Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca²⁺ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor

Matsuda T., Sakai S., Okazaki K.I., et al. Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca²⁺ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor. ACS Sensors 9, 1743 (2024); https://doi.org/10.1021/acssensors.3c02398.To monitor the Ca²⁺ dynamics in cells, various genetically encoded Ca²⁺ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca²⁺ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs

Gene expression of cell surface antigens in the early phase of murine influenza pneumonia determined by a cDNA expression array technique.

BACKGROUND: Influenza virus is a worldwide health problem with significant economic consequences. To study the gene expression pattern induced by influenza virus infection, it is useful to reveal the pathogenesis of influenza virus infection; but this has not been well examined, especially in vivo study. AIMS: To assess the influence of influenza virus infection on gene expression in mice, mRNA levels in the lung and tracheal tissue 48 h after infection were investigated by cDNA array analysis. METHODS: Four-week-old outbred, specific pathogen free strain, ICR female mice were infected by intra-nasal inoculation of a virus solution under ether anesthesia. The mice were sacrificed 48 h after infection and the tracheas and lungs were removed. To determine gene expression, the membrane-based microtechnique with an Atlas cDNA expression array (mouse 1.2 array II) was performed in accordance with the manual provided. RESULTS AND CONCLUSIONS: We focused on the expression of 46 mRNAs for cell surface antigens. Of these 46 mRNAs that we examined, four (CD1d2 antigen, CD39 antigen-like 1, CD39 antigen-like 3, CD68 antigen) were up-regulated and one (CD36 antigen) was down-regulated. Although further studies are required, these data suggest that these molecules play an important role in influenza virus infection, especially the phase before specific immunity

Natural killer cytolytic activity is associated with the expression of killer cell immunoglobulin-like receptors on peripheral lymphocytes in human.

Although it has been shown that killer cell immunoglobulin-like receptors (KIRs) on peripheral lymphocytes are upregulated by interleukin-2 (IL-2), which activates natural killer (NK) activity, it has not been demonstrated whether the expression of KIRs is related to NK activity. Therefore, we investigated the association between the KIR expression on lymphocytes and NK activity. CD158a/b expression on lymphocytes obtained from 37 subjects was analyzed using flow cytometry. Simultaneously, NK activity was measured each sample using a 51Cr-release assay. Additionally, lymphocytes were cultured in RPMI 1640 medium with or without IL-2 for 48 h, and then their CD158a/b expression and NK activity was analyzed. CD158a/b expression was significantly correlated with NK activity. Especially, the percentage of CD16+CD158a+ and CD8+CD158a/b+ cells in lymphocytes showed a highly significant correlation with NK activity. However, analysis of CD8+ and CD16+ cells revealed that there was only a significant correlation between the percentage of CD8+CD158a+ cells among only CD8+ cells and NK activity. The upregulation of CD16+CD158a+/b+ cells in response to IL-2 tended to be related to the increase of NK activity, but the relationship was not significant. In conclusion, the level of KIR expression was correlated with NK activity, and IL-2 treatment resulted in an increase of NK activity as well as KIR expression, suggesting that upregulation of KIRs enhances the ability to sort target cells, such as virus-infected cells from uninfected cells, according to major histocompatibility complex class I expression

A case of rheumatoid arthritis complicated by bucillamine-induced nephropathy satisfactorily treated with Kampo medicines

We reported a case of rheumatoid arthritis complicated by bucillamine-induced nephropathy satisfactorily treated with Kampo (Chinese/Japanese traditional) medicines. The patient was a 56-year-old male, who suffered from polyarthralgia from December 1996. In February 1997, he was diagnosed with RA in another hospital and treated with loxoprofen sodium, bucillamine and prednisolone. He visited our hospital in May 1997, and we began to treat him with a combination of modern Western and Kampo medicines. During his clinical course, proteinuria induced by bucillamine developed. After bucillamine was discontinued, his polyarthralgia and inflammatory parameters became worse. However, Kampo formulations, Keishi-bukuryo-gan mixed with Toki-shakuyaku-san and Dai-bofu-to, were effective for reducing the activity of RA. The clinical course of this case suggests that Kampo medicines might be useful for the treatment of RA patients, especially for those who have adverse reaction to disease-modifying antirheumatic drugs (DMARDs).　ブシラミン腎症を併発した慢性関節リウマチ(RA)に対して,漢方治療が奏功した1例を報告した。症例は56歳,男性。1996年12月,多関節痛出現。1997年2月,他院にてRAの診断のもと,ロキソプロフェン,プレドニゾロン,ブシラミンにて加療を受けた。1997年5月,当科初診以来,現代西洋医学的治療に漢方治療を併用して加療にあたった。経過中,ブシラミンによる蛋白尿が出現。ブシラミンを中止したところ,多関節痛と炎症反応が悪化した。しかし,桂枝茯苓丸合当帰芍薬散および大防風湯が奏功し,RAの活動性が低下し蛋白尿も消失した。このことから,副作用によりDMARDsが使用困難なRAの治療に対して,漢方治療が有効である可能性が示唆された

A case of rheumatoid arthritis with a decrease in the serum concentration of soluble CD23 by traditional herbal medicine

Traditional herbal (Kampo) medicine for the treatment of rheumatoid arthritis (RA) has not been evaluated for changes in immune status during its use, although several anti-inflammatory effects have been studied in vitro. In this case report, we describe a 32-year-old Japanese female with RA who was effectively treated with the Kampo medicine, Hochu-ekki to (Bu-Zhong-Yi-Qi-Tang, 補中益気湯) . This treatment decreased the serum concentration of soluble CD23 (sCD23) and RA activity, in addition to tumor necrosis factor-α. These findings suggest that the therapeutic action of herbal medicine might be attributable to suppression of B cell activation. 和漢薬治療は慢性関節リウマチ(RA)に対して極めて有用な治療法であるが,その作用機序に関しては,In Vitroによる生薬の抗炎症効果は解析されているものの,In Vivoでの免疫学的な評価はいまだ未着手な部分が多い。われわれは補中益気湯などが有効であったRAの一例を経験した。この患者は,RAの疾患活動性の低下に加えて血清中可溶性CD23(sCD23)が低下し,さらに,血清TNF-α濃度も,漸次低下していた。このことからRAの治療において和漢薬は単に抗炎症効果を有するだけでなくImmunomodulatorとして作用している可能性が示唆された。その一部としてRA患者でのB細胞の活性化を抑制している可能性がある

The examination of endoscopic findings in 221 cases of rheumatoid arthritis treated with Japanese Oriental (Kampo) medicine

OBJECTIVES: To evaluate the efficacy of Japanese Oriental (Kampo) medicine on treatment of rheumatic arthritis (RA) patients from the point of view of preventing the complication of gastroduodenal ulcer. METHODS: We examined endoscopic findings and contents of treatment in 221 rheumatoid arthritis (RA) cases treated with Japanese Oriental (Kampo) medicine retrospectively. RESULTS: A gastroduodenal ulcer was found in 10 patients (4.5%), an ulcer scar was found in 21 patients (9.5%) and a cancer was found in 6 cases (2.7%). Next, we compared the incidence of gastroduodenal ulcers in the group of patients who were receiving nonsteroidal antiinflammtory drugs (NSAIDs) therapy with the group of patients treated with Kampo medicine alone. The incidence of gastroduodenal ulcers was 9.3 % in NSAIDs group and 2.1 % in the group of patients treated with Kampo medicine alone. CONCLUSIONS: The incidence of gastroduodenal ulcers in RA patients treated with Kampo medicine was lower than that in other studies carried out in patients with RA. These findings suggested that treatment with Kampo medicine for RA is beneficial from the point of view of preventing gastroduodenal complication. 和漢薬治療中の関節リウマチ(RA)患者の胃十二指腸病変を検討した。【方法】当科で和漢薬治療中のRA患者のうち,1980年1月から2000年10月までに上部消化管内視鏡検査を施行した221例について内視鏡所見とRAの活動度,治療内容との関連を検討した。【結果】対象221例中,活動性の消化性潰瘍は10例(4.5%)。内訳は胃潰瘍9例,十二指腸潰瘍3例であった。潰瘍瘢痕は21例(9.5%)。内訳は胃潰瘍瘢痕16例,十二指腸瘢痕5例であった。その他,びらん性変化が12例(5.4%),早期胃癌が4例(1.8%)に認められた。非ステロイド性消炎鎮痛剤(NSAIDs)またはステロイド剤(ス剤)併用治療群(107例)と和漢薬単独治療群(96例)で比較検討すると,前者の活動性胃十二指腸潰瘍合併例が10例(9.3%)であるのに対し,後者では2例(2.1%)とNSAIDsまたはス剤併用群に有意に多く活動性潰瘍が合併していた。【考察】1999年CheatumらはNSAIDs服用中のRA患者の胃十二指腸病変を内視鏡検査で検討し,1009例中239例(23.6%)に胃または十二指腸潰瘍があったことを報告している。RA患者の胃十二指腸潰瘍の合併頻度については,Farahらの185例中67例(1988年),塩川らの1008例中175例(1991年)などがあり,いずれもNSAIDs投与との関連性が指摘されている。今回の検討では,221例中10例とこれらの報告と比較して少ない傾向にあった。和漢薬を中心とした治療で,NSAIDs使用を最小限にとどめることにより,消化性潰瘍の合併を減らしうる可能性が示唆された

Protective effect of the hooks and stems of Uncaria sinensis against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells

We have previously shown that an aqueous extract of the hooks and stems of Uncaria sinensis (O_.) H_., Uncariae Uncus Cum Ramulus, protects against glutamate-induced neuronal death in vitro. Nitric oxide (NO) free radicals are also implicated in the process of neuronal death. In this study, we investigated the protective effects of Uncaria sinensis extract (USE) and its phenolic and alkaloid fractions against NO donors, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), -induced neuronal death in cultured rat cerebellar granule cells. MTT assay showed cell viability to be significantly increased by the addition of USE (10, 30 and 100 μg/ml) compared with exposure (6, 12 and 24 h) to SNP (30 μM) only, and by the addition of USE (10 and 30 μg/mi) compared with exposure (6, 12 and 24 h) to SIN-1 (300μM) only. Phenolic fraction of USE (10 and 30μg/ml) significantly protected against SNP (30 μM, 24 hr)-induced cell death, and 3 and 10 μg/ml of this fraction significantly protected against SIN-1 (300 μM, 24 hr)-induced cell death. Alkaloid fraction of USE (30 and 100 μg/ml) significantly protected against SNP (30 μM, 24 hr) and SIN-1 (300 μM, 24 hr)-induced cell death. These results appear to indicate that Uncaria sinensis has a protective effect against NO-mediated neuronal death in cultured cerebellar granule cells and that its active components are included in phenolic and alkaloid fractions. 培養ラット小脳顆粒細胞を用いて,NO donor誘導神経細胞死に対する釣藤鈎エキスの保護作用を検討した。NO donorにはsodium nitroprusside(SNP) と 3-morpholinosydnonimine (SIN-1)を用い,細胞生存率の評価にはMTT法を用いた。釣藤鈎エキス(10,30,100μg/ml)は,SNP(30μM)6,12,24時間添加による神経細胞死を有意に抑制した。釣藤鈎エキス(10,30μg/ml)は,SIN-1(300μM)6,12,24時間添加による神経細胞死を有意に抑制した。釣藤鈎エキスのフェノール画分は,10,30μg/mlの濃度でSNP(30μM,24時間)添加による神経細胞死を有意に抑制し,同じく3,10μg/mlの濃度でSIN-1(300μM,24時間)添加による神経細胞死を有意に抑制した。釣藤鈎エキスのアルカロイド画分(30,100g/ml)は,SNP(30μM,24時間)およびSIN-1(300μM,24時間)添加による神経細胞死を有意に抑制した。以上より,釣藤鈎エキスはNO donorによって誘導される神経細胞死に対して保護作用を有し,その活性は釣藤鈎のフェノール画分およびアルカロイド画分にあることが示唆された