Structure of l-rhamnose isomerase in complex with l-rhamnopyranose demonstrates the sugar-ring opening mechanism and the role of a substrate sub-binding site

Abstractl-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. Previously determined X-ray structures of l-RhI showed a hydride-shift mechanism for the isomerization of substrates in a linear form, but the mechanism for opening of the sugar-ring is still unclear. To elucidate this mechanism, we determined X-ray structures of a mutant l-RhI in complex with l-rhamnopyranose and d-allopyranose. Results suggest that a catalytic water molecule, which acts as an acid/base catalyst in the isomerization reaction, is likely to be involved in pyranose-ring opening, and that a newly found substrate sub-binding site in the vicinity of the catalytic site may recognize different anomers of substrates

Prenatal and Peripartum Management of Patients with Hypofibrinogenemia Resulted in Two Successful Deliveries

Fibrinogen is an essential agent involved in maintaining pregnancy and coagulation. Since inherited fibrinogen disorders introduce greater risks for conditions such as placental abruption and postpartum hemorrhage, careful prenatal and perinatal management is essential for this patient population. We report two cases of successful deliveries in patients with hypofibrinogenemia. Case 1 is of a 26-year-old (gravida 1, para 1) woman. The patient’s fibrinogen level increased spontaneously to higher than 300 mg/dL during pregnancy, without treatment. She delivered at week 38 of gestation, with no complications. Case 2 is of a 30-year-old (gravida 3, para 1) woman. We performed repeated infusions of fibrinogen to maintain the level higher than 100 mg/dL during pregnancy and at least 200 mg/dL in the perioperative period; the patient delivered a healthy infant. We identified a new mutation, Hiroshima I (γ278Tyr→His). It is important to maintain appropriate fibrinogen levels in cases of inherited fibrinogen disorders for successful prenatal and peripartum management

Xenoantigen, an αGal epitope-expression construct driven by the hTERT-promoter, specifically kills human pancreatic cancer cell line

BACKGROUND: We previously reported the usefulness of the αGal epitope as a target molecule for gene therapy against cancer. To induce cancer cell specific transcription of the αGal epitope, an expression vector which synthesizes the αGal epitope under the control of a promoter region of the human telomerase reverse transcriptase (hTERT), NK7, was constructed. METHODS: NK7 was transfected into a human pancreatic carcinoma cell line, MIA cells, and telomerase-negative SUSM-1 cells served controls. Expression of the αGal epitope was confirmed by flow cytometry using IB4 lectin. The susceptibility of transfected MIA cells to human natural antibodies, was examined using a complement-dependent cytotoxic cross-match test (CDC) and a flow cytometry using annexin V. RESULTS: The αGal epitope expression was detected only on the cell surfaces of NK7-transfected MIA cells, i.e., not on naive MIA cells or telomerase negative SUSM-1 cells. The CDC results indicated that MIA cells transfected with NK7 are susceptible to human natural antibody-mediated cell killing, and the differences, as compared to NK-7 transfected telomerase negative SUSM-1 cells or telomerase positive naïve MIA cells, were statistically significant. The flow cytometry using annexin V showed a higher number of the apoptotic cells in NK-7 transfected MIA cells than in naïve MIA cells. CONCLUSIONS: The results suggest that αGal epitope-expression, under the control of the hTERT-promoter, may be useful in cancer specific gene therapy

Cytochromec−Crown Ether Complexes as Supramolecular Catalysts: Cold-Active Synzymes for Asymmetric Sulfoxide Oxidation in Methanol

A series of supramolecular complexes of various cytochrome c proteins with 18-crown-6 derivatives behave as cold-active synzymes in the H_2O_2 oxidation of racemic sulfoxides. This interesting behavior contrasts with native functionality, where the employed proteins act as electron transfer carriers. ESI-MS, UV, CD, and Raman spectroscopic characterizations reveal that four or five 18-crown-6 molecules strongly bind to the surface of the cytochrome c and also that nonnatural low-spin hexacoordinate heme structures are induced in methanol. Significantly, crown ether complexation can convert catalytically inactive biological forms to catalytically active artificial forms. Horse heart, pigeon breast, and yeast cytochromes c all stereoselectively oxidize (S)-isomers of methyl tolyl sulfoxide and related sulfoxides upon crown ether complexation. These supramolecular catalysts show the highest efficiency and enantiomer selectivity at −40 °C in the H_2O_2-dependent sulfoxide oxidation, while oxidative decomposition of the heme moieties predominantly occurs at room temperature. The oxidation reactivity of the employed sulfoxides is apparently related to steric constraints and electrochemical oxidation potentials of their S O bonds. Among the cytochrome c complexes, yeast cytochrome c demonstrates the lowest catalytic activity and degradation reactivity. It has a significantly different protein sequence, suggesting that crown ether complexation effectively activates heme coordination but may additionally alter the native backbone structure. The proper combination of cytochrome c proteins, 18-crown-6 receptors, and external circumstances can be used to successfully generate “protein-based supramolecular catalysts” exhibiting nonbiological reactivities

Japanese Lung Cancer Society Guidelines for Stage IV NSCLC With EGFR Mutations

Patients with NSCLC in East Asia, including Japan, frequently contain EGFR mutations. In 2018, we published the latest full clinical practice guidelines on the basis of those provided by the Japanese Lung Cancer Society Guidelines Committee. The purpose of this study was to update those recommendations, especially for the treatment of metastatic or recurrent EGFR-mutated NSCLC. We conducted a literature search of systematic reviews of randomized controlled and nonrandomized trials published between 2018 and 2019 that multiple physicians had reviewed independently. On the basis of those studies and the advice from the Japanese Society of Lung Cancer Expert Panel, we developed updated guidelines according to the Grading of Recommendations, Assessment, Development, and Evaluation system. We also evaluated the benefits of overall and progression-free survival, end points, toxicities, and patients’ reported outcomes. For patients with NSCLC harboring EGFR-activating mutations, the use of EGFR tyrosine kinase inhibitors (EGFR TKIs), especially osimertinib, had the best recommendation as to first-line treatment. We also recommended the combination of EGFR TKI with other agents (platinum-based chemotherapy or antiangiogenic agents); however, it can lead to toxicity. In the presence of EGFR uncommon mutations, except for an exon 20 insertion, we also recommended the EGFR TKI treatment. However, we could not provide recommendations for the treatment of EGFR mutations with immune checkpoint inhibitors, including monotherapy, and its combination with cytotoxic chemotherapy, because of the limited evidence present in the literature. The 2020 Japanese Lung Cancer Society Guidelines can help community-based physicians to determine the most appropriate treatments and adequately provide medical care to their patients

Cricotracheostomy for patients with severe COVID-19: A case control study

BackgroundTracheostomy is an important procedure for the treatment of severe coronavirus disease-2019 (COVID-19). Older age and obesity have been reported to be associated with the risk of severe COVID-19 and prolonged intubation, and anticoagulants are often administered in patients with severe COVID-19; these factors are also related to a higher risk of tracheostomy. Cricotracheostomy, a modified procedure for opening the airway through intentional partial cricoid cartilage resection, was recently reported to be useful in cases with low-lying larynx, obesity, stiff neck, and bleeding tendency. Here, we investigated the usefulness and safety of cricotracheostomy for severe COVID-19 patients.Materials and methodsFifteen patients with severe COVID-19 who underwent cricotracheostomy between January 2021 and April 2022 with a follow-up period of ≥ 14 days were included in this study. Forty patients with respiratory failure not related to COVID-19 who underwent traditional tracheostomy between January 2015 and April 2022 comprised the control group. Data were collected from medical records and comprised age, sex, body mass index, interval from intubation to tracheostomy, use of anticoagulants, complications of tracheostomy, and decannulation.ResultsAge, sex, and days from intubation to tracheostomy were not significantly different between the COVID-19/cricotracheostomy and control/traditional tracheostomy groups. Body mass index was significantly higher in the COVID-19 group than that in the control group (P = 0.02). The rate of use of anticoagulants was significantly higher in the COVID-19 group compared with the control group (P < 0.01). Peri-operative bleeding, subcutaneous emphysema, and stomal infection rates were not different between the groups, while stomal granulation was significantly less in the COVID-19 group (P = 0.04).ConclusionsThese results suggest that cricotracheostomy is a safe procedure in patients with severe COVID-19

慢性期移植腎機能低下例におけるIV型コラーゲンの分布の変化に関する検討

IV型コラーゲンは基底膜の主要構成成分であり,生体内に広く分布している.移植後急性期腎機能低下例においては,腎臓のIV型コラーゲン分布に変化が認められる.今回我々は移植腎機能廃絶の主原因である慢性期移植腎機能低下症(chronic allograft nephropathy: CAN)におけるIV型コラーゲンの変化について検討した.当院において施行された腎移植患者を,移植腎機能に基づいて以下の群に分類し,組織所見について検討した.なおCAN症例は,光学顕微鏡所見で診断された症例を用いた.Group A(n=5):腎機能正常群,Group B(n=10):急性期移植腎機能低下群,Group C(n=35): CAN症例で血清クレアチニン値2～4mg/dl,Group D(n=15): CANで血清クレアチニン値>4mg/dl.各症例におけるIV型コラーゲン分布について,IV型コラーゲンのα1鎖に対するモノクローナル抗体であるJK199,JK132を用いて,蛍光顕微鏡により検討した.Group AではJK199,JK132ともにメサンギウム基質(MM),ボーマン嚢基底膜(BBM),尿細管基底膜(TBM)に対する反応のみを認めた.Group Bでは,JK199はMM,BBM,TBMに加え,糸球体基底膜(GBM)および腎間質(INS)に対する反応を認めた.JK132の反応性はGroup Aと同様であった.一方,Group CおよびGroup Dでは,JK199においてはMM,GBM,BBM,TBM,INSに対する反応性が増強していた.JK132についてはMM,BBM,TBMに加え,GBMおよびINSにおいても反応が認められた.また,JK199およびJK132の反応性はGroup CよりもGroup Dにおいて強い傾向が認められた.GBMおよびINSにおけるJK132の反応陽性化はCANに特異的な所見であることが示唆された.Previously, we demonstrated the altered formation of collagen IV, which is the main constituent of the basement membrane, in renal allografts by staining with two monoclonal antibodies against the α1 chain of collagen IV. In the present study, we investigated the alteration of collagen IV in chronic allograft nephropathy (CAN), which is an irreversible change that can occur in renal allografts. Biopsy specimens of normal kidneys (Group A: n=5) and acute rejection (Group B: n=10) were studied as controls. Fifty biopsy specimens from 41 patients who had been diagnosed as having CAN were divided into two groups, according to renal function: Group C (n=35), sCr 2～4 mg/dl, and Group D (n=15), sCr>4 mg/dl. Two monoclonal antibodies, JK199 and JK132, those recognize the α1 chain of collagen IV were used. In Group A, JK199 reacted with the glomerular basement membrane (GBM), the mesangial matrix (MM), the basement membrane of Bowman\u27s capsule (BBM) and the tubular basement membrane (TBM). JK132 only reacted with the MM, BBM and TBM. In Group B, JK199 reacted with GBM, MM, BBM, TBM and the interstitium (INS). JK132 only reacted with MM, BBM and TBM. In Group C and Group D, JK199 and JK132 reacted universally with GBM and INS in addition to MM, BBM and TBM. The intensity of the reaction was higher in Group D than in Group C. Thus, the reactivity of JK132 with GBM and INS was a unique finding for the CAN specimens. These results suggest that collagen IV is upregulated in CAN and the reactivity of JK132 in GBM and INS may represent the point of irreversible dysfunction of renal allografts

慢性期移植腎機能低下例におけるIV型コラーゲンの分布の変化に関する検討

IV型コラーゲンは基底膜の主要構成成分であり,生体内に広く分布している.移植後急性期腎機能低下例においては,腎臓のIV型コラーゲン分布に変化が認められる.今回我々は移植腎機能廃絶の主原因である慢性期移植腎機能低下症(chronic allograft nephropathy: CAN)におけるIV型コラーゲンの変化について検討した.当院において施行された腎移植患者を,移植腎機能に基づいて以下の群に分類し,組織所見について検討した.なおCAN症例は,光学顕微鏡所見で診断された症例を用いた.Group A(n=5):腎機能正常群,Group B(n=10):急性期移植腎機能低下群,Group C(n=35): CAN症例で血清クレアチニン値2～4mg/dl,Group D(n=15): CANで血清クレアチニン値>4mg/dl.各症例におけるIV型コラーゲン分布について,IV型コラーゲンのα1鎖に対するモノクローナル抗体であるJK199,JK132を用いて,蛍光顕微鏡により検討した.Group AではJK199,JK132ともにメサンギウム基質(MM),ボーマン嚢基底膜(BBM),尿細管基底膜(TBM)に対する反応のみを認めた.Group Bでは,JK199はMM,BBM,TBMに加え,糸球体基底膜(GBM)および腎間質(INS)に対する反応を認めた.JK132の反応性はGroup Aと同様であった.一方,Group CおよびGroup Dでは,JK199においてはMM,GBM,BBM,TBM,INSに対する反応性が増強していた.JK132についてはMM,BBM,TBMに加え,GBMおよびINSにおいても反応が認められた.また,JK199およびJK132の反応性はGroup CよりもGroup Dにおいて強い傾向が認められた.GBMおよびINSにおけるJK132の反応陽性化はCANに特異的な所見であることが示唆された.Previously, we demonstrated the altered formation of collagen IV, which is the main constituent of the basement membrane, in renal allografts by staining with two monoclonal antibodies against the α1 chain of collagen IV. In the present study, we investigated the alteration of collagen IV in chronic allograft nephropathy (CAN), which is an irreversible change that can occur in renal allografts. Biopsy specimens of normal kidneys (Group A: n=5) and acute rejection (Group B: n=10) were studied as controls. Fifty biopsy specimens from 41 patients who had been diagnosed as having CAN were divided into two groups, according to renal function: Group C (n=35), sCr 2～4 mg/dl, and Group D (n=15), sCr>4 mg/dl. Two monoclonal antibodies, JK199 and JK132, those recognize the α1 chain of collagen IV were used. In Group A, JK199 reacted with the glomerular basement membrane (GBM), the mesangial matrix (MM), the basement membrane of Bowman's capsule (BBM) and the tubular basement membrane (TBM). JK132 only reacted with the MM, BBM and TBM. In Group B, JK199 reacted with GBM, MM, BBM, TBM and the interstitium (INS). JK132 only reacted with MM, BBM and TBM. In Group C and Group D, JK199 and JK132 reacted universally with GBM and INS in addition to MM, BBM and TBM. The intensity of the reaction was higher in Group D than in Group C. Thus, the reactivity of JK132 with GBM and INS was a unique finding for the CAN specimens. These results suggest that collagen IV is upregulated in CAN and the reactivity of JK132 in GBM and INS may represent the point of irreversible dysfunction of renal allografts