage clamp technique was applied to characterize the MVTR expressed in Xenopus laevis oocytes. AVT induced Ca2+-dependent Cl- currents in Xenopus oocytes injected with MVTR cRNA. On the other hand, [Arg8] vasopressin, oxytocin and isotocin did not induce such currents. RT-PCR showed that MVTR mRNA was specifically expressed in the brain. In situ hybridization analysis demonstrated significant expression of MVTR mRNA in suprachiasmatic nucleus, arcuate nucleus and medial habenular nucleus of mouse brain. These results suggest that MVTR may mediate a variety of physiological functions in mouse.

We electrophysiologically investigated the effect of some fragrant compounds in oolong tea on the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABAA receptors) which were expressed in Xenopus oocytes. Of the tested fragrances in oolong tea, cis-jasmone, jasmine lactone, linalool oxide and methyl jasmonate significantly potentiated the response. Among these, cis-jasmone and methyl jasmonate potently potentiated the response, having a respective dissociation constant of the compound (Kp) and maximum potentiation (Vm) of 0.49 mM and 322% for cis-jasmone, and 0.84 mM and 450% for methyl jasmonate. Inhalation of 0.1% cis-jasmone or methyl jasmonate significantly increased the sleeping time of mice induced by pentobarbital, suggesting that these fragrant compounds were absorbed by the brain and thereby potentiated the GABAA receptor response. Both of these compounds may therefore have a tranquillizing effect on the brain.

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic gamma-aminobutyric acid (GABA) receptors, GABA(A) receptors were expressed in Xenopus oocytes by injecting both poly(A)+ RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of alpha1 and beta1 subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.

Mutations of the thyrotropin receptor (TSH-R) gene have been reported in some cases of hyperthyroidism. We report a case of a family that had a high incidence of hyperthyroidism (6/13) which strongly suggested hereditary factors. We then analyzed whether the family had mutations of the TSH-R gene. No significant mutations in exon 10 of the TSH-R gene were found in the patient by restriction fragment length polymorphism analysis and polymerase chain reaction direct sequencing, when compared with those with 4 normal subjects and 2 patients with Graves' disease. Unknown mutations in the extracellular region of the receptor or other genes in this family remain to be studied.

Background: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes., Results: The crystal structure of substrate-free LigAB was solved at 2.2 A resolution. The molecule is an alpha2beta2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the beta subunit, which is covered by the alpha subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site., Conclusions: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism.

The exposure of Saccharomyces cerevisiae cells to 13-L-hydroperoxylinoleic acid (LOOH) caused their death, the degree of which was dependent on the growth phase of the cells. Pre-application of ethanol, hydrogen peroxide (H2O2) and LOOH to S. cerevisiae cells reduced the effect of LOOH on the cells, showing the transient cross adaptation to LOOH. Antioxidants such as N,N',-diphenyl-p-phenylenediamine (DPPD), melatonin and vitamin E, and inhibitors of permeability transition of mitochondria, cyclosporin A and trifluoperazine, inhibited the LOOH-triggered cell death, while an inhibitor of glutathione synthetase, buthionine sulfoximine (BSO), enhanced the cell death by LOOH. Reactive oxygen species (ROS) were detected by flow cytometry, using the ROS-specific fluorescent indicator. A ferric iron chelator, deferoxamine, inhibited the LOOH-triggered cell death, and peroxyl radicals (LOO.) were detected by a spin trapping method. These reactive radicals possibly induced the death of S. cerevisiae cells. However, the DNA fragmentation characteristic of apoptosis was not observed in S. cerevisiae cells after exposure to LOOH, staurosporine, dexamethasone or etoposide, which have been reported to cause apoptosis in mammalian cells.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.

Objective: To determine serum lipoprotein(a) (Lp[a]) concentrations and to analyze the apolipoprotein(a) (Apo[a]) phenotype in patients with rheumatoid arthritis (RA)., Methods: The subjects included 131 patients with RA and 200 healthy control subjects. Serum Lp(a) concentrations were measured by enzyme-linked immunosorbent assay, and the Apo(a) phenotype was determined by immunoblotting. HLA-DR typing was also done., Results: The mean serum Lp(a) level was significantly higher (P < 0.001) in the RA patients (27.5 mg/dl) than in the controls (15.0 mg/dl). The S3 allele was found in 70.0% of the patients versus 39.5% of the controls (P < 0.001). There was no significant difference in HLA-DR4 positivity between patients with and without the S3 phenotype., Conclusion: The serum Lp(a) level was increased in patients with RA, possibly partly because of S3 phenotype predominance.

To study the effects of 13-L-hydroxylinoleic acid (LOH) and food additives on gamma-aminobutyric acid (GABA) receptors, ionotropic GABA receptors were expressed in Xenopus oocytes by injecting mRNAs prepared from rat whole brain. LOH, which was prepared by reduction of 13-L-hydroperoxylinoleic acid (LOOH), inhibited the response of GABA receptors in the presence of high concentrations of GABA. LOH also inhibited nicotinic acetylcholine, glycine, and kainate receptors, while it had little effect on NMDA receptors expressed in Xenopus oocytes. However, LOH potentiated the response of GABA receptors as well as LOOH in the presence of low concentrations of GABA, possibly increasing the affinity of GABA for the receptors, while linoleic acid did not. Since some modification of the compounds seemed to change their effects on GABA receptors, the responses of GABA receptors elicited by 10 microM GABA were measured in the presence of compounds with various kinds of functional groups or the structural isomers of pentanol. Potentiation of GABA receptors depended strongly on the species of functional groups and also depended on the structure of the isomers. Then effects of various kinds of food additives on GABA receptors were also examined; perfumes such as alcohols or esters potentiated the responses strongly, while hexylamine, nicotinamide, or caffeine inhibited the responses, mainly in a competitive manner, and vanillin inhibited the responses noncompetitively. These results suggest the possibility that production of LOOH and LOH, or intake of much of some food additives, modulates the neural transmission in the brain, especially through ionotropic GABA receptors and changes the frame of the human mind, as alcohol or tobacco does.

We characterized the gntT gene encoding a high-affinity gluconate permease of Escherichia coli K-12. Primer extension and lacZ-operon fusion analyses revealed that gntT has one strong and two weak promoters, all of which are regulated positively by cAMP-CRP and negatively by GntR. The weak promoters became constitutive when separated from the upstream region including the strong promoter that overlaps a putative GntR-binding sequence. Gluconate-specific uptake activity was observed with cells harboring the gntT plasmid clone, which was enhanced by the presence of gntK encoding gluconate kinase.

A cDNA for a Na+/glucose cotransporter was cloned from rat jejunum cDNA library. This transporter was expressed in Xenopus oocytes by injection of cRNA synthesized from the cDNA, and the transporter ability was electrophysiologically examined. The cotransporter had a very narrow sugar specificity. Only D-glucose, D-galactose, and some of their derivatives elicited significant electrical responses. These results of sugar specificity were compared with those of the H+/hexose cotransporter of Chlorella. Dose-response relationships of several sugars followed a simple Michaelis-Menten type of kinetics. Both Vm and Km were dependent on the sugars. Not only the affinity of sugars to the cotransporter but also the rate of conformational change of the cotransporter loaded with the sugar and Na+, which translocates them from outside to inside, possibly depends on the sugar structure. The rate-limiting step of the transportation may be the conformational change, i.e., isomerization, of the cotransporter that translocates both the sugar and Na+ from outside to inside.

The nucleic acids of the helical and coccoid forms of Helicobacter pylori were studied to determine if the coccoid forms are "viable (capable of growing) but nonculturable." Using a reference strain (NCTC 11638) and five clinical strains, the nucleic acid contents, DNA integrity, and results of PCR and reverse transcription-PCR (RT-PCR) were compared for helical H. pylori and coccoid forms induced using glycochenodeoxycholic acid or bismuth citrate. The DNA and RNA contents of the coccoid forms were respectively 6.8- and 8.1-fold lower than those of helical H. pylori after 3 days of induction and 11.5- and 14.7-fold lower after 7 days. Agarose gel electrophoresis of DNA extracted from the coccoid forms after 3 days of induction showed a smear pattern indicating DNA cleavage, whereas DNA from helical H. pylori showed a single band with a high molecular mass. After 12 days of induction, all RNA samples from 100% coccoid cultures were negative for the mRNA of urease A or the 26-kDa species-specific protein by RT-PCR. However, most RNA samples obtained after 3 or 7 days of induction were positive at low levels despite the lack of recovery from these cultures. These results suggest that the coccoid form of H. pylori has impaired genomic DNA and is in the process of cellular degeneration, thus being still alive but nonincreasable.

To study the effects of food additives on glutamate receptors, they were expressed in Xenopus oocytes that received an injection of poly(A)+ mRNAs prepared from rat brain. The response of the receptors elicited by kainate (KA) and N-methyl-D-aspartate (NMDA) was measured electrophysiologically in the presence and absence of food additives. Both responses elicited by KA and NMDA were inhibited similarly by addition of additives such as caffeine, vanillin or saccharin. However, inhibition of KA-elicited response by food additives followed a competitive inhibition scheme with two binding sites, while that of NMDA-elicited response followed a simple noncompetitive inhibition scheme. Inhibition constants of food additives for both responses were more than 1 mM. So it is unlikely that food additives taken with processed food interrupt signal transmission under physiological conditions. The specificity of inhibition of both responses was examined by adding various compounds to the bathing solutions containing the agonist. Increase of the number of hydroxyl groups in alcohols with the chain of three carbon atoms decreased the potency of inhibition. Potency of the inhibition depended on the species of functional groups. The order of potency of the inhibition by compounds with a chain of six carbon atoms was alcohol = diamine > aldehyde > carboxylic acid. Hexanol inhibited the receptors more strongly than (3Z)-hexen-1-ol. NMDA-elicited response showed little selectivity in inhibition by structural isomers of pentanol, while KA-elicited response showed some selectivity in inhibition by the structural isomers.

To study the effects of food additives on nicotinic acetylcholine receptors (nAChR), they were expressed in Xenopus oocytes that received an injection of mRNA prepared from electroplax of Electrophorus electricus. The response of nAChR elicited by acetylcholine (ACh) was measured electrophysiologically in the presence and absence of aliphatic alcohols and food additives. All compounds examined inhibited nAChR non-competitively in a concentration-dependent way. The inhibition was stronger when the inhibitors were perfused 1 min before ACh, than when they were perfused simultaneously with ACh. The inhibition of nAChR by aliphatic alcohols (propanol to hexanol) increased as the number of carbon chains increased. The addition of alcohols and food additives did not affect the desensitization of nAChR caused by 2 microM ACh. These results suggest that alcohols and food additives bind to the anesthetic binding site in nAChR and inhibit it noncompetitively. However, these compounds will not hinder signal transmission in neuromuscular junctions under physiological conditions, because their inhibition constants are more than 1 mM and muscles usually have more receptors than the number necessary for signal transmission.

The Maillard protein cross-link pentosidine is a fluorescent condensation product of lysine, arginine and ribose. It accumulates in human tissues with age, and the accumulation process is accelerated in the tissues of diabetic patients. Using SP-Sephadex C-25 in the pretreatment for HPLC, we examined levels of pentosidine in urine without hydrolysis (free form) and levels of pentosidine in urine after hydrolysis (total forms), from 23 diabetic patients and 21 control subjects. The mean percentages of the values of free form per total forms (+/- SD) were 89 +/- 15% in diabetic patients, 88 +/- 16% in control subjects and 89 +/- 15% in total populations of diabetic patients and control subjects. There was a significant correlation between the values of free form and total forms in diabetic patients (r = 0.983, p = 0.0001), in control subjects (r = 0.820, p < 0.02) and in total populations of diabetic patients and control subjects (r = 0.951, p = 0.0001). The mean level of pentosidine per mol creatinine (+/- SD) was significantly elevated in urine from diabetic patients as compared to the level in control subjects (8.8 +/- 4.3 mumol/mol creatinine vs 4.2 +/- 1.4 mumol/mol creatinine, p = 0.0001 in free form; 10.1 +/- 5.3 mumol/mol creatinine vs 4.7 +/- 1.4 mumol/mol creatinine, p = 0.0001 in total forms). These results demonstrate that urinary pentosidine, especially in free form, could be a useful marker for the assessment of diabetes and diabetic complications.

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.

The effects of cocaine and of phencyclidine and procaine on acetylcholine receptor-controlled ion flux were measured in the millisecond-to-minute time region. Chemical kinetic measurements of ion flux were made in membrane vesicles prepared from the electric organ of Electrophorus electricus and in PC-12 cells, a sympathetic neuronal cell line. A quench-flow technique was used to measure ion flux in the millisecond-to-second range in membrane vesicles. Cocaine and phencyclidine both inhibit acetylcholine receptor-controlled ion flux, but by different mechanisms. Both compounds decrease the initial rate of ion flux, an effect observed with the local anesthetic procaine. This inhibition cannot be prevented by saturating concentrations of acetylcholine (1 mM). These results from chemical kinetic experiments are consistent with electrophysiological measurements which indicate that local anesthetics act by interfering with the movement of ions through receptor-formed channels. The chemical kinetic experiments, however, give additional information about the action of phencyclidine. They indicate that phencyclidine also increases the rate of receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptor conformations, effects not observed in the presence of cocaine or procaine.

The specific reaction rate (J) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 1 degrees C, J = 3 X 10(7) M-1 sec-1. J is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared from the electric organ of Electrophorus electricus) and a flow-quench technique that has a millisecond time resolution were used to measure the receptor-controlled ion translocation. Using the value of J and the molar concentrations of receptor sites and inorganic ions, we calculated that 6 X 10(3) ions are translocated per msec per receptor. Analysis of electrical noise in frog muscle cells at temperatures above 8 degrees C [Nether, E. & Stevens, C. F. (1977) Annu. Rev. Biophys. Bioeng. 6, 345-381] gave a value of about 1 X 10(4) ions msec-1 per channel. Thus, each technique gives essentially the same result. It is now possible, therefore, to correlate the results obtained when receptor function is measured in two different ways in membrane vesicles and in muscle cells: (i) chemical kinetic measurements, using membrane vesicles, which relate the ligand binding and ion translocation processes and (ii) analysis of acetylcholine noise in muscle cells [Katz, B. & Miledi, R. (1972) J. Physiol. (London) 224, 665-699], which allows one to measure elementary steps in the formation of ion channels through the cell membrane.

Acetylcholine-induced flux of inorganic ions across membranes and inactivation of the acetylcholine receptor were measured at pH 7.0, 1 degrees C, over a 5000-fold concentration range of acetylcholine. Receptor-containing electroplax membrane vesicles prepared from Electrophorus electricus and a quench-flow technique were used, allowing flux to be measured in the 2-msec to 1-min time region. Five different measurements were made: (i) rate of ion translocation with the active state of the receptor, (ii) rate of the slower ion translocation after equilibration of active and inactive receptor states, (iii) rate of inactivation, (iv) equilibrium between active and inactive forms of the receptor, and (v) reactivation of inactivated receptor. The kinetics of the steps in the receptor-controlled ion flux follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence can be used. Thus, the rate and equilibrium constants in a scheme that relates the ligand binding steps to ion translocation could be evaluated. It was found that the dependence of the receptor-controlled ion translocation over the concentration range investigated obeys the integrated rate equation based on the proposed mechanism. The flux rate before inactivation was approximately 10(7) ions sec-1 per receptor, which is comparable with that measured electrophysiologically in muscle cells. The half-time of inactivation is approximately 100 msec when the receptor is saturated with acetylcholine. The specific reaction rate of the ion translocation (J) is 3 X 10(7) M-1 sec-1. The results support a minimum reaction mechanism previously proposed on the basis of experiments in which carbamylcholine was used.