Bipolar Clark-Type Oxygen Electrode Arrays for Imaging and Multiplexed Measurements of the Respiratory Activity of Cells.

Various miniature Clark-type oxygen electrodes (COEs), which are typically used to measure dissolved oxygen (DO) concentration in cellular respiration, have been developed since the 1980s. Arrays with individually addressable electrodes that constitute the sensor were used for various applications. However, the large number of leads and contact pads required for connecting the electrodes and the external instrument complicate the electrode layout and make the operation of integrated COE arrays challenging. Here, we fabricated closed bipolar electrochemical systems comprising 6 × 8 and 4 × 4 arrays of COEs for imaging and multiplexed detection. The cathodic compartment was sealed with a hydrophobic oxygen-permeable membrane to separate the internal electrolyte solution from the sample solutions. Using the bipolar Clark-type oxygen electrode (BCOE) arrays and electrochemiluminescence (ECL), we measured the DO concentration at each cathode. The results revealed that the ECL intensity changed linearly with the DO concentration. In addition, we used ECL imaging to investigate the respiratory activity of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in suspensions with different cell densities. The ECL images showed that the ECL intensity changed noticeably with the bacterial density. The bacterial respiratory activity was then qualitatively analyzed based on the ECL images acquired successively over a time duration. Further, we measured the antibiotic efficacy of piperacillin, oxacillin, gentamicin, and cefmetazole against E. coli and P. aeruginosa using the BCOE. We found that the ECL intensity increased with the antibiotic concentration, thus indicating the suppression of the bacterial respiratory activity.

l-Carnitine pretreatment ameliorates heat stress-induced acute kidney injury by restoring mitochondrial function of tubular cells.

A major complication of heat-related illness is the development of acute kidney injury (AKI) and damage to kidney tubular cells. Because kidney tubular cells use fatty acids as a major energy source, impaired fatty acid oxidation (FAO) may be associated with kidney injury due to heat stress. Carnitine is essential in the transportation of fatty acid into mitochondria for FAO. To date, there has been little attention given to the role of carnitine in heat-related illness and AKI. To evaluate the relationship between carnitine inadequacy and heat-related illness severity or AKI, we examined serum carnitine levels in patients with heat-related illness. We also used heat-stressed mice to investigate the effect of l-carnitine pretreatment on various kidney functions such as mitochondrial activity, proinflammatory changes in kidney macrophages, and histological damage. We observed an elevation in serum acylcarnitine levels, indicating carnitine insufficiency in patients with severe heat-related illness and/or AKI. l-Carnitine pretreatment ameliorated ATP production in murine tubular cell mitochondria and prevented a change in the kidney macrophage population dynamics observed in AKI: a decrease in tissue-resident macrophages, influx of bone marrow-derived macrophages, and change toward proinflammatory M1 polarization. In conclusion, carnitine insufficiency may be closely associated with severe heat-related illness and related AKI. Enhancement of the FAO pathway by l-carnitine pretreatment may prevent heat stress-induced AKI by restoring mitochondrial function.NEW & NOTEWORTHY Enhancing fatty acid oxidation (FAO) after acute kidney injury (AKI) improves renal outcomes. This report shows that carnitine insufficiency, which could inhibit FAO, correlates to severe heat-related illness and AKI in a clinical study. We also demonstrate that administering l-carnitine to mice improves mitochondrial respiratory function and prevents deleterious changes in renal macrophage, resulting in improved renal outcomes of heat-induced AKI. l-Carnitine may be an effective preventive treatment for severe heat-related illness and related AKI.

Novel phenotypical and functional sub-classification of liver macrophages highlights changes in population dynamics in experimental mouse models.

Liver macrophages are critical components of systemic immune system defense mechanisms. F4/80high Kupffer cells (KCs) are the predominant liver-resident macrophages and the first immune cells to contact pathogens entering the liver. F4/80low monocyte-derived macrophages (MoMφs) are essential macrophages that modulate liver immune functions. Here we report a novel method of identifying subpopulations of these two populations using traditional flow cytometry and examine each subpopulation for its putative roles in the pathogenesis of an experimental non-alcoholic steatohepatitis model. Using male C57BL/6 mice, we isolated and analyzed liver non-parenchymal cells by flow cytometry. We identified F4/80high and F4/80low macrophage populations and characterized subpopulations using uniform manifold approximation and projection. We identified three subpopulations in F4/80high macrophages: CD163(+) KCs, CD163(-) KCs, and liver capsular macrophages. CD163(+) KCs had higher phagocytic and bactericidal activities and more complex cellular structures than CD163(-) KCs. We also identified four subpopulations of F4/80low MoMφs based on Ly6C and MHC class II expression: infiltrating monocytes, pro-inflammatory MoMφs, Ly6C(-) monocytes, and conventional dendritic cells. CCR2 knock-out mice expressed lower levels of these monocyte-derived cells, and the count varied by subpopulation. In high-fat- and cholesterol-diet-fed mice, only one subpopulation, pro-inflammatory MoMφs, significantly increased in count. This indicates that changes to this subpopulation is the first step in the progression to non-alcoholic steatohepatitis. The community can use our novel subpopulation and gating strategy to better understand complex immunological mechanisms in various liver disorders through detailed analysis of these subpopulations.

Assessing avian leukosis virus proviral load and lesion correlates in fowl glioma-inducing virus-infected Japanese bantam chickens.

The fowl glioma-inducing virus prototype (FGVp) and its variants, which belong to avian leukosis virus subgroup A (ALV-A), induce cardiomyocyte abnormalities and gliomas in chickens. However, the molecular mechanisms underlying these myocardial changes remain unclear, and ALV-induced tumorigenesis, which is caused by proviral insertional mutagenesis, does not explain the early development of cardiac changes in infected chickens. We established a quantitative PCR (qPCR) assay to measure ALV-A proviral loads in the brains and hearts of FGV-infected Japanese bantam chickens and compared these results with morphologic lesions. Four of 22 bantams had both gliomas and cardiac lesions. Hearts with cardiac lesions had a higher proviral load (10.3 ± 2.7 proviral copies/nucleus) than those without cardiac lesions (0.4 ± 0.4), suggesting that the proviral load in hearts is correlated with the frequency of myocardial changes. Our qPCR method may be useful in the study of ALV-induced cardiomyocyte abnormalities.

Postpartum recovery of nulliparous women following scheduled cesarean delivery and spontaneous vaginal delivery: a prospective observational study.

BACKGROUND: Inpatient postpartum recovery trajectories following cesarean delivery and spontaneous vaginal delivery are underexplored. OBJECTIVE: This study primarily aimed to compare recovery following cesarean delivery and spontaneous vaginal delivery in the first postpartum week, and secondarily to evaluate psychometrically the Japanese version of the Obstetric Quality of Recovery-10 scoring tool. STUDY DESIGN: Following institutional review board approval, the EQ-5D-3L (EuroQoL 5-Dimension 3-Level) questionnaire and a Japanese version of the Obstetric Quality of Recovery-10 measure were used to evaluate inpatient postpartum recovery in uncomplicated nulliparous parturients delivering via scheduled cesarean delivery or spontaneous vaginal delivery. RESULTS: A total of 48 and 50 women who delivered via cesarean delivery and spontaneous vaginal delivery, respectively, were recruited. Women delivering via scheduled cesarean delivery experienced significantly worse quality of recovery on days 1 and 2 compared with those who had spontaneous vaginal delivery. Quality of recovery significantly improved daily, plateauing at days 4 and 3 for cesarean delivery and spontaneous vaginal delivery groups, respectively. Compared with cesarean delivery, spontaneous vaginal delivery was associated with prolonged time to analgesia requirement, decreased opioid consumption, reduced antiemetic requirement, and reduced times to liquid/solid intake, ambulation, and discharge. Obstetric Quality of Recovery-10-Japanese is a valid (correlates with the EQ-5D-3L including a global health visual analog scale, gestational age, blood loss, opioid consumption, time until first analgesic request, liquid/solid intake, ambulation, catheter removal, and discharge), reliable (Cronbach alpha=0.88; Spearman-Brown reliability estimate=0.94; and intraclass correlation coefficient=0.89), and clinically feasible (98% 24-hour response rate) measure. CONCLUSION: Inpatient postpartum recovery is significantly better in the first 2 postpartum days following spontaneous vaginal delivery compared with scheduled cesarean delivery. Inpatient recovery is largely achieved within 4 and 3 days following scheduled cesarean delivery and spontaneous vaginal delivery, respectively. Obstetric Quality of Recovery-10-Japanese is a valid, reliable, and feasible measure of inpatient postpartum recovery.

Generation of pulsatile ERK activity in mouse embryonic stem cells is regulated by Raf activity.

The extracellular signal-regulated kinase (ERK) is a serine/threonine kinase that is known to regulate cellular events such as cell proliferation and differentiation. The ERK signaling pathway is activated by fibroblast growth factors, and is considered to be indispensable for the differentiation of primitive endoderm cells, not only in mouse preimplantation embryos, but also in embryonic stem cell (ESC) culture. To monitor ERK activity in living undifferentiated and differentiating ESCs, we established EKAREV-NLS-EB5 ESC lines that stably express EKAREV-NLS, a biosensor based on the principle of fluorescence resonance energy transfer. Using EKAREV-NLS-EB5, we found that ERK activity exhibited pulsatile dynamics. ESCs were classified into two groups: active cells showing high-frequency ERK pulses, and inactive cells demonstrating no detectable ERK pulses during live imaging. Pharmacological inhibition of major components in the ERK signaling pathway revealed that Raf plays an important role in determining the pattern of ERK pulses.

Avian retroviral cardiomyopathy induced by infectious molecular clones of avian leukosis viruses (fowl glioma-inducing virus variants).

We previously described cardiomyocyte abnormality caused by Km_5666 strain, a variant of fowl glioma-inducing virus (FGV) prototype, which is an avian leukosis virus (ALV). However, the cardiac involvement appeared to be eradicated from the flock after a few years. An epidemiological survey from 2017 to 2020 was performed to elucidate the current prevalence of the cardiopathogenic strains in this flock. Four of the 71 bantams pathologically examined showed both glioma and cardiomyocyte abnormality, from which three ALV strains were detected. DNA sequencing revealed that several different ALV strains coexisted in each bantam and that the conserved Km_5666 virus fluid also contained at least two different ALV strains. We generated three infectious molecular clones from these samples, named KmN_77_clone_A, KmN_77_clone_B, and Km_5666_clone. The envSU of KmN_77_clone_A shared high sequence identity with that of Km_5666 (94.1%). In contrast, the envSU of KmN_77_clone_B showed >99.2% nucleotide similarity with that of an FGV variant without cardiopathogenicity. Furthermore, Km_5666_clone experimentally reproduced both gliomas and cardiomyocyte abnormality in chickens. From these results, it is suggested that the pathogenic determinant of cardiomyocyte abnormality is located in envSU similar to that of Km_5666. The cloning technique described here is beneficial for evaluating the viral pathogenicity in cases where affected birds are coinfected with several different ALV strains.

Mouse Liver B Cells Phagocytose Streptococcus pneumoniae and Initiate Immune Responses against Their Antigens.

Recent studies have revealed that mammalian B cells ingest particulate Ags, such as bacteria, although little is known about the effect of this function on acquired immunity. We investigated the role of bacterium-phagocytosing B cells in acquired host immune responses. Cultured mouse liver B cells substantially phagocytosed serum-opsonized Streptococcus pneumoniae and produced IgM. On adoptive transfer of liver B cells that phagocytose S. pneumoniae labeled with pHrodo Red succinimidyl ester, recipient mice showed elevated plasma levels of IgG specific for bacterial Ags. In particular, the levels of IgG2a and IgG2b specific for pneumococcal surface protein A, as well as IgG3 for pneumococcal polysaccharide, were markedly increased compared with total IgG specific for each Ag. When phagocytic liver B cells were cultured with spleen CD4+ T cells obtained from mice primed with heat-killed S. pneumoniae 7 d before, they induced IL-2 production and proliferation of the CD4+ T cells, along with Th1 cytokine production. However, they induced neither the CD4+ T cell production of IL-21, a suggested marker promoting B cell proliferation and differentiation, nor the expression of genes important for somatic hypermutation or isotype switching; such responses were particularly evident when splenic B cells merely capturing S. pneumoniae without processing them were cultured with spleen CD4+ T cells. These findings suggest that phagocytic liver B cells may be involved in acquired immune responses by presenting derivative peptides to CD4+ T cells without their own somatic hypermutation or isotype switching.

Repetitive short-pulsed illumination efficiently activates photoactivatable-Cre as continuous illumination in embryonic stem cells and pre-implantation embryos of transgenic mouse.

The Cre-loxP system has been widely used for specific DNA recombination which induces gene inactivation or expression. Recently, photoactivatable-Cre (PA-Cre) proteins have been developed as a tool for spatiotemporal control of the enzymatic activity of Cre recombinase. Here, we generated transgenic mice bearing a PA-Cre gene and systematically investigated the conditions of photoactivation for the PA-Cre in embryonic stem cells (ESCs) derived from the transgenic mice and in a simple mathematical model. Cre-mediated DNA recombination was induced in 16% of the PA-Cre ESCs by 6 hr continuous illumination. We show that repetitive pulsed illumination efficiently induced DNA recombination with low light energy as efficient as continuous illumination in the ESCs (96 ± 15% of continuous illumination when pulse cycle was 2 s), which was also supported by a minimal mathematical model. DNA recombination by the PA-Cre was also successfully induced in the transgenic mouse pre-implantation embryos under the developed conditions. These results suggest that strategies based on repetitive pulsed illumination are efficient for the activation of photoactivatable Cre and, possibly other photo-switchable proteins.

Effect of L-Leucine Therapy on Hematopoietic Function in Elderly Myelodysplastic Syndrome Patients.

Patients with myelodysplastic syndrome (MDS) often require blood transfusion and anticancer therapy; however, elderly patients are intolerant to the associated side effects of anticancer therapy. Because L-leucine can be used to treat Diamond-Blackfan anemia, which is caused by defects in ribosomal protein (RP) genes, resulting in increased in vivo hemoglobin synthesis, it is possible that some MDS patients who have aberrations in their RP genes could also be effectively treated with L-leucine. In the present study, we investigated the effects of L-leucine on hematopoietic function (reticulocyte count), red blood cell count, and hemoglobin level in MDS patients. We administered L-leucine (1.8 g, twice daily, 3 d/week) with oral vitamin B6 supplements to a final cohort of eight MDS patients for 15 (interquartile range: 11-18) weeks. We assessed the patients at 10 ± 2 weeks after therapy initiation. Only the absolute reticulocyte count was affected, improving in 6/8 (75%) patients. The median absolute reticulocyte count was 3.5 × 104 (range: 2.7-6.4 × 104) cells/µL, an increase of 0.5 × 104 (range: 0.2-0.7 × 104) cells/µL. At 10 weeks, there was only one case of an improved hemoglobin level. Non-hematological adverse events of grade 3 were observed one raised triglycerides. These data suggest that L-leucine has little effect on MDS. However, it may contribute to the recovery of hematopoietic function, futher study be desired.

Clinical and Laboratory Markers Associated With Relapse in Cutaneous Polyarteritis Nodosa.

Importance: In cutaneous polyarteritis nodosa (CPAN), less aggressive treatments can be selected, because CPAN is not associated with life-threatening or progressive outcomes. Although patients with a recurrent clinical course may require additional immunosuppressive therapies, no pretreatment factors associated with a worse prognosis in CPAN have been reported. Objective: To identify clinical or laboratory markers associated with relapse of CPAN. Design, Setting, and Participants: This retrospective case series was performed at a dermatology clinic of a tertiary referral center in Okayama, Japan, from October 1, 2001, through April 30, 2017. Of 30 patients identified with CPAN, the 21 with histopathologic evidence of disease were eligible and enrolled in the study. Main Outcomes and Measures: The medical database was examined for sex, age at diagnosis, affected anatomical sites, type and extent of skin lesion, laboratory data, initial therapies, duration of follow-up, and current status. Relapse was defined as the first reoccurrence or new onset of cutaneous disease that required further escalation of treatment with prednisolone at a dosage of greater than 20 mg/d and/or add-on use of immunosuppressant therapy, more than 6 months after initial treatment. The pretreatment factors were statistically evaluated between the groups without and with relapse. Results: The 21 patients included 5 males and 16 females with a median age of 49 years (range, 11-74 years) at diagnosis. The median follow-up was 42 months (range, 8-374 months). Pretreatment cutaneous ulcer was significantly associated with relapse between the 2 groups (0 of 11 in the nonrelapse group vs 4 of 10 in the relapse group; χ21 = 4.67; P < .05). In the laboratory test results, significantly higher mean (SD) values were observed in the relapse group for C-reactive protein level (0.23 [2.00] vs 6.03 [3.10] mg/dL; standard error of the mean [SEM], 3.40 mg/dL; 95% CI, 0.01-10.8 mg/dL; P = .01), absolute neutrophil count (ANC) (3.4 × 103/µL [1.1 × 103/µL] vs 6.0 × 103/µL [3.2 × 103/µL]; SEM, 2.9 × 103/µL; 95% CI, 1.9 × 103/µL to 14.6 × 103/µL; P = .001), neutrophil-to-lymphocyte ratio (1.4 [0.8] vs 2.8 [0.9]; SEM, 1.2; 95% CI, 1.1-4.9; P = .002), and systemic immune-inflammation index (5.1 × 105 [3.9 × 105] vs 11.7 × 105 [7.7 × 105]; SEM, 7.3 × 105; 95% CI, 3.3 × 105 to 31.1 × 105; P = .007). The estimated 2-year cumulative relapse rate was significantly high in the patients with blood ANC of greater than 4.9 × 103/µL compared with 4.9 × 103/µL or less (9 of 10 [90%] vs 2 of 11 [18%]; 95% CI, 6%-72%). Conclusions and Relevance: Pretreatment status of cutaneous ulcer, the serum C-reactive protein level, the blood ANC, the neutrophil-to-lymphocyte ratio, and the systemic immune-inflammation index are associated with a worse prognosis in CPAN.

Assessment of the Latent Adverse Events of Antipsychotic Treatment Using a Subjective Questionnaire in Japanese Patients with Schizophrenia.

OBJECTIVE: The adverse effects of antipsychotic agents can have a marked influence on medication adherence. In this study, we.investigated the adverse events of antipsychotics that are less likely to be reported by patients and the reasons why such symptoms remain latent. METHODS: Data were collected by interviewing patients using a subjective questionnaire, and the associations between unreported symptoms and background factors were investigated. RESULTS: A total of 306 patients with schizophrenia or schizoaffective disorder were examined. Their major symptoms were daytime sleepiness (50.0%), weight gain (42.2%), and sexual dysfunction (38.9%). Sexual dysfunction was nominal significantly more common among the patients that had been treated with antipsychotic agent polypharmacy (odds ratio [OR], 2.14; 95% confidence interval [CI], 1.07 to 4.30), and was nominal significantly more common among outpatients (OR, 1.78; 95% CI, 1.02 to 3.13). Only approximately 30% of the patients had reported their symptoms to their physicians. CONCLUSION: Patients receiving antipsychotic treatment tolerate some symptoms and do not feel able to report them to their physicians. The most common reason for this is an insufficient patient-physician relationship. Sexual dysfunction is especially hard to identify because it is a delicate problem, and our findings demonstrate that subjective questionnaires are helpful for detecting such symptoms.

A Stable, Soluble, and Crystalline Supramolecular System with a Triplet Ground State.

A supramolecular complex was constructed by encapsulation of a 3 O2 molecule inside an open-cage C60 derivative. Its single-crystal X-ray diffraction analysis revealed the presence of the 3 O2 at the center of the fullerene cage. The CV measurements suggested that unprecedented dehydrogenation was promoted by the encapsulated 3 O2 after two-electron reduction. The ESR measurements displayed the triplet character as well as the anisotropy of the 3 O2 . Additionally, the SQUID measurements also demonstrated the paramagnetic behavior above 3 K without an antiferromagnetic transition. Upon photoirradiation with visible light, three phosphorescent bands at the NIR region were observed, arising from the exited 1 O2 generated by self-sensitization with the outer cage, whose lifetimes were not affected by the environments. These studies confirmed that the complex is a crystalline triplet system with incompatible "high spin density" but "small interspin interaction" properties.

[Comparison of the efficacy of ropivacaine and that of levobupivacaine for postoperative epidural analgesia in patients undergoing gynecological open abdominal surgery].

BACKGROUND: Postoperative analgesia is important in patients undergoing open abdominal surgery. We prospectively compared the efficacy of ropivacaine and that of levobupivacaine for postoperative epidural analgesia in patients undergoing gynecological open abdominal surgery. METHODS: Fifty-two ASA 1-2 patients scheduled for gynecological open abdominal surgery under combined general and epidural anesthesia were enrolled and randomized into two groups. In ropivacaine group (n = 26) the patients received 0.187% ropivacaine and fentanyl 3.2 microg x ml(-1) for postoperative epidural analgesia. In levobupivacaine group (n = 26) the patients received 0.187% levobupivacaine and fentanyl 3.2 microg ml(-1). Visual analogue scale (resting pain and pain on mobilization), the amount of rescue analgesics and epidural anesthesia related adverse events such as hypotension, nausea and vomiting were observed for 48 hours after surgery. RESULTS: There were no differences in visual analogue scale at all intervals between the two groups. In levobupivacaine group the patients used less amounts of rescue analgesics than ropivacaine group (P = 0.01). There were no differences in the incidences of postoperative hypotension, nausea and vomiting between the two groups. CONCLUSIONS: Both 0.187% ropivacaine and levobupivacaine similarly provide appropriate postoperative epidural analgesia for patients undergoing gynecological open abdominal surgery.

Characterization and usefulness of stool antigen tests using a monoclonal antibody to Helicobacter pylori catalase.

BACKGROUND AND AIM: Two types of stool antigen tests have been used in the management of Helicobacter pylori infection. Testmate Pylori Antigen enzyme immunoassay (TPAg EIA) is a direct sandwich enzyme immunoassay (EIA) while Testmate Rapid Pylori Antigen (Rapid TPAg) is performed using immunochromatography. The aim of this study was to study the characterization and usefulness of these tests. METHODS: Accuracy of both tests was studied using 111 fecal samples obtained from H. pylori-positive or -negative patients. Cross-reactivity was examined with four other Helicobacter spp. and five fecal bacteria in humans. To estimate the sensitivity of both kits, we tested H. pylori clinical strains. We also examined the diagnostic performances of both tests after the storage for 12 months. RESULTS: The accuracy of both Testmate kits was 100% in fecal samples from 111 patients. No cross-reactivity was observed in both Testmate kits in five fecal bacteria and four other Helicobacter spp. TPAg EIA and Rapid TPAg showed positive results in 1342 of 1344, and 483 of 485 clinical strains, respectively. Diagnostic performances was maintained for 12 months when TPAg EIA was stored at 4°C and Rapid TPAg at 30°C. CONCLUSIONS: We examined the details of high accuracy of TPAg EIA and Rapid TPAg. The diagnostic performance of both kits was maintained after storage for up to 1 year. The two types of tests would be useful in the management of H. pylori infection.

Quantitative analysis of perfluorinated chemicals in media for in vitro fertilization and related samples.

The actual standard in vitro fertilization (IVF) protocol recommends an overnight gamete co-incubation. All of the culture media used for human IVF are supplemented with serum or albumin. In the present study, we determined the concentrations of perfluorinated chemicals (PFCs) in IVF media (IVFM) and related samples by liquid chromatography with tandem mass spectrometry (LC/MS/MS). The results indicated that the concentrations of PFOS and PFOA in the protein source were higher than those in the IVFM samples. Compared with human plasma concentrations of PFCs, PFCs in all of the IVFM samples, such as PFBS, PFHxS and PFOA, were either not detected or present at only trace levels, even when protein source was added. LC/MS/MS could be used to determine PFCs in IVFM samples in future studies of the effects of PFC exposure on intrauterine insemination.

[Patient whose surgery was postponed due to complete atrioventricular block on arrival at operating theater].

An 86-year-old woman with low cardiac function was scheduled to undergo hip fracture surgery. Preoperative electrocardiogram showed complete left bundle brunch block, first degree atrioventricular block, left axis deviation and bigeminy. However, her electrocardiogram had changed to complete atrioventricular block on arrival at operating theater. ACC/AHA guideline on perioperative cardiovascular evaluation and care for non cardiac surgery indicates the assessment of both the urgency of the surgery and cardiac complications. Because complete atrioventricular block is classified to "active cardiac conditions", we decided to postpone the surgery for more detailed evaluation and treatment of cardiac conditions. In spite of the discontinuation of digoxin and carvegilol, complete atrioventricular block continued for a week, and the permanent pacemaker was inserted. The surgery was performed 2 weeks following the insertion of the pacemaker without any problems under combined general and lumbar epidural anesthesia.

A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS.

The anti-MRSA antibiotic, WAP-8294A, was isolated from the fermentation broth of Lysobacter sp. The major component, WAP-8294A2, is composed of 1 mol of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-ß-hydroxyasparagine, N-Me-D-Phe and N-Me-L-Val, and 2 mol of L-Ser, D-Orn and D-3-hydroxy-7-Me-octanoic acid. The structure of the WAP-8294A2 was mainly determined as a cyclic depsipeptide by 2D NMR experiments. However, it was difficult to use the NMR experiment to determine the minor components, A1, A4 and Ax13, isolated in small amounts. In the present study, ESI MS/MS was applied to the structure elucidation of these minor components. The structures of these minor components were determined on the basis of the fragmentation pattern of the product ions of WAP-8294A2 in the ESI MS/MS. As a result, it was confirmed that A1 and A4 had the same amino acid sequence as A2, while A1 and A4 had the 3-OH-octanoic acid and 3-OH-8-Me-nonanoic acid, respectively, in the place of the 3-OH-7-Me-octanoic acid in A2. In the structure of Ax13, it was found that Gly of A2 was changed to ß-Ala of Ax13.

Prepro-tachykinin gene expression in the brain of the honeybee Apis mellifera.

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization. The prepro-AmTRP gene is expressed predominantly in the MBs and in some neurons located in the optic and antennal lobes. cDNA microarray studies have revealed that AmTRP expression is enriched in the MBs compared with other brain regions. There is no difference in AmTRP-expressing cells among worker, queen, and drone brains, suggesting that the cell types that express the prepro-AmTRP gene do not change according to division of labor, sex, or caste. The unique expression pattern of the prepro-AmTRP gene suggests that AmTRPs function as neuromodulators in the MBs of the honeybee brain.

A microarray-based method for detecting methylated loci.

CpG island DNA methylation plays an important role in regulating gene expression in development and carcinogenesis. We developed a new microarray-based method called methylation amplification DNA chip (MAD) for detecting differences in methylation. In this method, only methylated CpG islands from the two samples that we wanted to compare were amplified and used for hybridization. The resource material for the microarray was derived from the methylated DNA library of the sample in which we wanted to detect hypermethylation. Choosing the methylated DNA library as the resource material of the microarray increased the percentage of DNA fragments derived from hypermethylated loci on the microarray.