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Although the dosage of oral antibiotics (OA) for the mandibular third molar extraction (MTME) varies among the administration periods according to the current guideline, our previous reports suggested that it might be possible to further shorten the administration period without increasing the incidence of surgical site infection (SSI). In the present study, we retrospectively evaluated the relationship between the incidence of SSI and the administration period of OA in patients who underwent the MTME in our hospital. This retrospective cohort study included 348 patients who underwent the MTME in our dental outpatient clinic from June 2020 to March 2022. The administrated antibiotic was amoxicillin (AMPC) in all patients. Patients were divided into two groups based on the administration period of AMPC single and three times before the surgery. The following information was collected: (1) patient factors (age, gender, body mass index, diagnosis, mandibular third molar status); (2) surgical factors (operation time, presence/absence of wound closure, presence/absence of hemostat, experience of surgeons); (3) relationship between administration period of OA and SSI occurrence; and (4) details of SSI. There were 217 cases in the single group and 131 cases in the three times group. The incidence of SSI was 1.1% (4/348), with 1.4% (3/217) in the single group and 0.8% (1/131) in the three times group; there was no significant difference between the two groups. Our result suggests that single administration of AMPC before the MTME would be sufficient for the prevention of SSI in Japanese patients without risk factors.

Introduction We investigated the use of oral antibiotics (OA) and surgical site infection (SSI) related to extractions of ordinary teeth and mandibular wisdom teeth in a dental outpatient clinic from January 2015 to December 2019. Methods The following information were surveyed: (1) presence/absence of OA, (2) timing, (3) type, (4) administration period, and (5) SSI rates. Results The use of OA during ordinary tooth extraction decreased from 68.3% to 41.3%, but SSI rate did not change during this period of time. Total SSI rate was 0.8% (122/14,832) on average. For mandibular wisdom tooth extraction, preoperative administration of third-generation cephalosporins decreased from 70.4% to 0.3% while that of penicillin (AMPC) increased from 0% to 98%. SSI rate was not changed after these improvements. Total SSI rate was 3.5% (180/5106) on average. The duration of OA was slightly decreased to two days in 2018 and 2019, and it was found that there was no significant difference in SSI rates between 2- and 3-day durations. Preoperative administration had 0.37 odds ratio (OR) (95% confidence interval (95%CI): 0.22–0.63) of SSI compared with postoperative administration. AMPC had 0.76 OR (95% CI: 0.55–1.04) of SSI compared with Third-generation cephalosporins and others. Timing of OA was P Conclusions SSI rates did not change over time, administration period of OA decreased and the use of AMPC increased. Therefore, it seems necessary to continue to investigate the effects of SSI risk factors proactively in the future and to make efforts in the advocacy of appropriate antimicrobial use.

Introduction: Because of the increasing age of the general population, there is an increasing number of older patients with lung cancer. Cancer chemotherapy often causes severe hematological toxicity in older patients. Objective: This study aimed to explore the risk factors affecting the hematological toxicity of cytotoxic anticancer drugs in patients with lung cancer. Methods: Data were retrospectively collected from 194 patients with lung cancer at Niigata University Medical and Dental Hospital, Japan, between April 2011 and March 2016, when the patients underwent their first round of cytotoxic chemotherapy. The patients were divided into three groups on the basis of age: Results: Patients aged ≥75 years were significantly more likely to experience grade 3 or 4 neutropenia, compared with patients aged Conclusion: We found that age ≥75 years, male sex, and a performance status of ≥2 were independent risk factors for grade 3 or 4 neutropenia.

Severe hepatotoxicity from combination chemotherapy with cisplatin and 5-fluorouracil is a rare adverse effect. In this case report, we present a case with severe hepatotoxicity immediately following chemotherapy with cisplatin and 5-fluorouracil. This female patient had previously been treated with this combination with no hepatotoxicity. The elevated liver enzymes quickly normalized after chemotherapy was stopped. There were no specific changes in liver imaging. As hepatotoxicity occurred after repeated administration of cisplatin, we suggest that this hepatotoxicity might represent a case of allergic hepatitis caused by cisplatin. Severe hepatotoxicity should be watched for with repeated administration of cisplatin.

Objective: Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonate that induces osteoclast apoptosis and inhibits bone resorption. Adding ZOL to neoadjuvant chemotherapy has been shown to have potential anticancer benefits in women with HER2-negative breast cancer. The objective of the present study was to investigate ZOL’s cost-effectiveness from the perspective of health care payers in Japan. Methods: A Markov model was developed to evaluate the costs and effectiveness associated with ZOL + chemotherapy (CTZ) and chemotherapy (CT) alone over a 10-year time horizon. Monthly transition probabilities were estimated according to JONIE1 (Japan Organization of Neoadjuvant Innovative Expert) Study data and an extrapolated Weibull model. Health outcomes were measured in quality-adjusted life years (QALYs). Costs were calculated using year-2018 Japanese yen (JPY) (1.00 US dollars (USD) = 110.4 JPY). Model robustness was addressed through one-way and probabilistic sensitivity analysis. The costs and QALYs were discounted at a rate of 2% per year. Results: In the base case, the use of CTZ was associated with a gain of 3.94 QALYs. The incremental cost per QALY of the CTZ gain was 681,056.1 JPY (6168.99 USD) per QALY. Conclusion: It is convincing that neoadjuvant CTZ for patients with breast cancer would be expected to have statistically significant clinical efficacy. Addition of ZOL to CT might be a cost-effective option compared with CT alone.

Objectives This retrospective study examined how a pharmacist-involved education program in a multidisciplinary team (PEMT) for oral mucositis (OM) affected head-and-neck cancer (HNC) patients receiving concurrent chemoradiotherapy (CCRT). Materials and methods Total samples data of 53 patients during the stipulated timeframe were retrospectively collected from electronic medical records from February 2017 to January 2019. We compared the presence/absence of OM (OM: yes/no) between patients with and without PEMT (PEMT: yes/no) as the primary endpoint and OM severity as the secondary endpoint. The following information was surveyed: age, gender, weight loss, steroid or immunosuppressant use, hematological values (albumin, white blood cell count, blood platelets, and neutrophils), cancer grade, primary cancer site, type and use of mouthwash and moisturizer, opioid use (yes/no, days until the start of opioid use, and dose, switch to tape), and length of hospital day (LOD). The two groups were compared using Fisher’s exact test for qualitative data and the Mann-Whitney U test for quantitative data, and a significance level of p.05 was set. Results The group managed by PEMT had significantly lower weight loss and a significantly lower incidence of local anesthetic and opioid use and switch to tape compared with the group not managed by PEMT (p.05). The two groups showed no significant difference in OM (yes/no) or OM severity. The PEMT group had significantly shorter LOD at 57 (53–64) days compared with the non-PEMT group at 63.5 (57–68) days (p.05). Conclusions Our results showed that PEMT did not improve OM (yes/no) or OM severity in HNC patients undergoing CCRT. However, the PEMT group had a lower incidence of grades 3 and 4 OM than the non-PEMT group, although not significantly. In addition, PEMT contributed to oral pain relief and the lowering of the risk for OM by reduction in weight loss.

Introduction: Postoperative infection is a major cause of morbidity and prolonged hospitalization in patients undergoing gastrointestinal surgery. This observational study aimed to investigate the risk factors associated with postoperative infection and to develop a prediction model for postoperative infections that occur after gastrointestinal surgery. Methods: The study population comprised 1637 patients who underwent gastrointestinal surgery at Niigata University Medical and Dental Hospital between June 2013 and May 2017. Observational data from 1883 surgical procedures were used in the statistical analyses (including 198 patients who underwent several operations). Results: The generalized estimating equation (GEE) was used to detect significant risk factors, including older age, history of smoking, body temperature greater than 38 &deg, C, non-endoscopic surgical procedures, surgery in the thoracic or lower gastrointestinal tract, and use of medical nutritional products during surgery. The sensitivity and specificity of the GEE model were 88.2% and 55.1%, respectively. Conclusion: This study established a predictable GEE model, incorporating the data of patients who were hospitalized several times into a prediction analysis, even though the sensitivity was not sufficiently high. The GEE model, which is considered clinically useful, can be constructed using a variety of variables, including those obtained from electronic health records.

Introduction According to the guidelines, the dosage for mandibular wisdom tooth extraction (MWTE) varies within the administration period. There is a 24-fold difference between the minimum and maximum doses. If an appropriate antimicrobial can be administered without increasing incidence of surgical site infection (SSI), it may lead to a global action plan on antimicrobial resistance (AMR). Therefore, we prospectively surveyed incidence of SSI post-operatively and use of oral antibiotics (OA) for MWTE. Methods Subjects were patients who underwent MWTE in our dental outpatient clinic from May 2019 to April 2020. Two groups were formed depending on type of administration period they received: 24 h and 48 h after surgery. The following information was collected: (1) patient factors (age, gender, body mass index, presence/absence of preoperative medication, diagnosis, impacted wisdom tooth status; (2) surgical factors (operative time, presence/absence of closure, presence/absence of hemostat, doctor career, type and frequency of painkiller); (3) relationship between administration period of OA and SSI occurrence; and (4) details of SSI. Results Three hundred forty subjects were analyzed, all of which used amoxicillin. There were 106 cases in 24 h group and 234 cases in 48 h group. The total incidence of SSI was 1.1% (4/340 cases), with 0.9% (1/106 cases) in 24 h group and 1.3% (3/234 cases) in 48 h group; there was no difference between the two groups. Conclusion Our study suggests that amoxicillin (250 mg/dose every 8 h x 3 doses beginning 1 h before surgery) might be sufficient in preventing SSI in Japanese dental patients without SSI risk factors.

Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice, we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx4Cv mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx4Cv mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx4Cv recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function and the utility of the NSG-mdx4Cv model for studies on muscle regeneration and Duchenne muscular dystrophy therapy.

The lymphocyte immunosuppressant sensitivity test (LIST) with 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay procedure has been used to predict the pharmacological efficacy of immunosuppressive agents to prevent acute rejection episodes for renal transplant recipients. In this study, mycophenolic acid (MPA) pharmacological efficacies were evaluated by LIST at both prior to and just after renal transplantation. We compared the efficacies to the clinical outcome of these recipients. MPA's pharmacological efficacy was evaluated by LIST not only before the operation but also at 2, 4, and 6 weeks after transplantation in 16 renal transplant recipients. These recipients were divided into high- and low-sensitivity groups according to peripheral blood mononuclear cell (PBMC) sensitivity to MPA in vitro. The MPA sensitivities were compared to cytomegalovirus (CMV) infection and acute rejection episodes in these recipients under MPA immunosuppressive therapy. The rate of CMV infection episodes in the low-MPA pharmacological efficacy group categorized at 2 weeks after renal transplantation was 5/6 (83.3%), which was significantly higher than the rate of 1/10 (10.0%) ( p < 0.01) in the high-MPA sensitivity group. However, the MPA pharmacological efficacy evaluated both before and after transplantation had no relationship with the incidence of rejection episodes. These findings suggest that the MPA pharmacological efficacy evaluated by LIST at 2 weeks after operation is a useful biomarker for predicting the following occurrence of CMV infection episodes in renal transplant recipients.

The lymphocyte immunosuppressant sensitivity test (LIST) with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay procedure can predict the pharmacological efficacy of immunosuppressive agents. A previous study reported the pharmacological efficacy of tacrolimus evaluated by LIST just before renal transplantation significantly correlated with the incidence of acute rejection episodes. However, the pharmacological efficacy of tacrolimus has not been estimated after renal transplantation. Therefore, the present study evaluated the pharmacological efficacy of tacrolimus by LIST using the MTT assay procedure before and 1, 3, and 12 months after transplantation in 17 renal transplant recipients that received tacrolimus-based immunosuppressive therapy. The tacrolimus pharmacological efficacies before and after the procedure were also compared with incidence of acute rejection and cytomegalovirus (CMV) infection episodes. The individual values of tacrolimus 50% inhibition of lymphocyte proliferation (IC50) varied widely before transplantation, and the mean value of the IC50 was 126.4 ± 337.7 ng/ml. The patients were divided into two groups according to the tacrolimus IC50 values evaluated before transplantation. The rate of acute rejection episodes in the tacrolimus high-sensitivity group was significantly lower than that in the tacrolimus low-sensitivity group (p = 0.005). The tacrolimus IC50 deviation between patients expanded further at one and three months after surgery. However, the sensitivity deviation almost converged at 1 year after surgery. Moreover, the pharmacological efficacy of tacrolimus evaluated at 1, 3, and 12 months after transplantation did not significantly correlate with the incidence of acute rejection episodes. The pharmacological efficacies of tacrolimus evaluated at both before and after surgery were not significantly correlated with the episodes of CMV infection. These findings suggest that the pharmacological efficacy of tacrolimus evaluated with LIST before surgery is a useful biomarker for predicting the occurrence of acute allograft rejection in renal transplantation.

It is obvious that pharmacists play a critical role as risk managers in the healthcare system, especially in medication treatment. Hitherto, there is not a single multicenter-survey report describing the effectiveness of clinical pharmacists in preventing medical errors from occurring in the wards in Japan. Thus, we conducted a 1-month survey to elucidate the relationship between the number of errors and working hours of pharmacists in the ward, and verified whether the assignment of clinical pharmacists to the ward would prevent medical errors between October 1-31, 2009. Questionnaire items for the pharmacists at 42 national university hospitals and a medical institute included the total and the respective numbers of medication-related errors, beds and working hours of pharmacist in 2 internal medicine and 2 surgical departments in each hospital. Regardless of severity, errors were consecutively reported to the Medical Security and Safety Management Section in each hospital. The analysis of errors revealed that longer working hours of pharmacists in the ward resulted in less medication-related errors; this was especially significant in the internal medicine ward (where a variety of drugs were used) compared with the surgical ward. However, the nurse assignment mode (nurse/inpatients ratio: 1 : 7-10) did not influence the error frequency. The results of this survey strongly indicate that assignment of clinical pharmacists to the ward is critically essential in promoting medication safety and efficacy.

The lymphocyte immunosuppressant sensitivity test (LIST) can predict the pharmacological efficacy of immunosuppressive agents. We herein estimated the mycophenolic acid efficacy using LIST before and 1, 3, and 12 months after the operation in 15 renal transplant recipients. The pharmacological efficacy of mycophenolic acid as assessed before transplantation showed small individual variations, whereas the variability greatly increased at 1 and 3 months after transplantation. Furthermore, the individual IC50 variation among these subjects at 1-year after operation was closely similar to that observed before surgery. No significant implications of these individual differences in the mycophenolic acid sensitivity were noted in regard to the clinical courses of these recipients.

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.

Drug counseling during pregnancy plays an important role in eliminating anxiety concerning the adverse effects of drugs on fetuses and explaining the importance of drug administration during pregnancy. In the Outpatient Department of Obstetrics and Gynecology at the Niigata University Medical and Dental Hospital, together with obstetricians, pharmacists have provided information on the adverse effects of drugs administered during pregnancy on fetuses (teratogenicity) since March 22, 1995. Up to March 31, 2006, 269 patients had received such counseling.In this study, we investigated the timing of a decrease in the work of searching for information on drugs, and evaluated strategies for the development of drug counseling.According to the order in which drug counseling was requested by obstetricians, 250 patients were first divided into groups of 50 patients each, and then the quantity of drugs with general names as a percentage of the total number of drugs with trade names was determined. For 247 patients (3 excluded because they were women who had received counseling on drugs prescribed to their husbands), the percentage of drugs with general names of the total of those with trade names was 37.4% (413/1,103).Our results suggested that there would need to be about 650 drugs with general names in the database of information on the adverse effects of drugs taken during pregnancy on the fetus to provide effective drug counseling in the future. They also suggested that the work of searching for information on drugs decreased after counseling 100 patients.

The physiological function of the prion protein (PrP) remains enigmatic despite its established involvement in the pathogenesis of spongiform encephalopathies. PrP is a glycolipid-anchored membrane protein, which constitutively recycles between the cell surface and an endosomal compartment. The N-terminal region of PrP contains a four tandem repeat (OP4) of the octapeptide PHGGGWGQ (OP) that binds and reduces Cu(II) ions. We have examined the kinetic properties of the OP4-mediated Cu(II) reduction and found that OP4 exhibits the highest reduction activity around pH 6.5, close to the pH in early endosomes. All four OP units and at least one tryptophan side chain are essential for Cu(II) reduction. The reaction is described by an uncompetitive substrate inhibition mechanism involving a 1:1 Cu(II)-OP4 active intermediate. Structural analysis by Raman spectroscopy has revealed that the Cu(II) ion is coordinated by four histidine Ntau atoms in the active intermediate and the feasibility of formation of this intermediate correlates with the Cu(II) reduction over a pH range from 5.0 to 8.2. Molecular mechanics calculations suggest that two tryptophan residues of OP4 are located near the Cu(II) site, being consistent with the importance of redox-active tryptophan in the Cu(II) reduction. PrP has been proposed to capture Cu(II) ions in the extracellular space and release them in the endosome. The results of this study strongly suggest that PrP also plays a role in the reduction of captured Cu(II) ions prior to their transfer to Cu(I)-specific intracellular copper trafficking proteins.

The absorption and UV resonance Raman spectra of 2′,3′,5′-tri- O -acetyladenosine (AcAdo) were investigated in two non-hydrogen-bonding, three proton acceptor, and four proton donor/acceptor solvents. The molar extinction coefficient at the absorption maximum around 260 nm ( e max ) changed up to ∼10% with change of the solvent. The intensities of 257 nm-excited Raman bands varied dramatically (up to ∼1000%) from solvent to solvent. The e max of UV absorption and the intensities of six Raman bands of AcAdo in the nine solvents were plotted against the Kamlet–Taft solvent parameters α , β , and π *, which represent the hydrogen-bond donor acidity, acceptor basicity, and the polarity–polarizability of the solvent, respectively. Both e max and the Raman band intensities increased with α . Similar but weaker correlations were seen for β . π * had the least influence. These observations strongly suggest that hydrogen bonding is the main factor that affects the intensities of UV absorption and resonance Raman scattering. Multiple regression analysis has shown that the correlations with α and β are improved by taking linear combinations of Raman band intensities. We propose a new method to evaluate the environmental α and β values of the adenine ring from the relative intensities of three strong Raman bands at ∼1500, 1480, and 1330 cm −1 . UV resonance Raman intensities of adenine ring vibrations are useful in analyzing the strength of hydrogen bonding at the proton acceptor (N1, N3, and N7) and donor (C6–NH 2 ) sites.

Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism.

Isotope-edited Raman spectroscopy, a combination of site-selective isotopic labeling and Raman difference spectroscopy, is a useful method for studying the structure and interaction of individual nucleic acid residues in oligonucleotides. To obtain basic data for applying isotope-edited Raman spectroscopy to guanine residues, we studied the vibrational modes of UV resonance Raman bands of the C8-deuterated guanine ring by examining the wavenumber shifts upon seven isotopic substitutions (2-13C, 2-15N, 6-18O, 7-15N, 8-13C, 9-15N and 1′-13C). The hydrogen bond sensitivities of the Raman bands were also investigated by comparing the Raman spectra recorded in several solvents of different hydrogen bonding properties. Some of the Raman bands were found to be markers of hydrogen bonding at specific donor or acceptor sites on the guanine ring. The Raman bands, which shift on C8-deuteration, remain in the difference spectrum between the unlabeled and C8-deuterated guanine rings. Among them, a negative peak around 1525 cm−1 and a strong positive/negative peak pair around 1485/1465 cm−1 serve as markers of hydrogen bonding at N7 and C6O, respectively. Another weak positive/negative peak pair around 1025/1040 cm−1 is sensitive to hydrogen bonding at the proton donor sites (N1—H and N2—H2). The applicability of the hydrogen bond markers has been tested by using a 22-mer oligonucleotide duplex containing eight guanine residues and its analog in which a single guanine residue is C8-deuterated. The difference spectrum shows that the hydrogen bonding state of the guanine residue at the labeled position is consistent with the Watson–Crick base pair structure of DNA. Isotope-edited Raman spectroscopy is a useful tool for studying the hydrogen bonding state of selected guanine residues in oligonucleotides. Copyright © 2002 John Wiley & Sons, Ltd.

The adenosine triphosphate assay using peripheral lymphocytes may be useful to evaluate the risks of acute rejection and infection in kidney transplant patients. We used the adenosine triphosphate assay to evaluate differences between recipients who were treated with cyclosporine- or tacrolimus-based immunosuppressive therapy.Adenosine triphosphate levels were measured in peripheral CD4+ cells before and after transplant and were correlated with clinical outcomes in 45 kidney transplant recipients. These recipients received immunosuppressive therapy with either cyclosporine (23 patients) or tacrolimus (22 patients).Adenosine triphosphate levels were significantly lower in the cyclosporine- than tacrolimus-based therapy groups from 2 to 6 weeks after transplant. Adenosine triphosphate levels were similar between these groups before and 1 week after transplant. The frequency of cytomegalovirus infection was greater in the recipients who received cyclosporine (17 patients [74%]) than tacrolimus (6 patients [27%]; P ≦ .003). The frequency of acute rejection episodes was similar between the cyclosporine and tacrolimus groups.These observations suggest that cyclosporine-based immunosuppressive therapy causes excessive immunosuppression compared with tacrolimus-based therapy, evidenced by the lymphocyte adenosine triphosphate levels. The adenosine triphosphate assay using peripheral CD4+ cells may be a useful method for predicting the occurrence of cytomegalovirus infections in kidney transplant recipients.

The imidazole ring of histidine exists in two tautomeric forms in neutral-to-basic aqueous solution, and the tautomerism of the histidine residue sometimes plays a key role in catalytic reactions of enzymes. We have investigated the molecular vibrations of two tautomers of 4-methylimidazole (4-MeIm), a model compound for the histidine side chain, by Raman spectroscopy and ab initio calculations based on the density functional theory (DFT) approach. Examination of the temperature dependence of Raman intensity revealed nine pairs of bands characteristic of the N1-protonated and the N3-protonated tautomers of 4-MeIm at 1576/1596, 1452/1427, 1304/1344, 1265/1259, 1229/1234, 1165/1149, 1088/1104, 996/1014, and 942/934 cm-1. Five to six pairs of tautomerism-sensitive Raman bands were also identified for each of the C2-, N-, and C2,N-deuterated analogues of 4-MeIm. The observed Raman wavenumbers were used to determine nine scaling factors for the in-plane force constants derived from DFT calculations using the 6...

Raman spectroscopy has been combined with site-selective isotopic labeling techniques to obtain structural information on a selected nucleic acid residue in oligonucleotides. In the difference spectrum between the unlabeled and site-selectively labeled oligonucleotides, the Raman signals from the residue at the labeled position show up, while the signals from the residues at unlabeled positions are canceled out. To demonstrate the utility of this new method, we have prepared a self-complementary tetradecamer DNA, d(AGTGCTCGAGCACT)2, containing single C8-deuterated adenine at position 9 (A9) or 12 (A12). UV (257 nm) resonance Raman difference spectra between the unlabeled and labeled oligonucleotides in solution reveal slightly different microenvironments around A9 and A12 as expected for a double-stranded helical structure of the tetradecamer DNA. In the presence of an antitumor antibiotic, actinomycin D, which intercalates into the 5′-GC-3′ sequence of DNA, the Raman signals of A9 on the 5′-side of the intercalation site become significantly weaker, indicating an increased base stacking with adjacent guanine bases. In contrast, the Raman signals of A12 on the 3′-side are not affected by the binding of actinomycin D. This observation provides the first experimental evidence that the intercalation of actinomycin D induces an asymmetric structural alteration of DNA in solution.

UV resonance Raman spectra of guanosine and its seven isotope-substituted analogs (2-13C, 2-15N, 6-18O, 7-15N, 8-13C, 9-15N and 1′-13C) were measured with 257 nm excitation in H2O and D2O solutions. In-plane vibrations of the guanine ring were selectively enhanced in the UV resonance Raman spectra, and most Raman bands showed significant wavenumber shifts upon isotopic substitution. The observed isotope shifts were used to assign the Raman bands to vibrations of the peripheral sites (N1— H, C2—NH2 and C6O), the pyrimidine ring and/or the imidazole ring. Previous assignments for some Raman bands were shown to be inconsistent with the isotopic data and they were revised. Relationships between the vibrational modes and the sensitivities to hydrogen bonding or conformation are discussed for known Raman marker bands. Each hydrogen bond marker arises from a vibration that involves, at least partly, the proton donor or acceptor atom. All the marker bands of glycosidic bond orientation and ribose ring puckering actually involve atomic displacements around the N9—C1′ moiety connecting the guanine ring to ribose, permitting vibrational coupling between them. The isotopic wavenumber shifts reported here may be useful in improving the force field for the 9-substituted guanine ring and in interpreting the vibrational spectra of guanine nucleoside and nucleotides. Copyright © 1999 John Wiley & Sons, Ltd.

Ultraviolet (UV) resonance Raman spectra of an acetyl derivative of adenosine and its C8-deuterated analog were measured in solvents with varied hydrogen bonding properties. The Raman spectrum of the acetyl derivative in H 2 O solution is almost identical to that of adenosine in H 2 O solution, indicating that the acetyl derivative is a good model for the adenine nucleotide in DNA. Most of the Raman bands increase or decrease in wavenumber upon hydrogen bonding at the proton-donor (C6-NH 2 ) and proton-acceptor sites (N1, N3 and N7) of the adenine ring. Among them, the ν 1 , ν 2 , ν 3 , ν 5 and ν 9 bands of the adenine ring and the ν 1 ′, ν 2 ′, ν 3 ′, ν 5 ′, ν 7 ′ and ν 9 ′ bands of the C8-deuterated adenine ring show wavenumber shifts larger than 5 cm −1 . Relative intensities of some Raman bands change as well. The wavenumber and intensity changes can be used as markers of hydrogen bonding at the proton donor and/or acceptor sites of the adenine ring. The Raman spectral difference between the non-deuterated and C8-D labeled adenine rings reflects the hydrogen-bonding state very sensitively and is expected to be useful in studying the hydrogen-bonding state of a particular adenine residue in oligonucleotides by a combination of site-selective C8-D labeling and UV resonance Raman spectroscopy.

SummaryMesp1 is regarded as the master regulator of cardiovascular development, initiating the cardiac transcription factor cascade to direct the generation of cardiac mesoderm. To define the early embryonic cell population that responds to Mesp1, we performed pulse inductions of gene expression over tight temporal windows following embryonic stem cell differentiation. Remarkably, instead of promoting cardiac differentiation in the initial wave of mesoderm, Mesp1 binds to the Tal1 (Scl) +40 kb enhancer and generates Flk-1+ precursors expressing Etv2 (ER71) and Tal1 that undergo hematopoietic differentiation. The second wave of mesoderm responds to Mesp1 by differentiating into PDGFRα+ precursors that undergo cardiac differentiation. Furthermore, in the absence of serum-derived factors, Mesp1 promotes skeletal myogenic differentiation. Lineage tracing revealed that the majority of yolk sac and many adult hematopoietic cells derive from Mesp1+ precursors. Thus, Mesp1 is a context-dependent determination factor, integrating the stage of differentiation and the signaling environment to specify different lineage outcomes.

Ultraviolet resonance Raman spectra of 2′-deoxy-3′,5′-bis(triisopropylsilyl)guanosine (TPS-dGuo) were recorded in non-hydrogen bonding, proton acceptor, and proton donor/acceptor solvents. Raman spectral changes observed on going from inert to proton acceptor solvents were ascribed to the hydrogen bonding at the proton donor sites of the guanine ring (N1H and C2NH 2 ), and the spectral changes associated with the solvent change from proton acceptor to donor/acceptor were ascribed to the hydrogen bonding at the proton acceptor sites (N3, C6O, and N7). A Raman band appearing at 1624 cm −1 in inert solvents is assigned mainly to the NH 2 scissors mode and its frequency changes to ≈ 1640 cm −1 in acceptor solvents, reflecting the hydrogen bonding at C2NH 2 . Another band at 1581 cm −1 , arising largely from the N1H bend, shows an upshift of ≈ 10 cm −1 upon hydrogen bonding at either N1H or acceptor sites. Hydrogen bonding at the acceptor sites also produces frequency shifts of other Raman bands (at 1710, 1565, 1528, 1481, and 1154 cm −1 in 1,2-dichloroethane solution). Among the Raman bands listed above, the 1710 cm −1 band due to the C6O stretch decreases in frequency, whereas the others increase. The downshift of the C6O stretching frequency is correlated with the strength of hydrogen bonding at C6O. The frequency of the 1481 cm −1 band increases with a decrease of the C6O stretching frequency, indicating that the 1481 cm −1 band is also a marker of hydrogen bonding at C6O. This finding is in sharp contrast to the previously proposed correlation with the hydrogen bonding at N7. The 1565 cm −1 band is assigned to a vibration mainly involving the N1C2N3 linkage, and its frequency increases with increasing strength of the hydrogen bond at N3. Three bands around 1315, 1180, and 1030 cm −1 , which are known to be sensitive to the ribose ring puckering and glycosidic bond orientation, also show small frequency changes upon hydrogen bonding. The Raman marker bands for the hydrogen bonds at C2NH 2 , N1H, C6O, and N3 are expected to be useful in studying the structures of nucleic acids and nucleic acid-protein complexes containing guanine residues.

Basiliximab is a recently developed immunosuppressive agent for the prevention of acute allograft rejection in renal transplant recipients. The combination use of basiliximab and a calcineurin inhibitor was suggested to be more effective in comparison to immunosuppressive therapy using calcineurin inhibitor without basiliximab. Cyclosporine has been generally administered with basiliximab for renal transplant recipients. However, in cases of tacrolimus-based immunosuppressive regimen, the clinical efficacy and safety of combined use of tacrolimus and basiliximab remains to be elucidated. This study evaluated the tacrolimus pharmacological efficacy using a lymphocyte immunosuppressant sensitivity test (LIST) with MTT assay procedures in 16 cases of renal transplant recipients treated by tacrolimus without basiliximab and in 13 cases treated by tacrolimus in combination with basiliximab. The rate of acute rejection episodes in the recipients treated with tacrolimus plus basiliximab was 1/13 (7.7%), whereas the rate in the recipients treated with tacrolimus without basiliximab was 6/16 (37.5%). The recipients were divided into two groups according to their peripheral blood mononuclear cell (PBMC) sensitivity to tacrolimus [i.e., including a tacrolimus high sensitivity group (IC50 50 >1.0 ng/ml). In the recipients treated with tacrolimus without basiliximab, the rate of acute rejection episodes in the tacrolimus high sensitivity group was 1/10 (10.0%), which was significantly lower than the rate in the low sensitivity group of 5/6 (83.3%; p = 0.008). The incidence of cytomegalovirus infection was not significantly different between the tacrolimus high and the low sensitivity groups of the recipients treated with tacrolimus with and without basiliximab. Therefore, in the case of selected tacrolimus-based immunosuppressive therapy for renal transplant recipients, the tacrolimus pharmacological efficacy should be evaluated using LIST at a time just before the transplant procedure in order to accurately predict allograft rejection. The data also suggested that low tacrolimus sensitivity recipients should be treated with tacrolimus-based immunosuppressive therapy in combination with basiliximab.

Infrared, Raman and UV resonance Raman spectra of adenosine and its 1,3-15N2, 2–13C, and 8–13C isotopic analogues were measured in neutral aqueous solution (Raman and UV Raman) and in the crystalline state (infrared and Raman). The observed isotopic wavenumber shifts are useful in distinguishing adenine ring vibrations from ribose vibrations. In-plane modes of the adenine ring are selectively enhanced in UV resonance Raman spectra, which facilitates the assignment of the in-plane vibrations. In addition to the in-plane modes, a ribose vibration coupled with adenine in-plane vibrations was identified in the UV resonance Raman spectra. The fundamental wavenumbers for 22 in-plane normal modes of the 9-substituted adenine ring of adenosine in the 1700–250 cm−1 region are proposed. Although the fundamental wavenumbers of adenosine correspond well with those of adenine above 1350 cm−l and below 800 cm−1 the vibrations in the 1350–800 cm−1, region are appreciably affected by the presence of the N-9—C-1′ glycosidic bond and the couplings between ribose and adenine ring vibrational motions. The adenosine fundamental wavenumbers and their isotopic shifts reported here may be useful in analysing vibrational spectra of adenine nucleosides and nucleotides and in improving the force field of the 9-substituted adenine ring.

Ultraviolet resonance Raman spectra of ribosyl C(1')-deuterated guanosine and adenosine were measured. Most in-plane vibrations of the purine rings in the 1420-1100-cm -1 region showed frequency upshifts upon C(1')-deuteration, while those in the 1100-700-cm -1 region showed downshifts. The purine ring vibrations above 1450 cm -1 were unaffected. The frequency shifts associated with the C(1')-deuteration are explained by assuming couplings of the purine vibrations with a ribose C(1')-H bending mode, which involves a hydrogen motion in the plane of N(9)-C(1')-H

Ultraviolet resonance Raman spectra of phytochrome in the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr) are reported. The spectra excited at 240-nm provide structural information about the protein part of phytochrome. The protein contains only a very small amount of β-sheet structure and most of the tyrosine side chains are located in hydrophobic environments. Indole rings of tryptophan (Trp) interact with neighboring groups in the Pr form and these interactions become weaker with the conversion from Pr to Pfr. Some Trp side chains of Pfr are surrounded by aliphatic groups but such is not the case in Pr. These changes in the environment occur at the same time as changes in orientation of Trp side chains. Our observations suggest that interactions between Trp residues and the tetrapyrrolic chromophore occur in the Pr form and that the strength of these interactions diminishes in the Pfr form.

Ultraviolet resonance Raman spectra of ribosyl C(1')-deuterated guanosine and adenosine were measured. Most in-plane vibrations of the purine rings in the 1420-1100-cm -1 region showed frequency upshifts upon C(1')-deuteration, while those in the 1100-700-cm -1 region showed downshifts. The purine ring vibrations above 1450 cm -1 were unaffected. The frequency shifts associated with the C(1')-deuteration are explained by assuming couplings of the purine vibrations with a ribose C(1')-H bending mode, which involves a hydrogen motion in the plane of N(9)-C(1')-H

Distamycin A (Dst) is an antibiotic which binds to the minor groove of double-stranded DNA at A/T-rich regions. We have examined the affinity and mode of Dst binding to DNA duplexes containing a conserved A/T core and variable terminal A/T regions by using circular dichroism spectroscopy. The observed circular dichroism spectra were analyzed by singular value decomposition and fitted to a two-step binding model. The result clearly shows a correlation between the affinity for Dst and the preference for Dst-DNA 1:1 binding over 2:1 binding. The A/T stretches that prefer 1:1 binding form high-affinity 1:1 complexes, whereas those preferring 2:1 binding form stable 2:1 complex with low overall affinities. The terminal A/T residues of the Dst binding region play an important role in the stabilization/destabilization of the 1:1 and 2:1 complexes, resulting in a terminal residue-dependent variation of the binding affinity and the binding mode preference.

Summary Objective: Lymphocyte immunosuppressant sensitivity test (LIST) is useful for predicting the pharmacological efficacy of immunosuppressive agents. In this study, the pharmacological efficacy of cyclosporine was estimated by LIST before and after renal transplantation. Methods: Lymphocyte immunosuppressant sensitivity test was performed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay before and at 1, 3, and 12 months after transplantation in 19 consecutive renal transplant recipients. Results: There was wide intersubject variability in cyclosporine IC50 before transplantation [Mean (SD) of 593·9 (1067·6) ng/mL]. This variability worsened 1 month after transplantation [525·7 (1532·7) ng/mL] but decreased at 3 months (193·5 (347·9) ng/mL) and 12 months (75·4 (95·4) ng/mL). In this small study, observed differences in IC50 values for the individual subjects at various time intervals was not associated with the occurrence of rejection, graft loss, and infection episodes. Conclusion: Lymphocyte sensitivity to cyclosporine assessed by the LIST assay showed a high level of inter-subject variability particularly before and 1 month after transplantation. The observed difference in IC50 values was not associated with clinical outcome in this small study.

The BM2 protein of influenza B virus forms a transmembrane proton channel essential for the virus infection. We investigated the structure and mechanism of the BM2 proton channel by using a 31-mer peptide (BM2-TMP) representing the putative transmembrane domain of BM2, with special focus on His19, Trp23 and His27. Like the full-length protein, BM2-TMP formed a transmembrane proton channel activated at acidic pH with a midpoint of transition at pH 6.4 +/- 0.1. Mutation of His19 to Ala almost abolished the channel activity, whereas the His27-to-Ala mutant retained partial activity. The proton selectivity of the channel was lost upon substitution of Phe for Trp23. Comparison of CD, fluorescence and Raman spectra measured for wild-type and mutated BM2-TMP at varied pH showed the pK(a) of the imidazole ring to be approximately 6.5 for His19 and approximately 7.6 for His27. Analysis of the pH-dependent fluorescence and Raman intensities suggested the occurrence of cation-pi interaction between the protonated imidazole ring of His and the indole ring of Trp. The His19-Trp23 cation-pi interaction below pH 6.5 is likely to trigger the opening of the proton channel, whereas His27 is not essential but enhances the channel activity through interaction with Trp23, which constitutes the proton-selective gate.

Ultraviolet resonance Raman spectra (266-nm excitation) were studied for adenine, uracil and thymine derivatives in several solvents at low concentration (0.5−10 mM) where hydrogen bonding between solute molecules is negligible. The Raman spectra patterns depend critically on the proton-accepting/donating properties of the solvents. Raman bands which show large solvent effects are expected to be useful in studying the hydrogen-bonding states of the nucleic acid bases.

The lymphocyte immunosuppressant-sensitivity test (LIST) has been reported extensively as being able to estimate the pharmacological efficacy of immunosuppressive drugs in individual patients. This study measured the IC50 values for 6 drugs, cyclosporine, tacrolimus, methylprednisolone, 6-mercaptopurine, mycophenolic acid, and mizoribine, against mitogen-induced proliferation of peripheral-blood lymphocytes in 29 renal transplant recipients. We also examined relationship between the IC50 values and 4 factors; age, duration of dialysis, percentage of lymphocytes in total white blood cells, and blastogenesis stimulation index by mitogen. There were no significant correlations between the IC50 values and these factors, except that the tacrolimus IC50 value was correlated weakly with the stimulation index (p

The cation-pi interaction, a noncovalent interaction of electrostatic nature between a cation and an electron-rich pi system, is increasingly recognized as an important force that influences the structures and functions of molecules including proteins. Unlike other metal cations, the transition metal cation Cu2+ is not regarded to take part in a cation-pi interaction because Cu2+ tends to oxidize the pi electron system, in particular that of Trp, and to introduce covalency in the metal-pi electron interaction. This paper reports the first spectral evidence for the cation-pi interaction between Cu2+ and Trp. The Cu2+ ion bound to the amino N-terminal Cu2+/Ni2+ binding motif composed of three amino acid residues interacts with the indole ring of the fourth Trp residue in a noncovalent manner. The Cu2+-Trp interaction produces a distinct negative band at 223 nm in circular dichroism (CD), which disappears upon mutation or depletion of the Trp residue or upon replacement of the Cu2+ ion by Ni2+. In UV absorption, a pair of negative/positive intensity changes is generated at 222/231 nm by the Cu2+-Trp interaction, being consistent with the previous observations on the indole ring interacting with K+ or a cationic His imidazole ring. The negative CD band around 223 nm is characteristic of the Cu2+-Trp pair and may be useful as a marker of the Cu2+-Trp cation-pi interaction. Coordination of negatively charged ligands to Cu2+ is suggested to be important for the cation to be involved in a cation-pi interaction.

We developed 10 Gbps burst-mode clock recovery IC with synchronization time of 50 ps. Using the IC with PIN-TIA PDs, an optical packet receiver which requires no preamble is also developed.

We demonstrate IP-packet/10-Gbit-Ethernet transporting by novel 80-Gbit/s/port optical packet switch prototype with IP/OP converters, packet-transceiver of instantaneous- locking (≪1 ns), and multiple-label-processor of ultra-fast processing (/spl Gt;100 ps) to achieve 7.2- Gbps IP-packet throughput with low packet-loss-rate (

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H + –Cl − exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open–close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H + , Na + , K + , or H 3 O + in the intracellular region to elucidate the open–close mechanism, and to clarify the role of H + ion an H + –Cl − exchange transporter. Our simulations revealed that H + and Na + caused channel opening and the passage of Cl − ions. Na + induced a bead-like string of Cl − –Na + –Cl − –Na + –Cl − ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.

Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability.