Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.     

Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells

Inhibition of whole-cell calcium currents in enzymatically dispersed frog atrial myocytes by D-600, diltiazem, and nifedipine was studied using a single-micropipette voltage-clamp technique. The objective of these experiments was to test the applicability of a modulated-receptor hypothesis similar to that proposed for local anesthetic interactions with sodium channels to account for the tonic and frequency-dependent interactions of these organic compounds with myocardial calcium channels. Data consistent with such a hypothesis include: (a) prominent use-dependent block of iCa by D-600 and diltiazem, which are predominantly charged at physiological pH; (b) iCa block by an externally applied, permanently charged dihydropyridine derivative is greatly attenuated; (c) all three antagonists produce large negative shifts in the voltage dependence of iCa availability; (d) block of iCa by these compounds is state-dependent; (e) reactivation of iCa in the presence of all three antagonists is biexponential, which suggests that drug-free channels recover with a normal time course and drug-bound channels recover more slowly; and (f) the kinetics of the drug-induced slow iCa recovery process may be determined largely by factors such as size and molecular weight, in addition to lipid solubility of the compounds. Experiments in which the pH was modified, however, reveal some important differences for the interaction of organic calcium antagonists with myocardial calcium channels. Acidification, in addition to changing the proportion of charged and neutral antagonist in solution, was found to selectively antagonize tonic inhibition of iCa by diltiazem and nifedipine, without changing the kinetics of the drug-induced slow iCa reactivation process. It is concluded that two distinct receptor sites may be involved in block of iCa by some of these compounds: a proton-accessible site and a proton-inaccessible site.     

Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells.

IL-18, a potent IFN-gamma-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-gamma-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-gamma-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-gamma-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.     

Potent induction of the adaptive response by a weak mutagen, methyl iodide, in Escherichia coli

Methyl iodide (MeI), a weakly mutagenic and highly chemoselective chemicals, was tested for its abilities to induced the adaptive and SOS responses in E. coli CSH26/pMCP1000 (alkA′-lacZ′) and CSH26/psK1002 (umuC′-lacZ′). MeI induced the adaptive response effectively but gave a very weak SOS response. Its potent ability in inducing the adaptive response was also demonstrated by adaptation to both the mutagenic and killing effects of N-methyl-N-nitrosourea (MNU) in E. coli WP2 cells. Simultaneous treatment with MeI in a non-growth medium slightly increased the mutagenicity of MNU, probably as a result of depletion of the repair enzyme, O⁶-methylguanine-DNA methyltransferase, which is constitutively present in the cells. As MeI itself proved to be only weakly mutagenic, a small part of the adaptive response which we have observed may involve indirect methylation of the repair enzyme by methyl transfer from MeI-induced O⁶-methylguanine residues in DNA. But the extent of the induced adaptive response seems to be much higher than would be expected from the observed weak mutagenicity of MeI. It is therefore suggested that the mechanism of induction of the adaptive response may involve direct methylation of the O⁶-methylguanine-DNA methyltransferase itself.     

Genetic variants of the receptors for thromboxane A2 and IL-4 in atopic dermatitis

Thromboxane A2 (TXA2) is an arachidonate metabolite which is considered to relate to chronic inflammation in atopic diseases characterized by elevated immunoglobulin E productivity. The elevation of immunoglobulin E levels involves many molecules including interleukin-4 (IL-4) and interleukin-4 receptor alpha chain (IL-4R alpha). To assess whether genetic variants of TXA2 receptor, IL-4 and IL-4R alpha genes relate to the elevation of serum immunoglobulin E levels in patients with atopic dermatitis (AD), we conducted an association study of genetic polymorphisms of TXA2 receptor (795C/T), IL-4 (-589C/T), and IL-4R alpha (Ile50Val) in a Japanese population (n = 789). The TXA2 receptor 795TT genotype strongly related to AD with high serum immunoglobulin E concentrations. AD patients with both TXA2 receptor 795TT genotype and the IL-4R alpha Ile50/Ile50 genotype showed the greatest immunoglobulin E concentrations. These results suggest TXA2 receptor polymorphism strongly interacts with IL-4R alpha polymorphism as a major determinant of high serum immunoglobulin E levels in AD.     

Rhinoceros Auklet pair‐mates migrate independently but synchronize their foraging activity during the pre‐laying period

Pair bonds are considered important for successful breeding in monogamous birds but their maintenance may be challenging for migratory species, as mates can be separated for months during the non‐breeding period. To investigate whether mates of monogamous migratory seabirds stay together throughout the non‐breeding period and how and when they start synchronizing their activity before breeding, we tracked seven pairs and 22 individuals of Rhinoceros Auklets Cerorhinca monocerata with geolocators and saltwater immersion loggers. Mates migrated across similar areas during the non‐breeding period but with a sustained temporal shift, putting them an average of 377 km apart and resulting in an average difference of return date at the colony of 5.6 days, with no sex biases. These values did not differ significantly from those between ‘pairs’ of randomly selected, non‐mated birds. Mates showed synchronized on‐water/in‐air at‐sea activities once both birds returned and spent the first night together at the colony. The synchronization of activities was highest on the day following the nights when both mates visited the colony, and decreased with elapsed time. Mates then left the colony together for a pre‐laying exodus of 8–9 days and males returned 2–4 days earlier than females before incubation started. Mates kept synchronizing at‐sea activity during the early part of the exodus. We interpret this as the mates staying together at sea during the pre‐laying period, increasing the males’ chances of copulation at sea. The patterns of mate association observed in Rhinoceros Auklets contrast with those of the Procellariiformes, presumably reflecting differences in the place and timing of copulation.     

Pharmacokinetic Study of Adjuvant Gemcitabine Therapy for Biliary Tract Cancer following Major Hepatectomy (KHBO1101)

Background

Biliary tract cancer (BTC) patients who have undergone surgical resection with major hepatectomy cannot tolerate the standard gemcitabine regimen (1,000 mg/m² on days 1, 8, and 15 every 4 weeks) due to severe toxicities such as myelosuppression. Our dose-finding study of adjuvant gemcitabine therapy for biliary tract cancer following major hepatectomy determined that the recommended dose is 1,000 mg/m² on days 1 and 15 every 4 weeks. Here, we evaluate the pharmacokinetics and pharmacodynamics of gemcitabine in these subjects.

Methods

We evaluated BTC patients scheduled to undergo surgical resection with major hepatectomy followed by gemcitabine therapy. A pharmacokinetic evaluation of gemcitabine and its main metabolite, 2′,2′-difluorodeoxyuridine (dFdU), was conducted at the initial administration of gemcitabine, which was given by intravenous infusion over 30 min at a dose of 800–1,000 mg/m². Physical examination and adverse events were monitored for 12 weeks.

Results

Thirteen patients were enrolled from August 2011 to January 2013, with 12 ultimately completing the pharmacokinetic study. Eight patients had hilar cholangiocarcinoma, three had intrahepatic cholangiocarcinoma, and one had superficial spreading type cholangiocarcinoma. The median interval from surgery to first administration of gemcitabine was 65.5 days (range, 43–83 days). We observed the following toxicities: neutropenia (n = 11, 91.7%), leukopenia (n = 10, 83.3%), thrombocytopenia (n = 6, 50.0%), and infection (n = 5, 41.7%). Grade 3 or 4 neutropenia was observed in 25% (n = 3) of patients. There were differences in clearance of gemcitabine and dFdU between our subjects and the subjects who had not undergone hepatectomy.

Conclusion

Major hepatectomy did not affect the pharmacokinetics of gemcitabine or dFdU.

Trial Registration

UMIN-CTR in (JPRN) UMIN000005109     

Masticatory performance in 80-year-old individuals

Objective: To evaluate the masticatory performance of elderly people at the age of 80 years. Subjects: A total of 283 individuals of 80 years of age took part in a general and dental health survey. Main outcome measures: A dental examination including the number of remaining teeth, occlusion, prostheses, bite force recording, and a questionnaire regarding masticatory performance were recorded. Setting: Five municipalities (Okazaki city, Tokoname city, Tahara town, Atsumi town and Minami‐chita town) in Aichi prefecture, Japan. Results: There were 20 or more teeth in 7.4% subjects, and 44.5% were edentulous. Subjects with no occlusion accounted for 77.4% of the total. Subjects with prostheses accounted for 90.8%. Maximum bite force and masticatory ability score for patients with 20 or more teeth or not wearing prostheses were higher than other groups. The non‐wearing prostheses group had a low masticatory ability score. Conclusion: Most of the 80‐year‐old individuals recovered their masticatory ability with the assistance of prostheses. Several individuals with 20 or more remaining teeth or without removable dentures present in both jaws had a high score for bite forces and masticatory abilities.     

Involvement of the Calcium-sensing Receptor in Human Taste Perception

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca²⁺, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.     

Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.