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Nanodiscs have attracted considerable attention as structural scaffolds for membrane-protein research and as biomaterials in

Accelerated formation of fibrin clots in a tumor microenvironment can be used for targeted delivery of interferon gamma (IFNγ) to tumor cells. Here, we selected cysteine-arginine-glutamic acid-lysine-alanine (CREKA) as the fibrin clot-binding peptide and designed 2 types of fusion proteins for tumor targeting. The CREKA peptide was fused to IFNγ's C-terminus, with or without a matrix metalloproteinase-2 (MMP2)-cleavable linker (IFNγ-mmp-CREKA or IFNγ-CREKA, respectively). The former was designed to release IFNγ from IFNγ-mmp-CREKA bound to fibrin clots, to ensure IFNγ's function in the tumor milieu. IFNγ-activated sequence-dependent reporter gene expression in B16-BL6 cells revealed that the biological activities of IFNγ-CREKA and IFNγ were comparable, whereas that of IFNγ-mmp-CREKA was approximately 60% that of IFNγ. Plasma concentrations of IFNγ-CREKA and IFNγ-mmp-CREKA remained at effective levels for at least 4 weeks after gene transfer into mice. After gene transfer to tumor-bearing mice, intratumoral concentration of IFNγ in pCpG-IFNγ-mmp-CREKA group was tended to be higher than those of the other groups. Inhibition of colon-26 tumor growth was significantly more with gene transfer of IFNγ-mmp-CREKA than with IFNγ or IFNγ-CREKA. These results indicate that targeted delivery of IFNγ to fibrin clots through IFNγ-mmp-CREKA fusion can enhance the therapeutic efficacy of IFNγ in cancer gene therapy.

Integral membrane proteins (IMPs) modulate key cellular processes; their dysfunctions are closely related to disease. However, production of IMPs in active conformations for further study is hindered by aggregation and toxicity in living expression systems. IMPs are therefore produced in cell‐free systems employing liposome chaperoning, but membrane integration of the nascent IMPs is suboptimal and orientation of the integrated proteins remains uncontrollable. Thus, an artificial membrane protein sorting system is developed, based on polyhistidine/nickel‐chelate affinity, combined with cell‐free membrane protein synthesis. Its proof of concept is demonstrated with a N‐terminal hexahistadine‐fused conexin‐43 (NHis–Cx43) model IMP. Nickel‐chelating liposomes efficiently incorporate twofold newly synthesized NHis–Cx43 compared with Cx43. NHis–Cx43, when synthesized in this system, forms dye‐permeable hemichannels, similar to plasma membrane pores formed by Cx43 in cells. The topology of incorporated NHis–Cx43 indicates two orientations in the liposomal membranes. However, NHis–Cx43 orientation is controlled, resulting in single topology, by combination of the natural molecular chaperone DnaKJE. Successful synthesis and at least 4.5‐fold increase lipid incorporation are also achieved with three other NHis‐fused IMPs, including α‐helix and β‐barrel IMPs. Overall, this simple membrane protein sorting system is usable combined with molecular chaperones to prepare proteoliposomes for many applications.

Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression.

Interferon γ (IFN-γ), an anticancer agent, is a strong inducer of indoleamine 2, 3-dioxygenase 1 (IDO1), which is a tryptophan-metabolizing enzyme involved in the induction of tumor immune tolerance. In this study, we investigated the IDO1 expression in organs after IFN-γ gene transfer to mice. IFN-γ gene transfer greatly increased the mRNA expression of IDO1 in many tissues with the highest in the liver. This upregulation was associated with reduced L-tryptophan levels and increased L-kynurenine levels in serum, indicating that IFN-γ gene transfer increased the IDO activity. Then, Lewis lung carcinoma (LLC) tumor-bearing wild-type and IDO1-knockout (IDO1 KO) mice were used to investigate the effects of IDO1 on the antitumor activity of IFN-γ. IFN-γ gene transfer significantly retarded the tumor growth in both strains without any significant difference in tumor size between the two groups. By contrast, the IDO1 activity was increased only in the wild-type mice by IFN-γ gene transfer, suggesting that cells other than LLC cells, such as tumor stromal cells, are the major contributors of IDO1 expression in LLC tumor. Taken together, these results imply that IFN-γ gene transfer mediated IDO1 upregulation in cells other than LLC cells has hardly any effect on the antitumor activity of IFN-γ.

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. Although the combination therapy employing pegylated interferon (IFN)-α and ribavirin is effective, this treatment is effective in only approximately 50% patients with genotype 1 HCV infection. IFN-γ is a potent anti-HCV agent that exhibits its antiviral action through a receptor distinct from that for IFN-α. Therefore, IFN-γ application might provide an alternative approach to IFN-α-based therapies. However, recombinant IFN-γ protein exhibits a poor pharmacokinetic property, that is, a very short half-life. It is our hypothesis that sustained IFN-γ serum concentrations produced by gene transfer could effectively eliminate HCV in vivo. We examined the in vivo antiviral activity in human hepatocyte chimeric mice infected with genotype 1b HCV at high HCV RNA titers (10(5)-10(7) copies/ml). The human IFN-γ-expressing plasmid vector pCpG-huIFNγ exhibited prolonged transgene expression in mice compared with the plasmid vector pCMV-huIFNγ. Moreover, the gene transfer of pCpG-huIFNγ eliminated HCV from the liver of the chimeric mice for a sustained period. On the contrary, administration of pCMV-huIFNγ could not eliminate HCV. In conclusion, we found that a single pCpG-huIFNγ injection resulted in long-term elimination of HCV RNA in chimeric mice, providing, for the first time, direct evidence that chronic infection with high titer HCV in vivo can be treated by sustained IFN-γ treatment.

Type II diabetes is one of the most problematic metabolic disorders and is associated with secondary conditions such as heart disease and eye complications. Interleukin-6 (IL-6), a multifunctional cytokine, could influence conditions of altered glucose metabolism such as insulin resistance in diabetic patients. However, a consensus about the role of IL-6 on glucose metabolism has not been reached. The aim of the present study is to investigate whether the expression of IL-6 affects glucose metabolism in diet-induced obesity (DIO) mice, a model of type II diabetes and obesity, using gene delivery of IL-6. DIO mice received hydrodynamic or intramuscular injection of IL-6-expressing plasmid to investigate the importance of the site of IL-6 expression. DIO mice that received a sustained IL-6 gene transfer showed similar glucose levels to lean mice in a glucose tolerance test. DIO mice exhibited reduced food intake and low body and epididymal fat weights after IL-6 gene transfer. IL-6 gene delivery reduced the mRNA expression of metabolism-related genes in the liver, skeletal muscle, and adipose tissue of DIO mice. The metabolic status of DIO mice receiving intramuscular injections was moderately better than that of DIO mice receiving hydrodynamic injections. The infiltration of inflammatory cells into the sites where the IL-6-expressing plasmid DNA was delivered was observed. Transient expression of IL-6 had limited effects on all parameters examined. These results indicate that the expression of IL-6 has an effect on obesity and the metabolism of glucose and lipid in diabetic mice and that the expression site of IL-6 is not an important factor.

Type II interferon (IFNγ) is a representative Th1 cytokine and it possesses a variety of functions, including immune regulation, antiviral and antitumor activity. Because of its multifunctional nature, IFNγ is expected to be applied to the treatment of autoimmune diseases, cancer and viral infection. Although IFNγ has therapeutic potential for such diseases, the clinical use of IFNγ has been limited due to its short in vivo half-life and serious adverse effects. In contrast, gene delivery of IFNγ is an alternative approach to increasing the retention time of IFNγ. To extend transgene expression after plasmid DNA (pDNA) gene transfer, we designed and developed pDNA with varying numbers of CpG motifs. CpG-reduced pDNA resulted in more durable transgene expression than its CpG replete counterpart in mice. Comparison of the effect of promoter/enhancer elements on transgene expression showed that ROSA26 promoter-mediated IFNγ expression was safe because of the lack of an initial surge after hydrodynamic gene transfer. We also designed an IFNγ-mouse serum albumin (MSA) fusion protein, IFNγ-MSA. Gene transfer of this fusion protein resulted in a sustained concentration of IFNγ fusion protein in mouse serum, and inhibited tumor metastasis in mice. These results provide experimental evidence that IFNγ gene therapy can be a useful treatment for a variety of diseases.

Sustained expression of murine interferon (IFN)-γ (Muγ) was found to be effective in preventing tumor metastasis and atopic dermatitis in mouse models. However, our preliminary experiments suggested that the time-dependent decrease in the Muγ expression was not compensated for by repeated injections of Muγ-expressing plasmid. To identify the mechanism underlying this observation, a reporter plasmid was hydrodynamically injected into mice and the levels of the plasmid, mRNA and reporter protein were measured in mice receiving a pre- or co-administration of Muγ-expressing plasmid. Co-injection of Muγ-expressing plasmid had no significant effects on transgene expression from the reporter plasmid. In contrast, pre-injection of Muγ-expressing plasmid greatly inhibited the expression of the reporter protein. Moreover, pre-injection of Muγ-expressing plasmid also reduced the amount of the reporter plasmid in the nuclear fraction of mouse liver to < 10%, and that of reporter mRNA to < 1%. The degree of reduction in the expression of reporter protein was comparable with the reduction in mRNA. These results indicate that the difficulty in regaining the expression level of IFN-γ is due to the impaired delivery of plasmid to the nucleus and to the suppression of transcription from the plasmid.

Cell‐free membrane protein (MP) synthesis in the presence of liposomes is a spotlighting technique to prepare MP‐reconstituted liposomes. In article number https://doi.org/10.1002/advs.201800524, Kazunari Akiyoshi and co‐workers develop an artificial MP sorting system based on polyhistidine/nickel‐chelate affinity. This system enables efficient reconstitution of MPs into the liposomal membrane to be achieved, and supplementations of natural molecular chaperone can control the orientation of MPs.

Background The in vivo half-life of interferons (IFNs) is very short, and its extension would produce a better therapeutic outcome in IFN-based therapy. Delivery of IFN genes is one solution for providing a sustained supply. IFNs have a variety of functions, including the suppression of transgene expression, through interaction with IFN receptors (IFNRs). This suppression could prevent IFNs from being expressed from vectors delivered. Silencing the expression of IFNAR and IFNGR, the receptors for type I and II IFNs, respectively, in cells expressing IFNs may prolong transgene expression of IFNs. Methods Mouse melanoma B16-BL6 cells or mouse liver were selected as a site expressing IFNs (not a target for IFN gene therapy) and IFN-expressing plasmid DNA was delivered with or without small interfering RNA (siRNA) targeting IFNRs. Results Transfection of B16-BL6 cells with siRNA targeting IFNAR1 subunit (IFNAR1) resulted in the reduced expression of IFNAR on the cell surface. This silencing significantly increased the IFN-β production in cells that were transfected with IFN-β-expressing plasmid DNA. Similar results were obtained with the combination of IFN-γ and IFNGR. Co-injection of IFN-β-expressing plasmid DNA with siRNA targeting IFNAR1 into mice resulted in sustained plasma concentration of IFN-β. Conclusions These results provide experimental evidence that the RNAi-mediated silencing of IFNRs in cells expressing IFN, such as hepatocytes, is an effective approach for improving transgene expression of IFNs when their therapeutic target comprises cells other than those expressing IFNs. Copyright © 2010 John Wiley & Sons, Ltd.

Chaperoning functions of liposomes were investigated using cell-free membrane protein synthesis. KcsA potassium channel-reconstituted liposomes were prepared directly using cell-free protein synthesis. In the absence of liposomes, all synthesized KcsA protein aggregated. In the presence of liposomes, however, synthesized KcsA spontaneously integrated into the liposome membrane. The KscA-reconstituted liposomes were transferred to the planar bilayer across a small hole in a thin plastic sheet and the channel function of KcsA was examined. The original electrophysiological activities, such as voltage- and pH-dependence, were observed. These results suggested that in cell-free membrane protein synthesis, liposomes act as chaperones, preventing aggregation and assisting in folding and tetrameric formation, thereby allowing full channel activity.

The excretion, tissue distribution and metabolic fate of simeconazole [(RS)-2-(4-fluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-trimethylsilyl propane-2-ol] in rats were studied by administering 14C-labeled simeconazole orally to male and female rats at 5 mg/kg b.w. (low dose) and 70 mg/kg b.w. (high dose). The simeconazole was readily absorbed and most of the radioactivity was excreted in the urine and feces within three days. The blood radioactivity level reached a maximum at 4–8 hr and 1–2 hr post-dosing in male and female rats, respectively. The radioactivity in the tissues and organs rapidly decreased with time. Simeconazole was initially metabolized to M1, the hydroxymethyl metabolite. M1 was further metabolized to glucuronide, sulfate, silanols and various dessiloxane compounds. © Pesticide Science Society of Japan

Tumor-associated macrophages (TAMs) negatively affect the therapeutic effects of anticancer agents. To examine the role of TAMs in interferon (IFN)-γ gene therapy, we selected two types of solid tumors, which varied in the number of TAMs, and investigated the effects of IFN-γ gene transfer on tumor growth. Many TAMs were detected in the solid tumors of murine adenocarcinoma colon-26 cells, whereas few TAMs were detected in murine melanoma B16-BL6 cells. IFN-γ gene transfer hardly suppressed the growth of colon-26 tumors, whereas it was effective in suppressing the growth of B16-BL6 tumors. The antiproliferative effects of IFN-γ on cultured colon-26 cells were similar to those on cultured B16-BL6 cells. To evaluate the role of TAMs, we injected clodronate liposomes (CLs) modified with poly(ethylene glycol) (PEG) to functionally deplete TAMs in tumor-bearing mice. Repeated injections of PEG-CLs significantly retarded the growth of colon-26 tumors and combination with IFN-γ gene transfer further inhibited the growth. In contrast, PEG-CLs hardly retarded the growth of B16-BL6 tumors. These results clearly indicate that TAM depletion is effective in enhancing the therapeutic effect of IFN-γ in TAM-repleted and IFN-γ-resistant tumors.

Recently a huge amount of fluoride in coal has been released into indoor environments by the combustion of coal and fluoride pollution seems to be increasing in some rural areas in China. Combustion of coal and coal bricks is the primary source of gaseous and aerosol fluoride and these forms of fluoride can easily enter exposed food products and the human respiratory tract. Major human fluoride exposure was caused by consumption of fluoride contaminated food, such as corn, chilies and potatoes. For each diagnostic syndrome of dental fluorosis, a log-normal distribution was observed on the logarithm of urinary fluoride concentration in students in China. Urinary fluoride content was found to be a primary health indicator of the prevalence of dental fluorosis in the community. In the fluorosis areas, osteosclerosis in skeletal fluorosis patients was observed with a high prevalence. A biochemical marker of bone resorption, urinary deoxypyridinoline content was much higher in residents in China than in residents in Japan. It was suggested that bone resorption was stimulated to a greater extent in residents in China and fluoride may stimulate both bone resorption and bone formation. Renal function especially glomerular filtration rate was very sensitive to fluoride exposure. Inorganic phosphate concentrations in urine were significantly lower in the residents in fluorosis areas in China than in non-fluorosis area in China and Japan. Since airborne fluoride from the combustion of coal pollutes extensively both the living environment and food, it is necessary to reduce fluoride pollution caused by coal burning.

We studied exposures to higher daily maximum temperatures and concentrations of air pollutants in Tokyo during the summer months of July and August from 1980 to 1995 and their effects on hospital emergency transports for cardiovascular and respiratory diseases for males and females > 65 years of age. Cardiovascular diseases were angina, cardiac insufficiency, hypertension, and myocardial infarction. Respiratory diseases were asthma, acute and chronic bronchitis, and pneumonia. Except for pneumonia, daily maximum temperatures were not associated with hospital emergency transports. Increasing daily maximum temperatures, however, were associated with decreased hospital emergency transports for hypertension. Concentrations of nitrogen dioxide or particulate matter < or = 10 microm, however, were associated with daily hospital emergency transports for angina, cardiac insufficiency, myocardial infarction, asthma, acute and chronic bronchitis, and pneumonia. For cardiac insufficiency, hypertension, myocardial infarction, asthma, chronic bronchitis, and pneumonia, the expected daily number of emergency transports per million were greater for males than for females. For angina and acute bronchitis, there were no differences for the expected daily numbers of emergency transports per million between males and females.

Inhalation exposure to fluoride compounds has been associated with respiratory failure. We have addressed effects of fluoride on alveolar macrophages and lung responses to intratracheally (i.t.) instilled fluoride in rats. I.t. instillation of fluoride at doses of 200 and 400 microg F/rat caused significant polymorphonuclear leukocyte (PMN) infiltration in the rat lung at 20 h post-administration, while 100 microg fluoride did not recruit a significant number of PMNs in the alveolar space. Total RNA was extracted from the lung lavage cells obtained from 5 h post i.t. instillation and mRNA levels of chemokines and proinflammatory cytokines were semi-quantitatively evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). I.t. instillation of fluoride significantly enhanced mRNA expression of cytokines such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory proteins-1alpha and -2. Fluoride-induced augmentation in IL-1beta mRNA expression was also examined by Northern hybridization following in vitro exposure of alveolar macrophages to fluoride. However, the enhancement of IL-1beta mRNA expression following in vitro exposure to fluoride was observed only at 500 microM, a dose higher than the 50% lethal concentration (LC(50)). Non-specific adhesion of alveolar macrophages to the plastic dish was significantly increased following in vitro exposure to fluoride. The fluoride-induced non-specific adhesion was significantly reduced by anti-CD18, suggesting that beta(2) integrin played a role in the increase of adherence. Those results suggest that fluoride activates alveolar macrophages, enhances the production of chemokines and proinflammatory cytokines, and causes PMN infiltration in the lung.

To determine the effects of high-temperature exposure on antibody response to viral antigen in mice, male BALB/c mice were placed for 13 days in animal chambers at 23 degrees C, 32 degrees C, and 35.5 degrees C. Rectal temperature rose from 37 degrees C to 39 degrees C on day 1 in a 35.5 degrees C environment. The rectal temperature was kept constant throughout the exposure period. The IgG-antibody to Sendai virus (SV) antigen was inhibited to about 50% of the control value (23 degrees C). The serum corticosterone concentration indicating thermal stress increased steadily, peaking on day 1 and then gradually decreased and recovered to the normal level on day 13. Body weight decreased to about 72% of the controls on day 13. Thymus and spleen weight decreased to 31.7% and 61.5% respectively. At 32 degrees C, these effects were less than at 35.5 degrees C. Effects of high-temperature exposure at 35.5 degrees C appeared to noticeably decrease thymus and spleen weight. It is clear that IgG-antibody response to SV antigen is suppressed by high-temperature exposure.

石炭燃焼に由来するフッ素症を調査するため, 日中共同研究を行った。研究は1994年から5年計画で, 日本農村医学会のメンバーと中国衛生部予防医学科学院との問で, 屋内フッ素汚染の調査と健康影響に関する共同研究として実施した。大規模な中国現地調査期間は1995年から1997年の3年間である。調査地域は, 汚染のない対照地域1か所と汚染地域2か所の3地域である。いずれも飲料水にはフッ素汚染のない地域である。調査は, フッ素暴露集団における健康状態を把握し, フッ素症発生と健康状態を検討することを目的に, 屋内外大気汚染濃度の測定と, 小学生高学年50人, 中学生50人, その親100人, 患者50人をそれぞれの地域で選び, 尿中フッ素濃度の測定, 尿中成分分析, 歯牙フッ素症と骨フッ素症の確定診断を実施した。その結果, 水のフッ素汚染がない地域で, 石炭燃焼に由来するフッ素症発症の確認がされた。しかも, その発症は, 石炭燃焼によって汚染された屋内大気中フッ素を直接吸入することによるものではなく, 屋内大気で汚染された穀物の摂取によるものであった。フッ素は石炭だけでなく, 火力調整用に混ぜられる土壌にも多く含まれ, 汚染に大きく寄与していた。汚染の代表的な作物は唐辛子, トウモロコシ, ジャガイモであった。また, 汚染地区住民の尿中にフッ素が大量に検出された。

The combustion of high fluoride-content coal as an energy resource for heating, cooking, and food drying is a major exhaust emission source of suspended particulate matter and fluoride. High concentrations of these pollutants have been observed in indoor air of coal-burning families in some rural areas in China. Because airborne fluoride has serious toxicological properties, fluoride pollution in indoor air and the prevalence of fluorosis have been analyzed in a fluorosis area and a healthy nonfluorosis area in China and in a rural area in Japan. For human health, fluoride in indoor air has not only been directly inhaled by residents but also has been absorbed in stored food such as corn, chilies, and potatoes. In the fluorosis area in China, concentrations of urinary fluoride in the residents have been much higher than in the nonfluorosis area in China and in the rural area in Japan. In the fluorosis area, almost all elementary and junior high school students 10-15 years of age had dental fluorosis. Osteosclerosis in the skeletal fluorosis patients was very serious. Urinary deoxypyridinoline in rural residents in China was much higher than in rural residents in Japan. Data suggest that bone resorption was extremely stimulated in the residents in China and that fluoride may stimulate both bone resorption and bone formation. Because indoor fluoride from combustion of coal is easily absorbed in stored food and because food consumption is a main source of fluoride exposure, it is necessary to reduce airborne fluoride and food contamination to prevent serious fluorosis in China. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8

Although an excess intake of fluoride has been reported to cause skeletal fluorosis, very little is known about the mechanism of adverse effects of fluoride on bone. In the present study cytotoxic effects of fluoride were studied using the osteosarcoma cell line, UMR 106. The DNA ladder formation upon agarose electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed that UMR 106 underwent apoptosis following exposure to 5 mM fluoride for 8 h. On the other hand exposure to A23187, a calcium ionophore, caused necrosis while co-exposure to fluoride and A23187 inhibited fluoride-mediated apoptosis in UMR 106. The proliferation of UMR 106 cells cultured for 6 days in the presence of 0.5 mM fluoride was significantly decreased compared to the control culture. The cytotoxic effects of fluoride were modulated by both the cell density and the pH of the culture medium. The fluoride-induced viability loss in UMR 106 was enhanced in culture of high cell-density and inversely correlated with pH of the culture medium. Enhancement of fluoride cytotoxicity at acidic pH was also observed in rat alveolar macrophages and RAW 264, a macrophage cell line. The results suggest that fluoride-mediated apoptosis and culture conditions, including pH of the medium, should be taken into consideration to evaluate toxicity of fluoride in vitro.

Human activities, such as combustion of fossil fuels, agricultural activities and changes in land- use, are responsible for global climate change. Because of the rapid increase of anthropogenic greenhouse gas emissions (carbon dioxide, methane and nitrous oxide), climate models predict an increase in global surface temperatures of 1.0°C to 3.5°C by the year 2100. Using general circulation models, it is estimated that surface temperatures in and around Japan will rise 1°C to 3°C during the next 100 years. Because global warming may increase the frequency and length of high temperatures during hot summer months in temperate regions, various health risks caused by heat stress have been studied. According to an epidemiological survey, the incidence of heat-related illness was significantly correlated to hot environments in Tokyo, Japan and in Nanjing and Wuhan, China. The epidemiological results showed that the incidence of heat related mortality and morbidity, such as heat stroke in the elderly, increased very rapidly when daily maximum temperatures in summer months rose above 31°C in Tokyo and 35°C in Nanjing and Wuhan, respectively. J Epidemiol, 1996 ; 6 : S141-S144.

We have demonstrated that gene delivery of a fusion protein of mouse interferon (IFN) γ with mouse serum albumin (IFNγ-MSA) was effective in prolonging the circulation half-life of IFNγ in mice. However, the fusion to MSA greatly reduced the biological activity of IFNγ to less than 1%. In this study, we designed IFNγ fusion proteins with a 20 amino-acid long albumin-binding peptide (ABP) to prolong the in vivo half-life of IFNγ without reducing its biological activity. IFNγ-ABP and ABP-IFNγ, two fusion proteins with the ABP being fused to the C- or N-terminal of IFNγ, retained 40%-50% biological activities determined using a gamma-activated sequence-dependent luciferase assay. These fusion proteins exhibited the ability to bind to MSA. Gene delivery of IFNγ-ABP or ABP-IFNγ to mice using the hydrodynamic injection method resulted in a sustained concentration of IFNγ in the serum compared with gene delivery of IFNγ. In addition, the growth of mouse colon carcinoma CT-26 cells in the lung was efficiently inhibited by gene delivery of the IFNγ fusion proteins. These results indicate that the fusion of ABP is a useful approach to achieving prolonged retention in the blood circulation through binding to serum albumin and retaining biological activity.

Hydrodynamic injection of pmCMV(enh) -hEF-1(prom) -muIFN-γ, a plasmid DNA (pDNA) expressing murine interferon (IFN)-γ with a murine cytomegalovirus (mCMV) enhancer and a human elongation factor (EF)-1 promoter, has been proven effective for the treatment of cancer and atopic dermatitis in mice. However, the initial peak of IFN-γ soon after injection was quite high compared to the steady level for subsequent periods, which could cause unwanted adverse effects. Therefore, in the present study, aiming to optimize the efficacy/side-effect ratio of IFN-γ gene transfer, we have developed plasmid vectors encoding murine IFN-γ under the control of different combinations of promoter and enhancer sequences.The promoter and enhancer sequence of pmCMV(enh) -hEF-1(prom) -huIFN-γ, a prototype plasmid expressing human IFN-γ, was replaced or deleted to obtain various pDNAs. To assess the transgene expression profile, each pDNA was delivered to mice by hydrodynamic injection and the serum IFN-γ concentration was measured periodically. On the basis of the results obtained, murine IFN-γ expressing pDNAs were constructed and the body weight change was monitored as an indicator of adverse effects.The prototype pmCMV(enh) -hEF-1(prom) -huIFN-γ showed a high but declining concentration of IFN-γ. Those containing hROSA26 promoter expressed the transgene in a more constant manner with no initial high concentrations and scarcely reduced the body weight.These results indicate that hROSA26 promoter, irrespective of the presence and type of enhancers, is suitable for achieving constant and steady level of transgene expression and effective in avoiding the body weight loss caused by high concentrations of IFN-γ.

1. 1.|Significant lipid peroxidation occurred both in rat and guinea pig liver in passive hyperthermia caused by a hot environment. In guinea pig, lipid peroxidation was greatly induced not only in liver homogenate but also in intracellular structures, such as mitochondria and microsomes. 2. 2.|In rat, hepatic cytosolic selenium glutathione peroxidase activities were greatly induced in hyperthermia, while hepatic cytosolic glutathione peroxidase activities in guinea pig were not affected in hyperthermia. 3. 3.|In both animals, hepatic cytosolic glutathione S-transferase, cytosolic superoxide dismutase, and catalase activities were not affected. 4. 4.|Since the peroxidation of lipids in biological membranes is a destructive phenomenon and is associated with a variety of cellular damage, hyperthermia greatly developed hypertrophy and vacuolized degeneration in hepatic cells. 5. 5.|In guinea pig, the activities of hepatic mitochondrial electron transport system, such as cytochrome c oxidase and cytochrome c reductase system were simultaneously inhibited in hyperthermia. 6. 6.|On the other hand, hepatic microsomal electron transport system, such as cytochrome P450 and aminopyrine N-demethylase activities were not seriously affected by hyperthermia. 7. 7.|In nuclei and mitochondria, 90-kDa heat shock induced proteins markedly expressed in liver. 8. 8.|Since hyperthermia greatly induces lipid peroxidation in liver, heat stress may have a potential impact on peroxidative damage in humans and animals.

The mutagenicity of airborne particulates in Tokyo was evaluated by the Salmonella microsuspension procedure. Airborne particles were collected in 9 collection stages according to their aerodynamic diameter and tested for the mutagenicity with S. typhimurium strain TA 98. Because the microsuspension procedure is highly sensitive, it was possible to detect the mutagenic activity from only 10 μg of the particles of less than 2.5 μm in diameter. Moreover we could detect the mutagenic activity of the particles ranging from 2.5 to 10 μm, which had been difficult to be detected by the original Ames test. The particles collected in winter showed higher activity than those in summer. The mutagenic activity was high in the particles of less than 0.4 μm and of about 1 μm in winter. In the same season, the specific activity for the mutagenicity of the particulates did not differ in the sampling area. The concentration of polyaromatic hydrocarbons in airborne particulates correlated with the mutagenicity of those in every collection stage, and it was suggested that mutagens in particulate matter behave as polyaromatic hydrocarbons.

Gene delivery of mouse interferon (IFN) γ has been shown to inhibit metastatic tumor growth and onset of atopic dermatitis in mouse models. In this study, we tried to increase the circulation half-life of IFNγ after its gene delivery by designing a novel fusion protein of IFNγ with mouse serum albumin (MSA). Western blot analysis confirmed that IFNγ–MSA was expressed as a fusion protein, but hardly formed dimer as IFNγ did. The biological activity of IFNγ–MSA, which was examined using a plasmid expressing luciferase under the control of gamma-activated sequence elements, was about 200-fold lower than the activity of IFNγ. Intravenous injection of the proteins into mice confirmed that the circulation half-life of IFNγ was significantly prolonged by the modification. A hydrodynamic injection of a plasmid expressing IFNγ–MSA resulted in a sustained concentration in mouse serum; it resulted in about sixfold greater area under the concentration–time curve and about threefold longer mean residence time of IFNγ activity than those of IFNγ. Gene delivery of IFNγ–MSA inhibited tumor metastasis to a similar level to that of IFNγ despite the reduced activity of IFNγ–MSA. These results indicate that gene delivery of IFNγ–MSA is a promising approach to prolong the circulation half-life of IFNγ activity.

M.A3あるいはM.A4をNADPH存在下でラット肝ミクロゾームと反応させたところ, 主代謝物の生成が認められた. 主代謝物を分離精製後, そのMS, NMRスペクトルから, それらはM.A3およびM.A4が13 (β) 位に水酸化を受けたものであった. 13位水酸化反応のキネティックパラメーターを調べたところ, M.A3のVmax値はM.A4のそれより大きくM.A3の代謝がM.A4より速かった. このことは in vivo の実験結果とよく一致した. 一方, Km値は両者でほぼ同じ値であった. M.A3とM.A4の代謝の受けやすさの差は本研究結果から, 酸化酵素と基質の結合段階にあるのではなく, それ以降の過程にあるものと思われた. 13位水酸化反応はフェノバルビタールの処理により誘導される型のチトクロームP-450が関与する酸化反応と考えられた.

M.A3およびM.A4の雌雄ラットにおける代謝を5位を3Hで標識した化合物を用いて調べた. 3H-M.A3または3H-M.A4を5mg/kgの割合で経口投与したところ, いずれの化合物においても3Hは7日後までにほぼ完全に糞および尿中へ排泄された. 組織中の3H濃度は脂肪や肝臓において高かったが, 時間の経過とともに速やかに減少し, 7日後に組織中に残留する3Hはわずかであった. 3H-M.A3を投与したラットの組織中の3Hおよび親化合物の濃度は3H-M.A4のそれより低く, M.A3は M.A4より速く代謝を受けるようであった. 組織中には主代謝物として, 両化合物ともに13-ヒドロキシ体が検出され, 尿中にはジヒドロキシ体が検出された. 糞中にはこれらの代謝物のほかにさらに極性の大きい代謝物が検出された. M.A3およびM.A4は優先的に13位が水酸化された後. ジヒドロキシ体へと代謝され, さらにこれらは極性物質にまで代謝されるものと思われた. M.A3とM.A4のラットにおける代謝運命に本質的な違いは認められなかった. ラット小腸における吸収実験においてM.A3の吸収はM.A4よりやや速かった.

Background Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-β or IFN-γ, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA. Methods Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-γ-expressing plasmids were constructed and their transgene expression and inhibitory effect on pulmonary metastasis were evaluated. Results Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Muγ, the IFN-γ-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-γ concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of tumor cells. Conclusions The duration of transgene expression of IFN-γ was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in an increased anticancer activity of IFN gene transfer. Copyright © 2009 John Wiley & Sons, Ltd.

To study the concentration change of respirable suspended particulate (RSP) in indoor and outdoor air environment, a new portable sampler (AND-sampler) was made. The sampler could collect particles above 10 microns, 2 to 10 microns, and below 2 microns in aerodynamic diameter separately. The relationship between the airborne particulate concentration in indoor and outdoor air environment varied with the aerodynamic diameter of the particles. The concentration of RSP in indoor air environment increased in proportion to that in outdoor environment. Polycyclic aromatic hydrocarbons (PAH) concentration in airborne particulate also varies with the aerodynamic diameter of the particles. Fine particles below 2 microns in aerodynamic diameter contains high concentration of PAH. The PAH concentration in indoor air environment increases in proportion to that in outdoor air.

現在自動車排ガス, 産業廃棄物, 散布農薬など多数の有毒有機汚染物質の発生源が存在する。日本を含め多数の国で, 地球規模の農薬汚染が進行し, 農場においては, 殺虫剤, 除草剤, 殺菌剤といった農薬が, 頻繁に散布されている。現在, 農薬による中毒防止のため, 農薬汚染や暴露および健康リスクに関して, 世界中で研究が進行している。大気や自然生態系において高い濃度の農薬が検出されるため, 多くの国でその健康影響に強い関心が寄せられている。ここでは環境中の農薬汚染とその健康リスクについて要約した。さらに自然生態系や体内に残留する農薬のリスク評価についても検討した。

パラコート剤は日本においてもっとも広範に使用されてきている除草剤の一種である。パラコート剤の慢性的生体影響を検討するため, 1mg, 5mg, 25mg/kg/dayの投与量で1年2か月にわたりパラコートをラットに投与した。慢性的パラコート剤の投与により, 肺, 心臓, 肝臓等種々の臓器に顕著な組織化学的変化が認められた。肺部においては, 1mg/kg/dayの投与量において既に, 肺の維繊化が引き起こされた。血清生化学検査では, アルカリフォスファターゼ活性とクレアチニンの上昇が認められた。血清コレステロール, トリグリセライド, リン脂質は著しく減少した。パラコートの慢性投与により脳, 腎臓, 肝臓においては著しい過酸化脂質の蓄積が認められた。

The change in concentration of suspended particulate matter (SPM) in indoor and outdoor air in the Tokyo metropolitan area was studied using a new portable sampler (SPMP-sampler). The relationship between the airborne particle concentration in indoor and outdoor air varied with the aerodynamic diameters of the particles. The concentration of the SPM in indoor air increased in proportion to that in outdoor air. The polycyclic aromatic hydrocarbon (PAH) concentration in SPM also varied with the aerodynamic diameters of the particles. Fine particles with diameters of less than 2 microns contained high concentrations of PAH. The PAH concentration in indoor air increased in proportion to that in outdoor air. There were significant correlations between the concentrations of B(k)F, B(a)P, and B(ghi)P in indoor air. The mutagenic activities in the airborne particles also varied with the aerodynamic diameters of the particles. Fine particles with diameters of less than 2 microns had high mutagenic activities.

現在, 南極大陸上空を中心に成層圏オゾンの枯渇 (オゾンホール) の拡大が観察されており, 地球全域でのオゾン減少による紫外線B照射の増大が危倶されている。紫外線B照射によって, 雪盲, 白内障, 皮膚癌のような種々の障害が引き起こされる一方, ある種の感染症に対する免疫能が低下する。非黒色腫の基底細胞癌と有棘細胞癌は, 明らかに紫外線B照射量との関係が強い。悪性黒色腫も少なくとも一部は, 紫外線B照射と関係がある。アメリカ合衆国における調査では, 高緯度から低緯度になるにしたがい, 紫外線B照射が強くなり, 黒色腫を含む皮膚癌の発生率が高くなる。数量的予測は困難であるが, アメリカ合衆国の疫学データーから, UNEPとWHOは1%の成層圏オゾンの減少により白内障が0.3%から0.6%, 基底細胞癌が2.7%, 有棘細胞癌が4.6%, 悪性の黒色腫が0.6%増加すると予想している。このため, 紫外線B暴露量との関連で皮膚癌の発生率の検討を世界各国で行なう必要がある。さらに, 紫外線B照射の増大が免疫を抑制し, 感染症の発症に関与する可能性について充分な検討が必要である。

Chlorofluorocarbon-11 (CFC11) lasts for an average of 74 years in the atmosphere, CFC12 for an average of 111 years, and CFC113 for an average of 90 years. Every CFC molecule destroys thousands of molecules of stratospheric ozone. Recently, the extent of the Antarctic ozone hole has been recognized. The depletion of stratospheric ozone may lead to increase ultraviolet-B (UV-B) radiation. UV-B radiation has many damaging effects on human health, such as snow blindness, cataract and skin cancer. UV-B radiation also suppresses the immune defenses against certain infections. While it is difficult to estimate the numerical effect on the basis of epidemiologic data in the U.S. A., UNEP and WHO estimate that for every 1% decrease in stratospheric ozone, there will be between a 0.3 to 0.6% increase in cataract. They also estimate that for every 1% depletion of ozone, the incidences of basal cell carcinoma, squamous cell carcinoma and malignant melanoma will increase 2.7, 4.6 and 0.6%, respectively. There is also concern that increased UV-B radiation might lead to an increase of the incidence and severity of infectious diseases due to suppression of the immune system. Since the data on UV-B exposure are extremely limited, it is necessary to confirm the incidence rate of skin cancer in various countries in relation to UV-B exposure.

Since inhaled fluoride is implicated in the acute respiratory failure, cytotoxic effects of fluoride on alveolar macrophages, primary target cells of inhaled toxicants, were investigated. The LC50 of sodium fluoride was estimated to be 0.41 mM, while 1 mM sodium chloride, bromide and iodide had virtually no effects on the viability of alveolar macrophages. Photomicroscopic observation revealed that nuclei of the fluoride-exposed alveolar macrophages were fragmented. The ladder formation was observed when DNA isolated from fluoride-exposed alveolar macrophages was electrophoresed in agarose gel. These results suggest that cytotoxicity of fluoride is associated with apoptosis in rat alveolar macrophages.

[1] Using acoustic Doppler current profiler (ADCP) and conductivity-temperature-depth (CTD) data sets obtained from 1991 to 1993, volume, freshwater, and temperature transports through the Tsushima-Korea Straits are estimated. To remove tidal currents contained in raw ADCP data, tidal currents are calculated using harmonic constants obtained by ADCP surveys from 1998 to 1999. The annual mean and range of the volume transport through the straits are 2.4 Sv (1 Sv = 106 m3/s) and 0.8 Sv, respectively. These values are consistent with those of previous studies. The annually averaged freshwater transport is 33 × 106 kg/s, which is the same as the total river discharge around the Yellow and East China Seas. This suggests that the total river discharge and freshwater transport through the Tsushima-Korea Straits are the prominent two components in the freshwater budget, averaged annually in the Yellow and East China Seas. The annually averaged temperature transport through the Tsushima-Korea Straits is 0.17 PW.

In endemic fluorosis areas in China associated with coal burning, indoor airborne fluoride pollution is severe. To determine the effects of fluoride aerosols on pulmonary antibacterial defense mechanisms and lung damage, mice were exposed to various concentrations of fluoride aerosol (2, 5, or 10 mg/m3) or filtered air (control) for 14 d, 4 h/d in an inhalation chamber. Bactericidal activity against Staphylococcus aureus in the lung and the number and profile of free pulmonary cells, protein content, and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage (BAL) fluid were assessed. Urinary fluoride concentration, an indicator of fluoride exposure, increased in proportion to fluoride aerosol concentration in the chamber. Wet lung weight was significantly higher on d 14 in mice exposed to 10 mg/m3 than in controls. Pulmonary bactericidal activity against S. aureus was concentration-dependently suppressed at 5 and 10 mg/m3 fluoride. The number of alveolar macrophages (AMs) in the BAL fluid of the mice not bacterially challenged decreased significantly at 10 mg/m3 fluoride. The number of polymorphonuclear leukocytes and lymphocytes increased significantly at 10 mg/m3 fluoride exposure. The concentration of total protein (TP) and albumin in BAL supernatant increased significantly at 5 and 10 mg/m3 fluoride exposure, and LDH activity rose markedly at the higher fluoride concentration. Data indicate that fluoride inhalation produces pulmonary cellular alterations that are associated with a diminished ability to cope with infectious bacteria.

Heat stroke is associated with prolonged exposures to high air temperatures that usually occur in the summer months of July and August in Tokyo, Japan. Also during July and August, residents of Tokyo are often exposed simultaneously to high concentrations of air pollutants. To assess the impacts of these combined exposures, daily numbers of heat stroke emergency transport cases/million residents for Tokyo were stratified by gender and three groups: 0-14, 15-64; and > 65 years of age, for the months of July and August in 1980-1995. A regression model was constructed using daily maximum temperature (Tmax) and daily average concentrations of NO2 and O3 as model covariates. Classification indices were added to make it possible to compare the expected number of heat stroke cases by age and gender. Lag times of 1-4 days in Tmax and air quality covariates and terms to account for interactions between pairs of model covariates were also included as additional risk factors. Generalized linear models (GLMs), assuming a Poisson error structure for heat stroke emergency transport cases, were used to determine which covariates were significant risk factors for heat stroke for the three age groups of males and females. Same-day Tmax and concentrations of NO2 were the most significant risk factors for heat stroke in all age groups of males and females. The number of heat stroke emergency transport cases/million residents was greater in males than in females in the same age groups. The smallest number of heat stroke emergency transport cases/million residents occurred for females 0-14 years of age and the greatest number of heat stroke emergency transport cases/million residents occurred for males > 65 years of age. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5