A Ca2+- and Voltage-Dependent Cl- -Sensitive Anion Channel in the Chara Plasmalemma: A Patch-Clamp Study

The inside-out patch-clamp technique was applied to the plasmolyzedplasmalemma of inter-nodes of Chara corallina without enzymatictreatment. We found two different types of channel activitythat were CP^–-sensitive. Both types of channel were Ca²⁺-dependent.However, the one that exhibited greater dependence on Ca²⁺ ionswas the focus of our studies, and we named it the Ca²⁺-dependentCP^–-sensitive anion channel. When the concentration ofCa²⁺ ions on the cyto-plasmic side was 1.0 µM, the Ca²⁺-dependentCP^–-sensitive channel opened most frequently between approximately–80 and –100 mV. At 10 µM Ca²⁺, it openedless frequently, and at 0.1 µM Ca²⁺ it scarcely openedat all. These observations indicate that the anion channel ofinterest is voltage-dependent over a restricted range of concentrationsof Ca²⁺ ions. The dependence on Ca²⁺ and voltage of the channelcan explain the behavior of the excitable Ca²⁺-activated Cl^–channel in the Chara plasmalemma. The channel activity was blockedby several antagonists of calmodulin. ⁴ Present Address: Department of Biology, College of GeneralEducation, Osaka University, Toyonaka, 560 Osaka, Japan (Received October 8, 1990; Accepted April 4, 1991)     

False positive reaction for carcinoembryonic antigen in paget cells

Virchows Archiv B Cell Pathology - Paget cells from cases of mammary and extramammary Paget’s disease were examined for carcinoembryonic antigen (CEA) and CEA-related antigens by the...     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: II. Inductive property of membrane and effects of pH o and impermeable monovalent cations on breakdown phenomenon

Summary Changes in the chord conductanceG and the membrane electromotive forceE _m in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–V _m relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pH _o in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pH _o 7.In some cellsE _m shifted to a positive level as large as +50+70 mV during the breakdown phenomenon. Such a large positive shift ofE _m is caused mainly by the increase in conductance of Cl^– and partly Ca²⁺ and K⁺.     

Possible novel therapy for diabetes with cell-permeable JNK-inhibitory peptide

The JNK pathway is known to be activated in several tissues in the diabetic state, and is possibly involved in the development of insulin resistance and suppression of insulin biosynthesis. Here we show a potential new therapy for diabetes using cell-permeable JNK-inhibitory peptide. Intraperitoneal administration of the peptide led to its transduction into various tissues in vivo, and this treatment markedly improved insulin resistance and ameliorated glucose tolerance in diabetic mice. These data indicate that the JNK pathway is critically involved in diabetes and that the cell-permeable JNK-inhibitory peptide may have promise as a new therapeutic agent for diabetes.     

Corticosterone acutely prolonged N-methyl-d-aspartate receptor-mediated Ca2+ elevation in cultured rat hippocampal neurons

This work reports the first demonstration that corticosterone (CORT) has a rapid and transient effect on NMDA receptor-mediated Ca2+ signaling in cultured rat hippocampal neurons. Using single cell Ca2+ imaging, CORT and agonists of glucocorticoid receptors were observed to modulate the NMDA receptor-mediated Ca2+ signals in a completely different fashion from pregnenolone sulfate. In the absence of steroids, 100 micro m NMDA induced a transient Ca2+ signal that lasted for 30-70 s in 86.1% of the neurons prepared from postnatal rats (3-5 days old). After pre-treatment with 0.1-100 micro m CORT for 10-20 min, NMDA induced extremely prolonged Ca2+ elevation. This prolonged Ca2+ elevation was terminated by the application of MK-801 and followed by washing out of CORT. The proportion of CORT-modulated neurons within the NMDA-responsive cells increased from 25.1 to 95.5% when the concentration of CORT was raised from 0.1 to 50 micro m. Substitution of BSA-conjugated CORT produced essentially the same results. When hippocampal neurons were preincubated with 10 micro m cortisol and 1 micro m dexamethasone for 20 min, a very prolonged Ca2+ elevation was also observed upon NMDA stimulation. The CORT-prolonged Ca2+ elevation caused a long-lasting depolarization of the mitochondrial membrane, as observed with rhodamine 123. In contrast, incubation with 100 micro m pregnenolone sulfate did not considerably alter the time duration of NMDA-induced transient Ca2+ elevation, but caused a significant increase in the peak amplitude of Ca2+ elevation in hippocampal neurons. These results imply that high levels of CORT induce a rapid and non-genomic prolongation of NMDA receptor-mediated Ca2+ elevation, probably via putative membrane surface receptors for CORT in the hippocampal neurons.     

Structural change of ribosomes during apoptosis: degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells

Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [35S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, P0, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis.     

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.     

Soluble interleukin-18 receptor complex is a novel biomarker in rheumatoid arthritis

Introduction

There has been no report in the literature of a soluble form of interleukin (IL)-18 receptor α (IL-18Rα). In this study, we evaluated the levels and characteristics of soluble IL-18Rα (sIL-18Rα) in the sera of patients with rheumatoid arthritis (RA) and compared these results to control populations.

Methods

The sIL-18Rα complex was isolated from pooled human blood serum using an anti-IL-18Rα monoclonal antibody affinity column. The purified sIL-18Rα was then examined using Western blot analysis and used in experiments to evaluate the effects on an IL-18-responsive natural killer (NK) human cell line, NK0. An enzyme-linked immunosorbent assay was developed, and sera from 145 patients with RA, 6 patients with adult-onset Still's disease, 31 patients with osteoarthritis (OA), 39 patients with systemic lupus erythematosus (SLE) and 67 controls were tested, along with levels of immunoglobulin M, rheumatoid factor, anticyclic citrullinated peptide antibody, IL-18, IL-13 and interferon (IFN)-γ. Area under the receiver operating characteristic curve (ROC-AUC) analysis was used to evaluate the diagnostic utility of the sIL-18Rα complex.

Results

The isolated sIL-18Rα complex can be associated with IL-18 and the soluble form of the IL-18Rβ chain. The sIL-18Rα complex bound to the surface to the NK0 cell line, antagonized the stimulatory effects of IL-18 and IL-2 on the NK0 cell line and inhibited IFN-γ production by the cells. The serum levels of sIL-18Rα complex in RA (186.0 ± 33.5 ng/mL, n = 145) and adult-onset Still's disease (98.2 ± 8.9 ng/mL, n = 6) were significantly (P < 0.001) higher than those in the healthy controls (52.3 ± 8.5 ng/mL, n = 67), OA (38.6 ± 5.4 ng/mL, n = 31), SLE (44.6 ± 3.2 ng/mL, n = 39). The serum level of sIL-18Rα complex was not significantly different between RA and adult-onset Still's disease patients. The serum levels of IL-18, IL-13 and IFN-γ in the RA patients were significantly (P < 0.01) higher than in OA and SLE patients as well as healthy controls. ROC-AUC analysis of the serum concentration of sIL-18Rα indicated that it was significantly diagnostic of RA. Moreover, a tumor necrosis factor inhibitor, etanercept, significantly (P < 0.0001) decreased levels of sIL-18Rα in the sera of 29 RA patients 6 months after treatment.

Conclusions

The sIL-18Rα complex could be a potentially useful biomarker for the diagnosis of RA.