Quantification of polyprenyl diphosphates in Escherichia coli cells using high-performance liquid chromatography.

Dephosphorylation of undecaprenyl diphosphate is a crucial step in the synthesis of undecaprenyl phosphate, which is essential for cell wall synthesis. We have developed a method for the quantification of intracellular polyprenyl diphosphates, which have never before been measured directly. Polyprenyl phosphates and diphosphates prepared by chemical phosphorylation of polyprenols from Staphylococcus aureus were used to establish the conditions for fractionation by ion-exchange chromatography and high-performance liquid chromatography (HPLC). By using an elution solvent containing tetraethylammonium phosphate as an ion-pair reagent for HPLC, polyprenyl phosphate and polyprenyl diphosphate with carbon numbers from 40 to 55 could be detected as separate peaks from the reversed-phase column. This analytical method was applied to lipids extracted from Escherichia coli to determine the intracellular levels of octaprenyl phosphate, undecaprenyl phosphate, octaprenyl diphosphate, and undecaprenyl diphosphate. This is the first report of separate measurement of cellular levels of polyprenyl phosphates and polyprenyl diphosphates.

Nontuberculous mycobacteriosis oligoarthritis of the right hand misdiagnosed as rheumatoid arthritis: A case report.

Infection with Mycobacterium marinum has several different clinical presentations. Most commonly, it appears as a solitary papulonodular lesion on an extremity. A rare presentation of osteoarticular M. marinum involving multiple small joints and tenosynovitis of the hand, which was misdiagnosed as rheumatoid arthritis, is reported. The patient was initially treated for seronegative rheumatoid arthritis but failed to respond to methotrexate. Magnetic resonance imaging showed arthritis and tenosynovitis. Subsequently, synovial biopsy led to histological and microbiological diagnosis. Antimycobacterial treatment should be started promptly in such cases. The combined use of rifampicin, ethambutol, and clarithromycin appears to be effective, and debridement is indicated in patients with deep-seated infections.

Scope and limitations of absolute configuration determination of allenic natural products using the CCC stretching VCD signal.

The allene functional group in natural products isolated so far exists in a non-racemic form, but its axial chirality is difficult to elucidate. Allenes exhibit a characteristic antisymmetric CCC stretching mode at around 1950 cm-1, and their VCD properties have not been studied in detail. This work, for the first time, applied VCD spectroscopy to allenic natural products and allenic molecules with other asymmetric centers focusing on the antisymmetric CCC stretching mode. This vibrational mode yielded a negligibly weak VCD signal for several molecules, but in the presence of electron-withdrawing and/or conjugating substituents, it generated a stronger one. Its sign was found to be influenced by the nature of substituents. These findings should deepen the understanding of the VCD properties of the allene functional group and should be useful for future studies of chiral allenes.

Sialyltransferase Activity Assay for Ganglioside GM3 Synthase.

GM3 synthase (GM3S) is a sialyltransferase that transfers sialic acid from CMP-sialic acid to lactosylceramide. This reaction results in formation of ganglioside GM3 and is essential for biosynthesis of its downstream derivatives, which include a- and b-series gangliosides. Here, we describe a method for GM3S enzymatic assay using fluorescence-labeled alkyl lactoside as acceptor substrate, followed by HPLC for separation of enzymatic product. The method allows quantitative assay of GM3S sialyltransferase activity in cultured cells and mouse brain tissues.

Neutrophil extracellular traps are involved in enhanced contact hypersensitivity response in IL-36 receptor antagonist-deficient mice.

Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), have been implicated in the pathogenesis of skin disorders. We previously reported that Il36rn-/- mice exhibit an enhanced contact hypersensitivity (CHS) response through increased neutrophil recruitment. In addition, Il36rn-/- mice show severe imiquimod-induced psoriatic skin lesions and enhanced neutrophil extracellular trap (NET) formation. We hypothesized that NETs may play an important role in the CHS response. To confirm this, we examined the CHS response and NET formation in Il36rn-/- mice. Il36rn-/- mice showed enhanced CHS responses, increased infiltration of inflammatory cells, including neutrophils, CD4+ T cells, and CD8+ T cells, NET formation, and enhanced mRNA expression of cytokines and chemokines, including IL-1ß, C-X-C motif chemokine ligand (CXCL)1, CXCL2, and IL-36γ. Furthermore, NET formation blockade improved the CHS response, which consequently decreased inflammatory cell infiltration and NET formation. Consistently, we observed decreased expression of these cytokines and chemokines. These findings indicate that IL-36Ra deficiency aggravates the CHS response caused by excessive inflammatory cell recruitment, NET formation, and cytokine and chemokine production, and that NET formation blockade alleviates the CHS response. Thus, NET formation may play a prominent role in the CHS response.

Work-related factors related to forgetting to take oral diabetes medication during the working day among Japanese male employees with diabetes.

AIM: This study aimed to explore the work-related factors related to forgetting to take oral diabetes medication during the working day among Japanese employees with diabetes. METHODS: This worksite-based study was designed to clarify the important work-related factors for preventing the aggravation of diabetes among working-age people and support for coexisting diabetes treatment and work (Ryoritsu Shien). This cross-sectional survey was conducted in 2018. The participants were full-time employees with type 2 diabetes aged over 40 years who took oral diabetes medication during the working day. The participants were classified into forgetting and non-forgetting groups. Their characteristics and work-related factors were evaluated using a self-administered questionnaire and specific health checkup data. RESULTS: Of the 93 employees with diabetes, 22 (23.7%) were classified into the forgetting group. After adjusting for confounding factors, irregular meal times during the working day was positively associated with forgetting to take oral diabetes medication during the working day [multivariable-adjusted odds ratio (OR), 7.08; 95% confidence interval (CI): 1.38-36.32, irregular vs. regular]. Skipping meals during the working day was positively associated with forgetting to take oral diabetes medication during the working day (multivariable-adjusted OR 3.55, 95% CI 1.14-11.09 presence vs. absence). CONCLUSIONS: Our findings suggest that irregular meal times and skipping meals during the working day are important factors related to forgetting to take oral diabetes medication during the working day among Japanese male employees with diabetes.

Biomimetic Total Syntheses of (+)-Chloropupukeananin, (-)-Chloropupukeanolide D, and Chloropestolides.

Chloropupukeananin, chloropupukeanolides, and chloropestolides are a family of structurally complex bioactive natural products that possess highly functionalized tricyclo[4.3.1.03,7]decane or bicyclo[2.2.2]octane skeletons. Biosynthesis of the chloropupukeananin family is triggered by the intermolecular heterodimeric Diels-Alder reaction between maldoxin and iso-A82775C; however, the enzymes involved have not yet been identified. We herein report the one-pot biomimetic synthesis of chloropupukeananin and chloropupukeanolide D. Moreover, the effect of the solvent on the intermolecular Diels-Alder reaction of siccayne and maldoxin suggested that the biosynthesis of the chloropupukeananin family involves a Diels-Alderase-catalyzed heterodimeric Diels-Alder reaction.

Neutrophil extracellular traps are induced in a psoriasis model of interleukin-36 receptor antagonist-deficient mice.

Loss-of-function mutations in the interleukin (IL)-36 gene IL36RN are associated with psoriasis. The importance of neutrophil extracellular traps (NETs), web-like structures composed of neutrophil DNA, in the pathogenesis of psoriasis has been unclear. Here, we aimed to clarify the role of NET signaling in the deficiency of IL36 receptor antagonist (DITRA). We evaluated the severity of psoriasis-like lesions induced by imiquimod cream treatment in Il36rn-/- mice. The mRNA levels of psoriasis-related cytokines were measured via real-time reverse transcription polymerase chain reaction, and the effects of Cl-amidine, a peptidyl arginine deiminase 4 (PAD4) inhibitor, on psoriasis-like lesions were evaluated. PAD4 is a histone-modifying enzyme that is involved in NET formation. Psoriasis area and severity index scores, epidermal thickness, and infiltrated neutrophil counts were significantly increased in Il36rn-/- mice; NET formation was confirmed pathologically. Several cytokines and chemokines were upregulated in the skin lesions of Il36rn-/- mice and Cl-amidine treatment improved these psoriasis-like lesions. These results suggest that NET formation plays an important role in the pathology of psoriasis-like lesions in these mice and might represent a promising therapeutic target for DITRA.

IL-36 receptor antagonist deficiency resulted in delayed wound healing due to excessive recruitment of immune cells.

Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), have been implicated in the pathogenesis of various skin disorders. Previous findings showed that IL-36γ promoted wound healing in mice; however, the pathogenic role of IL-36Ra in wound healing remains unclear. We elucidated the role of IL-36Ra, a regulator of IL-36 in tissue repair by investigating the recruitment of inflammatory cells and cytokine production in the absence of IL-36Ra. Full-thickness excisional wounds were made on the back of Il36rn-/- mice and healing was assessed by monitoring macroscopic wound sizes, numbers of infiltrated cells, and gene expression of inflammatory cytokines. Macroscopic wound healing, re-epithelialization, and granulation tissue formation were delayed by 3 days post-injury in Il36rn-/- mice. This delay was associated with increased infiltrations of neutrophils and macrophages, and increased expression of cytokines, such as IL-36γ, C-X-C motif chemokine ligand 1 (CXCL1), and transforming growth factor (TGF)-ß. Importantly, administration of TAK-242, a toll-like receptor 4 (TLR4) inhibitor, caused normalization of wound healing in Il36rn-/- mice, abrogating the initial delay in tissue repair. These results showed that targeting TLR4- mediated infiltrations of immune cells and cytokine production could be beneficial in regulating wound healing in IL-36Ra-deficient skin disorders.

Work-related, personal, and diabetes-related factors relevant to dropout from outpatient diabetes treatment visits among Japanese male employees with diabetes.

AIM: In recent years, support for coexisting diabetes treatment and work (Ryoritsu Shien) has become increasingly important for employees with diabetes. It is important to identify factors associated with dropout from outpatient visits for diabetes treatment among employees with diabetes to determine appropriate support to prevent dropout. Therefore, we explored work-related, personal, and diabetes-related factors relevant for dropout from outpatient diabetes treatment visits among Japanese employees with diabetes. METHODS: This cross-sectional study was conducted in 2018. Participants were 140 full-time employees with type 2 diabetes aged over 40 years. Participants were classified into two groups: a dropout group and a continuation group. Work-related, personal, and diabetes-related factors were evaluated using a self-administered questionnaire and specific health checkup data. RESULTS: Of the 140 employees with diabetes, 12 (8.6%) were classified in the dropout group. Multivariable logistic regression analysis revealed three factors associated with a lower risk for dropout from diabetes treatment outpatient visits. These were: high supervisor support, as measured by the Job Content Questionnaire (multivariable-adjusted odds ratio [OR] 0.71, 95% confidence interval [CI] 0.52-0.97, per 1 score); older age (multivariable-adjusted OR 0.79, 95% CI: 0.66-0.95, per 1 year); and presence of metabolic syndrome (multivariable-adjusted OR 0.04, 95% CI 0.01-0.28, presence vs. absence). CONCLUSIONS: Our findings suggest that supervisor support, age, and metabolic syndrome are important factors related to dropout from outpatient diabetes treatment visits among Japanese male employees with diabetes.

Mechanism of eukaryotic serine racemase-catalyzed serine dehydration.

Eukaryotic serine racemase (SR) is a pyridoxal 5'-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-d-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.

Associations among work-related stress and mental fatigue, and regular exercise in Japanese employees with or without diabetes.

AIM: Understanding work-related factors associated with regular exercise in employees could be useful for determining appropriate interventions to prevent the onset and aggravation of diabetes. This study aimed to assess the associations among work-related stress and mental fatigue, and regular exercise in Japanese employees with or without diabetes. METHODS: This cross-sectional study was conducted in 2018, and included 2916 full-time employees aged older than 40 years without cardiovascular disease, cancer, respiratory disease, kidney disease, disc herniation, and depression. Work-related stress was measured by the Job Content Questionnaire and work-related mental fatigue was measured using the Numerical Rating Scale (NRS). RESULTS: Regular exercise was present in 23.9% of 117 employees with diabetes and in 21.3% of 2799 employees without diabetes. In employees with diabetes, the multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for regular exercise were 0.26 (0.07-0.96) in those with an NRS score of 4-6 (moderate) and 0.22 (0.06-0.85) in those with an NRS score of 7-10 (severe) compared with those with an NRS score of 0-3 (none or mild), after adjusting for confounding factors. Similarly, in employees without diabetes, the multivariable-adjusted ORs (95% CIs) for regular exercise were 0.77 (0.62-0.97) in those with an NRS score of 4-6 and 0.75 (0.59-0.94) in those with an NRS score of 7-10 compared with those with an NRS score of 0-3. CONCLUSIONS: Work-related mental fatigue was negatively associated with regular exercise in employees with and without diabetes.

TAK-242 ameliorates contact dermatitis exacerbated by IL-36 receptor antagonist deficiency.

Loss-of-function mutations in IL36RN cause generalized pustular psoriasis (GPP), which is characterized by neutrophil-infiltrated lesions. Neutrophils are important during contact hypersensitivity in mice. However, it has never been determined whether interleukin-36 receptor antagonist (IL-36Ra) deficiency is an exacerbating factor in contact dermatitis. We examined whether a loss-of-function IL36RN mutation exacerbates contact dermatitis and evaluated the changes in contact dermatitis-related cytokines. Wild-type and Il36rn-/- mice were treated with 1-fluoro-2,4-dinitorobenzene (DNFB) and evaluated for ear thickness, histopathological features, numbers of infiltrated neutrophils, and numbers of CD4 + and CD8 + T cells. Furthermore, mRNA levels of contact dermatitis-related cytokines were measured by real-time polymerase chain reaction, and effects of TAK-242, a toll-like receptor 4 (TLR4) inhibitor, on the contact hypersensitivity (CHS) response were evaluated. We found that the ear thickness, cytokine expression, and neutrophil infiltration significantly increased in Il36rn-/- mice compared with that in wild-type mice. TAK-242 alleviated CHS and prevented neutrophil infiltration, cytokine expression, and ear thickening in Il36rn-/- mice. These data indicate that Il36rn-/- mutations are an exacerbating factor for CHS and that TAK-242 can reduce the inflammatory responses that are associated with the CHS response.

Asymmetric Total Synthesis of (-)-Maldoxin, a Common Biosynthetic Ancestor of the Chloropupukeananin Family.

The total synthesis of pestheic acid based on an intramolecular SNAr reaction without a nitro group and the asymmetric synthesis of (-)-maldoxin by a catalytic enantioselective oxidative dearomatization of pestheic acid are described. The reactivity of (-)-maldoxin as a diene in the Diels-Alder reaction is also investigated.

Triple Helicene Cage: Three-Dimensional π-Conjugated Chiral Cage with Six [5]Helicene Units.

A three-dimensional π-conjugated chiral cage with six [5]helicene units (a triple helicene cage) was synthesized for the first time. Taking advantage of the Yamamoto coupling reaction, the triflate-substituted triple [5]helicene, a strained and preorganized precursor, was dimerized to afford the target compound. Single-crystal X-ray diffraction analysis revealed the unique structural features of the triple helicene cage: a cage-shaped rigid structure with outer helical grooves and an inner chiral cavity. All-P and all-M enantiomers were separated successfully by HPLC over a chiral column and their chiroptical properties were characterized by circular dichroism spectra.