Cofactor-specific covalent anchoring of cytochrome b 562 on a single-walled carbon nanotube by click chemistry †

International audienceRedox-active cytochrome b 562 with a tethered azide group on the heme propionate side chain is covalently linked to an acetylene moiety introduced on the sidewall of a single-walled carbon nanotube (SWNT) by copper-catalyzed click chemistry forming a triazole ring with the heme active site directly linked to the SWNT. The cytochrome b 562 –SWNT hybrid is characterized by electrochemistry and atomic force microscopy. Interfacing redox-active enzymes with electrode materials is a key technology used in the development of high performance biosensors and biofuel cells. 1–3 Recent advances in carbon nanomaterials have enabled us to design hybrid systems with linked enzymes. Carbon nanotubes (CNTs), of the single-or multi-walled type, are promising building blocks for fabrication of hybrid materials. 4–8 CNTs have large surface areas, high strength, chemical stability, and attractive electronic properties. CNTs have also provided a wide variety of synthetic tools applicable for introduction of a range of substituents for linking the enzymes. A copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction is a widely utilized method used to form a covalent linkage which includes a triazole ring between building blocks containing azide and alkyne groups. 9,10 The CuAAC reaction has thus been used in organic synthesis, bio-conjugation chemistry, and surface chemistry. This powerful coupling reaction can be applied in efforts to efficiently tailor the chemical modication of single-walled CNTs (SWNTs) to construct hybrid materials 11–13 including enzymes. 14 Redox-active hemoproteins form a major class of enzymes that are useful for constructing enzyme-immobilized electrodes due to their diverse functions including electron transfer, catal-ysis, and sensing. 15–25 Many hemoproteins possess a replaceable heme b cofactor in the heme pocket, enabling immobilization on the electrode via the heme–heme pocket interaction. 26–45 In this paper, we demonstrate specically oriented covalent immobili-zation of azide-linked cytochrome b 562 (CYT) on the sidewall of SWNT using the CuAAC reaction (Fig. 1). The advantage of this method which uses a replaceable heme tethered to an azide moiety, lies in the wide range of applications for functionaliza-tion of wild-type hemoproteins. The characterization and elec-trochemical properties of the covalently-linked hybrid materials of SWNT and cytochrome b 562 are described. Fig. 1 (a) SWNT with covalently-linked cytochrome b 562 and (b) the preparation scheme using a copper-catalyzed azide–alkyne cyclo-addition (CuAAC) reaction

Therapeutic Effects of a Sodium Glucose Cotransporter 2 Inhibitor in Diabetic Patients with Chronic Kidney Disease

Multiple large-scale clinical trials have indicated that sodium glucose cotransporter 2（SGLT2）inhibitors reduce the incidence of cardiovascular events, deterioration of renal function and mortality. However, the therapeutic effects of SGLT2 inhibitors are supposed to be limited in patients with reduced renal function considering the mechanism of their action. In this study, a SGLT2 inhibitor, ipragliflozin was given to 30 type 2 diabetic patients with nephropathy whose estimated glomerular filtration rate（eGFR）was not lower than 30 mL/min/1.73 m2. After 12 to 16 weeks, hemoglobin A1c decreased by 0.6％（p＜0.001）, body weight was reduced by 1.8 kg（p＜0.01）and blood pressure was lowered by －10/－6 mmHg（p＜0.001/p ＜0.001）. This was accompanied by reductions in serum uric acid（－0.7 mg/dL, p＜0.001）, triglycerides （－25 mg/dL, p＝0.028）and g-glutamyl transferase（－8 U/L, p＝0.001）. On the other hand, plasma B-type natriuretic peptide also decreased by 12％（p＝0.020）and urinary albumin excretion was reduced by 23％ （p＝0.018）although the eGFR was not significantly changed. It is concluded that ipragliflozin is effective in lowering blood glucose even in patients with diabetic kidney disease and is beneficial in improving theaccompanying obesity and hypertension. In addition, ipragliflozin is thought to have favorable influences on the metabolisms of uric acid and lipids. These properties of ipragliflozin is expected to bring about protective effects against the progression of nephropathy and the development of cardiovascular disease resulting in the improvement of prognosis in diabetic patients with mild to moderate chronic kidney disease

Immunotherapy by a Slow Delivery System of Interleukin-2 in Mice Model

A sustained release system for interleukin-2 (IL-2), and IL-2 mini-pellet (IL-2 mp), was developed by fusing IL-2 into a needle shaped collagen. Serum concentration of IL-2 after a single subcutaneous injection of the IL-2 mp into C57BL/6 mice remained elevated longer than after an injection of aqueous IL-2. IL-2 in the serum became undetectable by 6h after a subcutaneous injection of 1 x 10(6) unit of IL-2 in phosphate-buffered saline (PBS). In contrast, after a single subcutaneous injection of IL-2 mp containing the same amount of IL-2, the concentration of IL-2 increased to its maximum at 6h after injection, then began to decrease gradually. IL-2 was detected even on the third day after a single subcutaneous injection of one IL-2 mp. Augmentation of NK activity and generation of IL-2 activated killer cells were observed in the spleen from day 1--day 3 after a single subcutaneous injection of IL-2 mp into C57BL/6 mice. This activation was not observed following a single subcutaneous injection of the same amount of IL-2 in PBS. Adoptive immunotherapy by a single subcutaneous injection of IL-2 mp followed by intravenous injections of in vitro cultured IL-2 activated killer cells showed better results in decreasing the number of metastases of Lewis lung carcinoma in C57BL/6 mice than immunotherapy using IL-2 solution.(ABSTRACT TRUNCATED AT 250 WORDS)</p

Diagnosis of Gastric Cancer in Early Stage — The Clinical Ob­servation of Operated Cases

1. An attempt has been made to find the diagnostic criteria for early gastric cancer. It is most important to detect the evidences or suspected features of the malignant growth in incipient stage in order to attain the radical cure by surgical operation. 2. Twelve patients with early gastric cancer (groups A and B) were selected out of 476 patients who had undergone gastrectomy during the past three years in the Okayama Saiseikai General Hospital. The other 6 patients in the &#34;precancerous group&#34; (group C) were also studied, who had abnormal epithelial proliferation in the resected stomach membrane during the same period. 3. The processes of discovery of early cancer have been described. Fairly precise diagnosis can be made in the mucosal carcinoma, but it is not in the ulcer-carcinoma. It was generally difficult to estimate the degree of the malignancy and the extension of the growth preoperatively. 4. The details of the diagnostic aids are as follows. i. Negative occult blood of stool does not always mean the definite diagnostic aid. ii. The malignant gastric change may occur even in non-anacidity. Further investigations should be followed up on gastric ulcer patients if malignant alteration is under the consideration. iii. Minor roentgenological findings, such as the absence or irregularity of mucosal folds, rigid and/or overlapped contour, localized absence or decrease of the peristaltic waves and absence or bow-shaped deformity of the angulus, are of important significance. Such changes should be minutely sought for by X-ray film examination. iv. On gastroscopy and gastrocamera photography, such changes as erosion or irregular granular thickening of the membrane with abnormal reddening and edematous appearance, irregularity of ulcer edge, uneven swelling on ulcer margin with reddening and unsharpness of the edge of adherent coat on ulcer floor, must be noted in the early gastric cancer. v. It is not safe to leave a patient having stomach ulceration under a mere conservative management because it is often quite difficult to dissolve the question of malignancy of the lesion with all sorts of examinations. vi. So far as clinical examinations have indicated malignancy, histological examination must be carried out immediately at the time of operation, even when malignant lesion is absent in inspection and palpation on the exposure of the stomach. vii. On the gross observation of the resected stomach, a particular attention must be paid to erosion, depression or atrophy, irregular granular thickening and abnormal reddening on the restricted areas of the mucosal surface.</p

Specific inhibition of p38 mitogen-activated protein kinase with FR167653 attenuates vascular proliferation in monocrotaline-induced pulmonary hypertension in rats

AbstractObjectivesp38 mitogen-activated protein kinase is associated with many clinical entities characterized by inflammation. We postulated that inhibition of p38 mitogen-activated protein kinase with FR167653 attenuates inflammation and the development of pulmonary hypertension in monocrotaline-treated rats.MethodsRats were divided into 4 groups: (1) the control group (daily 0.9% saline), (2) the FR group (daily FR167653, 2 mg · kg−1 · d−1), (3) the MCT group (daily 0.9% saline the day after a single monocrotaline dose, 60 mg/kg), and (4) the MCT+FR group (daily FR167653, 2 mg · kg−1 · d−1, the day after a single MCT dose). Body weight, pulmonary artery pressure, and morphometric changes of the pulmonary artery with the histopathologic method were observed weekly for 4 weeks. Also, p38 mitogen-activated protein kinase activity and inflammatory cytokine expression in the lung were measured.ResultsFour weeks after monocrotaline administration, mean pulmonary artery pressure in the MCT+FR group was lower than in the MCT group (MCT+FR vs MCT: 24.7 ± 1.9 vs 36.5 ± 2.1 mm Hg; P < .05). In morphometric analysis the percentage of medial wall thickness and the percentage of muscularization in the MCT+FR group were reduced compared with those in the MCT group after 4 weeks (P < .05); however, the number of macrophages was not significantly different. p38 mitogen-activated protein kinase activity was significantly attenuated in the MCT+FR group compared with in the MCT group (7.2 ± 0.52 vs 2.1 ± 0.23 fold-increase, P < .05, at 1 week). Although mRNA levels of tumor necrosis factor α and interleukin 1β were reduced in the MCT+FR group compared with in the MCT group (tumor necrosis factor α: 1.18 ± 0.36 vs 3.05 ± 1.12 fold-increase, P < .05, at 2 weeks; interleukin 1β: 2.2 ± 0.34 vs 4.4 ± 1.09 fold-increase, P < .05, at 1 week), FR167653 did not suppress increased monocyte chemotactic protein 1 mRNA expression induced by monocrotaline (3.2 ± 0.62 vs 3.1 ± 0.42 fold-increase, at 1 week).ConclusionFR167653 significantly attenuates the expression of inflammatory cytokines, ultimately preventing the progression of pulmonary hypertension. These results suggest that p38 mitogen-activated protein kinase might play a central role in the molecular events that underlie the development and progression of pulmonary hypertension