Role of asparagine-linked oligosaccharides in protein folding, membrane targeting, and thyrotropin and autoantibody binding of the human thyrotropin receptor

The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.     

Development of antiviral therapeutic agents from traditional medicines

Traditional medicines contain various metabolites derived from nucleic acid, protein, and lipid metabolism. Some of these specific metabolites may recognize the differences between viral and host metabolism resulting in anti-viral activity; hence traditional medicines may be useful sources for new antiviral agents. Traditional medicines can be cheaply obtained and have been orally administered as hot-water extracts. Therefore, they may be used for the prophylactic and therapeutic treatment of viral infection by drinking them, such as coffee or tea. Here we describe how the antiviral activity of traditional medicines was screened in vitro and how their therapeutic antiviral activities were verified in vivo, to obtain traditional antiviral medicines that can be clinically used. Therefore, we have selected 12 herbal extracts, from more than 250 herbal medicines, that exhibit therapeutic activities against cutaneous herpes simplex virus (HSV) type 1 (HSV-1) infection in mice. Four of the 12 augmented the therapeutic efficacy of acyclovir (ACV) in mice and showed potent anti-HSV activity against infection with ACV-resistant HSV-1 mutants in mice. These herbal extracts selectively inhibited viral DNA synthesis and showed a different mode of anti-HSV-1 action from that of ACV. They were also effective against both recurrent HSV and cytomegalovirus infections, without toxicity. Such prophylactic and therapeutic antiviral activities of the traditional medicines were verified by the purification of major active compounds. We could show new indications of traditional medicines as antiviral agents. Thus, the drinking of the extracts, in a daily tea or coffee, may be used for prophylaxis and therapy of diseases caused by herpes virus infection and improve the quality of life.     

Mutation of the p53 gene in human astrocytic tumours correlates with increased resistance to DNA-damaging agents but not to anti-microtubule anti-cancer agents

Human brain cancers (gliomas) overexpress large numbers of a receptor for interleukin 13 (IL13), making this receptor an attractive target for anti-glioma therapies. We have recently proposed that the glioma-associated IL13 receptor is different from the one expressed on some hemopoietic and somatic cells. In an attempt to identify an even more glioma-specific target, we have used an antagonist of a related cytokine, IL4, which neutralizes the physiological effects of both IL13 and IL4 on normal cells. Here we demonstrate that the IL4 antagonist also counteracts the action of cytotoxins targeted to the IL13 receptor on normal human cells. Importantly, the IL4 antagonist does not inhibit IL13-based cytotoxins on glioma cells at all. Thus, the IL13 receptor on glioma cells can be categorized as tumor-specific in the presence of an IL4 antagonist. We conclude that IL13 receptor-directed cytotoxins can be delivered to glioma cells without being cytotoxic to normal cells.     

High prevalence of antibodies to glutamic acid decarboxylase in comparison to islet cell antibodies in patients with long-standing insulin-dependent diabetes mellitus

To elucidate the clinical significance of antibodies to glutamic acid decarboxylase (GAD Ab) compared to islet cell antibodies (ICA) in recent-onset and long-standing insulin-dependent diabetes mellitus (IDDM). We examined GAD Ab and ICA in 29 recent-onset and 85 long-standing patients with IDDM. GAD Ab was detected by a radioimmunoassay kit using purified pig brain GAD as an antigen. The prevalence of GAD Ab in the recent-onset diabetic patients was 55.2%, slightly lower than that of ICA (65.5%). In contrast, the prevalence of GAD Ab in long-standing diabetic patients was 42.4%, which was significantly higher than that of ICA (23.5%) (p < 0.01). GAD Ab were consistently detected in approximately 40% of patients with long-standing disease, while ICA decreased according to duration of disease. The GAD Ab titer in ICA-positive patients (mean +/- SD, 1588.2 +/- 6755.1; range, 6-38574) was significantly higher than that in ICA-negative patients (mean +/- SD, 13.4 +/- 17.9; and range, 5-72 units) (p < 0.001). These findings suggest that GAD Ab are more useful than ICA to know participation of immune disorders in long-standing patients with IDDM.     

Therapeutic usefulness of wild-type p53 gene introduction in a p53-null anaplastic thyroid carcinoma cell line

Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.     

Performance of multimedia broadcasting through ISDB transmissionsystem

Multimedia broadcasting has the benefits of versatile information representation, intelligent viewing environments, and economical systems. Integrated Services Digital Broadcasting (ISDB) presents a concept for future digital broadcasting systems. Full-scale multimedia broadcasting services can be realized by ISDB. There are two aspects in multimedia broadcasting from the viewpoint of ISDB. One is “transmission multimedia” and another is “presentation multimedia”. The former means that ISDB provides services by exploiting the characteristics of various types of broadcast channels. It also integrates all broadcast networks and is interoperable with communications and package media. The latter means that ISDB can flexibly combine and send various types of representation media that most appropriately convey the information. Multimedia broadcasting by ISDB is expected to achieve interactive viewing and to raise individual viewer satisfaction. ISDB will also make it possible to set up multiple small-scale broadcast stations and to provide a many-to-many information linking environment for transmitting information between an unspecified number of individuals     

Hydrogen recovery from cyclohexane as a chemical hydrogen carrier using a palladium membrane reactor

A palladium membrane reactor was applied to recover the hydrogen from cyclohexane as one of the promising chemical hydrogen carriers. The operation conditions of the palladium membrane reactor to obtain a higher hydrogen recovery were predicted by computer simulation. As a result, it was shown that the hydrogen recovery rate becomes higher as the pressure on the hydrogen permeation side is lowered below atmospheric pressure or as the reaction pressure increases. This was confirmed experimentally. As the perm-side pressure was lowered, the conversion as well as the hydrogen recovery rate at 573 K was found to increase. About 80% of the hydrogen contained in cyclohexane, depending on the operation condition was successfully recovered.     

Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1-34), PTHrP(1-34), PTHrP(1-141), PTHrP(100-114) and antisense oligonucleotides against PTHrP

Parathyroid hormone-related protein (PTHrP) is thought to be an important autocrine/paracrine factor for chondrocyte metabolism since mice lacking the PTHrP gene exhibit abnormal cartilage development. To determine the biological role of PTHrP in chondrocytes, we first compared the agonist potency of human (h) PTHrP(1-34) with hPTH(1-34) in cultured rat articular chondrocytes. Neither hPTHrP(1-34) nor hPTH(1-34) altered basal DNA synthesis, but attenuated the stimulatory effect of transforming growth factor beta (TGF-beta). Both agents suppressed the expression of alpha(1) type II collagen mRNA in a dose-response fashion with the same potency. In addition, the action of exogenously added hPTHrP(1-34) and hPTH(1-34) on intracellular cAMP and [Ca2+]i levels was similar. We next compared the effect of PTHrP within its entire amino acid sequence (1-141). With regard to thymidine incorporation, alpha(1) type II collagen gene expression and accumulation of cAMP and [Ca2+]i level, there was no significant difference between hPTHrP(1-34) and hPTHrP(1-141). PTHrP C-terminal (100-114) did not show any function. To further investigate PTHrP function, intracellular PTHrP translation was inhibited by a transgene of antisense oligonucleotides against PTHrP. Antisense oligonucleotides decreased PTHrP mRNA translation, specifically inhibited DNA synthesis in control as well as TGF-beta-treated chondrocytes and enhanced alpha(1) type II collagen mRNA expression in TGF-beta-treated chondrocytes. These results suggest that there is no significant difference between exogenously added hPTH(1-34), hPTHrP(1-34) and PTHrP(1-141) with regard to the biological action of these agents, including cell growth, differentiation and second messenger pathway. However, the result of DNA synthesis in the antisense PTHrP-inhibition study suggests that intracellular PTHrP may have an as yet unknown biological role, in addition to a classical PTH/PTHrP receptor-mediated function in the rat articular chondrocyte.