Biosynthesis of dillapiole/apiole in dill (Anethum graveolens): characterization of regioselective phenylpropene O-methyltransferase.

The phenylpropene volatiles dillapiole and apiole impart one of the characteristic aromas of dill (Anethum graveolens) weeds. However, very few studies have been conducted to investigate the chemical composition of volatile compounds from different developmental stages and plant parts of A. graveolens. In this study, we examined the distribution of volatile phenylpropenes, including dillapiole, in dill plants at various developmental stages. We observed that young dill seedlings accumulate high levels of dillapiole and apiole, whereas a negligible proportion was found in the flowering plants and dry seeds. Based on transcriptomics and co-expression approaches with phenylpropene biosynthesis genes, we identified dill cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase 1 (AgOMT1), an enzyme that can convert 6- and 2-hydroxymyristicin to dillapiole and apiole, respectively, via the methylation of the ortho-hydroxy group. The AgOMT1 protein shows an apparent Km value of 3.5 µm for 6-hydroxymyristicin and is 75% identical to the anise (Pimpinella anisum) O-methyltransferase (PaAIMT1) that can convert isoeugenol to methylisoeugenol via methylation of the hydroxy group at the para-position of the benzene ring. AgOMT1 showed a preference for 6-hydroxymyristicin, whereas PaAIMT1 displayed a large preference for isoeugenol. In vitro mutagenesis experiments demonstrated that substituting only a few residues can substantially affect the substrate specificity of these enzymes. Other plants belonging to the Apiaceae family contained homologous O-methyltransferase (OMT) proteins highly similar to AgOMT1, converting 6-hydroxymyristicin to dillapiole. Our results indicate that apiaceous phenylpropene OMTs with ortho-methylating activity evolved independently of phenylpropene OMTs of other plants and the enzymatic function of AgOMT1 and PaAIMT1 diverged recently.

An Ambidextrous Polyphenol Glycosyltransferase PaGT2 from Phytolacca americana.

The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.

C1 esterase inhibitor in pediatric cardiac surgery with cardiopulmonary bypass plays a vital role in activation of the complement system.

Our prospective study was therefore designed to determine which part of the systemic inflammatory response after cardiac operations resulted from Cardiopulmonary bypass (CPB) in neonates and infants. After approval by the human ethical committee of the Gunma Children's Medical Center (GCMC) and informed consent of the parents, 40 consecutive term congenital heart disease patients aged until 1 year who underwent long CPB time (> 3 h) at surgery were included in the prospective study between January 2012 and December 2014. C1 esterase inhibitor (C1-inh) drug (@Berinert) was generously provided by CSL Behring (King of Prussia, PA). The C1-inh (20 IU/kg) was given intravenously 60 min after CPB. Blood samples for complement factors were obtained before and 48 h after administration of C1-inh. Six patients did not survive and their data were not included. Of 34 patients included, median age was 6.5 months, median body weight was 6050 g, and 16 (47%) were female. According to the Mann-Whitney U test, there were no differences between the two groups concerning demographic and intraoperative data, postoperative chemical data. C1q concentration was only significant lower in patients with C1-inh non-treated group than in patients with C1-inh treated group. But, the consumption of C1q, C3, C4, CH50, and C1-inh in patients with C1-inhibitor non-treated group was observed early postoperatively. There is a significant difference in the values before and after C1-inh treatment between the two groups. The lower value in the C1-inh-treated group is explained by the activation of the classical pathway through the replenishment of complements by C1-inh treatment. This study proposes the administration of C1-inh is an effective therapy to reduce the activation and improve the clinical capillary leak syndrome.

Asanguineous Cardiopulmonary Bypass in Infants: Impact on Postoperative Mortality and Morbidity.

BACKGROUND: We routinely start cardiopulmonary bypass (CPB) for pediatric congenital heart surgery without homologous blood, due to circuit miniaturization, and blood-saving measures. Blood transfusion is applied if hemoglobin concentration falls under 8 g/dL, or it is postponed to after coming off bypass or after operation. How this strategy impacts on postoperative mortality and morbidity, in infants weighing ≤ 7 kg? METHODS: Six-hundred fifteen open-heart procedures performed from January 2014 to June 2018 were selected. One-hundred sixty-three patients (26.5%) were transfused on CPB (group 1), while 452 (73.5%) patients were not transfused on CPB (group 2). Operative risk and complexity were similar in both groups. Postoperative mortality and morbidity were compared. Multiple logistic regression was used to detect factors independently associated with outcome. RESULTS: Observed mortality in nontransfused group (0.7% = 3/452) was significantly lower than expected (4.2% = 19/452): p = 0.0007, and much lower than in transfused group (6.7% = 11/163): p < 0.0001. CPB transfusion (p = 0.001) was independently associated with mortality, either acting as the sole factor or in combination with the Society of Thoracic Surgeons morbidity score (p = 0.013). Patients not transfused during CPB required less frequently vasoactive inotropic drugs (p = 0.011) and duration of their mechanical ventilation was shorter (93 ± 134 hours) than for transfused patients (142 ± 170 hours): p = 0.0003. CPB transfusion was an independent determinant factor for morbidity (p = 0.05), together with body weight (p < 0.0001), vasoactive inotropic score (p < 0.0001), CPB duration (p = 0.001), and postoperative transfusion (p = 0.009). CONCLUSION: The strategy of transfusion-free CPB course, feasible in most patients ≤ 7kg, was associated with improved outcome. Asanguineous priming of CPB circuit should become standard, even in neonates and infants.

HmuS from Yersinia pseudotuberculosis is a non-canonical heme-degrading enzyme to acquire iron from heme.

Some Gram-negative pathogens import host heme into the cytoplasm and utilize it as an iron source for their survival. We report here that HmuS, encoded by the heme utilizing system (hmu) locus, cleaves the protoporphyrin ring to release iron from heme. A liquid chromatography/mass spectrometry analysis revealed that the degradation products of this reaction are two biliverdin isomers that result from transformation of a verdoheme intermediate. This oxidative heme degradation by HmuS required molecular oxygen and electrons supplied by either ascorbate or NADPH. Electrons could not be directly transferred from NADPH to heme; instead, ferredoxin-NADP+ reductase (FNR) functioned as a mediator. Although HmuS does not share amino acid sequence homology with heme oxygenase (HO), a well-known heme-degrading enzyme, absorption and resonance Raman spectral analyses suggest that the heme iron is coordinated with an axial histidine residue and a water molecule in both enzymes. The substitution of axial His196 or distal Arg102 with an alanine residue in HmuS almost completely eliminated heme-degradation activity, suggesting that Fe-His coordination and interaction of a distal residue with water molecules in the heme pocket are important for this activity.

Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene.

The stilbene compound resveratrol was glycosylated to give its 4'-O-ß-D-glucoside as the major product in addition to its 3-O-ß-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4'-O-ß-D-glucoside, and converted pinostilbene to its 4'-O-ß-D-glucoside as a major product and its 3-O-ß-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-ß-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-ß-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.

Experiences With Aggressive Cardiac Rehabilitation in Pediatric Patients Receiving Mechanical Circulatory Supports.

Although some patients with fulminant myocarditis can be rescued owing to the improvements in mechanical circulatory support therapy, there are few reports providing evidence of cardiac rehabilitation during mechanical circulatory supports, particularly among pediatric patients. We treated two pediatric patients who underwent aggressive cardiac rehabilitation during mechanical support. Five days after the initiation of extracorporeal membrane oxygenation therapy aggressive cardiac rehabilitation was started in a 10-year-old girl with fulminant myocarditis. After explantation of the device, she was discharged on postoperative day 23. A 6-year-old girl with fulminant myocarditis started receiving cardiac rehabilitation two days after the initiation of an extracorporeal left ventricular assist device, despite having hemiplegia due to a recent broad stroke. She achieved an exercise capacity of supported walking for 280 meters after 127 days of cardiac rehabilitation and then went abroad to undergo heart transplantation when she was in the best physical condition possible. Early initiation of cardiac rehabilitation may be safe and effective for successful pediatric mechanical circulatory support therapy; this acts as a bridge to explantation or heart transplantation.

Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities.

Resveratrol was glucosylated to its 3- and 4'-ß-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4'-ß-glucoside. P. americana cells converted piceatannol into its 4'-ß-glucoside. The 3- and 4'-ß-glucosides of resveratrol were further glucosylated to 3- and 4'-ß-maltosides of resveratrol, 4'-ß-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-ß-glucoside and 3-ß-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4'-ß-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4'-ß-glucoside showed high phosphodiesterase inhibitory activity.

Comprehensive evaluation of fibrin glue as a local drug-delivery system-efficacy and safety of sustained release of vancomycin by fibrin glue against local methicillin-resistant Staphylococcus aureus infection.

Infection of prosthetic grafts, especially with methicillin-resistant Staphylococcus aureus (MRSA), often becomes critical. Although topical administration of antibiotics has been applied empirically, no comprehensive data are available showing long-lasting effects and safety of local antibiotic usage. By means of animal experiments we assessed fibrin glue (FG) as a slow-release vehicle for vancomycin (VCM) against local MRSA infection. Preliminary in-vitro experiments were performed to confirm that the FG-VCM mixture maintained viscosity as a sealant and led to slow-release of VCM. We next created a subcutaneous pocket in a rodent back and implanted a 1 cm (2) woven graft with 1 ml FG alone, or with serial concentrations of VCM (0-120 mg/ml. n = 3 for each group). MRSA of 1 × 10(7) colony-forming units (CFU) was injected into the pocket after wound closure. The graft was explanted 7 days later and was submitted for culture ("Culture-graft"). Blood samples were obtained for regular blood work, serum VCM concentration measurements, and blood culture ("Culture-blood"). The pocket tissue was also submitted for measurement of local VCM concentration. There was a remarkable infectious response in the group without vancomycin; however, no other groups developed any sign of infection. "Culture-graft" resulted in MRSA growth for V0 only. "Culture-blood" was negative in all groups, and only minimal serum concentrations of vancomycin were detected. One-dose topical administration of VCM via FG was effective against localized MRSA graft infection without systemic VCM administration. Topical administration of antibiotics may help treat difficult graft infections and reduce systemic use of potent antibiotics.

Inhibition of heme uptake in Pseudomonas aeruginosa by its hemophore (HasA(p)) bound to synthetic metal complexes.

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana.

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-ß-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4'-O-ß-glucoside: 3-O-ß-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

Modulation of cystathionine beta-synthase activity by the Arg-51 and Arg-224 mutations.

Human cystathionine beta-synthase (CBS) catalyzes a pyridoxal 5'-phosphate (PLP) dependent beta-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 A away from heme. A possible explanation of our results is discussed on the basis of CBS structure.

HBV- and HCV- infected workers in the Japanese workplace.

Around three million Japanese are persistently infected with HBV or HCV. Though most of them work in various industries, little is known about the actual conditions in their workplaces. To clarify the workplace conditions of workers with hepatitis, three kinds of questionnaire surveys, answered by occupational health physicians and workers with hepatitis, were carried out. The rates of workers recognized as workers with hepatitis B or C by occupational health physicians were 0.82% and 0.48% of 130,092 workers, respectively. About 30% of workers with hepatitis were engaged in "hazardous work". The percentage of workers engaged in various types of hazardous work among workers with hepatitis was nearly the same as that among all Japanese workers. About 30% of occupational health physicians witnessed exacerbation of hepatitis in the workers at their workplaces, and 22% of workers with hepatitis experienced exacerbation of hepatitis. The rate of workers with hepatitis who had experienced exacerbation was not significantly different between workers with and without hazardous work. Workers with hepatitis have strong concerns about the relationship between work and exacerbation. As causes of exacerbation, occupational health physicians cited "unknown", "drinking" and "quit treatment" while workers with hepatitis answered "work-related causes", besides "unknown" and "drinking."

Smoking cessation program and CYP2A6 polymorphism.

The relationship between CYP2A6 genotype and smoking status remains unclear although several studies have been reported. In this study, we have investigated the significance of CYP2A6 genotype on smoking habit and treatment of nicotine patch. Sixty-one smokers (1.7%) working in a Japanese company (n = 3585) participated in this smoking cessation program. Forty-four of 61 (72.1%) smokers were treated by nicotine patch. A genotyping analysis was carried out for 41 (40 men and 1 women) of 61 participants (67.2%). The smoking cessation rate at 90 days was 54.1% (33/61). Age and smoking years in re-smoking group are significantly lower than those in smoking cessation group. The smoking cessation rate of participants treated with nicotine patch (63.6%; 28/44) was significantly higher than that of the group non-treated with nicotine patch (29.4%; 5/17) at 90 days (p < 0.05). The incidence of homozygotes of CYP2A6 gene deletion (CYP2A619934/19934) in 41 cases (9.8%; 4/41) could be higher than that in 894 healthy controls (3.7%; 33/894) (p = 0.12), while no other variant alleles (CYP2A619932, CYP2A619933 and CYP2A619936) were found. Age and smoking years of participants with CYP2A619934/19934 are significantly higher than those with CYP2A619931 positive. The scores of Fagerstrom test, an analysis for nicotine-dependence, were slightly different between participants with CYP2A619934/19934 and CYP2A619931 positive. Although treatment of nicotine patch is efficacious to smoking cessation, cases with CYP2A619934/19934 might be more sensitive to nicotine adverse effects and more difficult to quit smoking once they have smoking habit.

The crystal structure of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt): Roles of the axial ligand Tyr75 and two distal arginines in heme binding.

Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule. The heme-bound water molecule makes extensive hydrogen bond network that includes Arg40 and Arg144 on the distal heme pocket. Arg40, highly conserved for HasAs from Yersinia species, forms a salt bridge with the propionate side chain of heme, and makes π-π stacking and hydrophobic interactions with porphyrin plane. Interestingly, similar Arg-heme interactions are also found for periplasmic heme transporter, PhuT, suggesting that this is an example of a convergent evolution and one of the important interactions for bacterial heme transportation. Heme titration, heme binding kinetics, and the crystal structures of wild-type and Y75A proteins show that, although Tyr75 is primarily important for heme capturing, other interactions with Arg40, Arg144, and hydrophobic residues also contribute for heme acquisition. We also found that HasAypt can form a dimer in solution. The structure of the domain-swapped Y75A HasAypt dimer shows the presence of two low-spin heme molecules coordinated with His84 and His140, and displacement of the Arg40 loop of dimeric Y75A HasAypt results in deformation of the heme-binding pocket. A similar rearrangement of the distal heme loop might occur in heme transfer from HasAypt to HasRypt.

Enzymatic Synthesis of Quercetin Monoglucopyranoside and Maltooligosaccharides.

Quercetin 3-O-ß-monoglucopyranoside and quercetin 3-O-ß-maltooligosaccharide were synthesized from quercetin using glucosyltransferase-3 from Phytolacca americana and cyclodextrin glucanotransferase.

Expression of cytochrome P450 in tumor tissues and its association with cancer development.

CYPs (cytochrome P450s) catalyze the conversion of numerous numbers of xenobiotics including carcinogens and drugs. CYPs can be involved in metabolic pathways of activation of procarcinogens and/or inactivation of carcinogens during the tumorigenic processes. Recently, increasing number of cancer tissues as well as normal tissues have been found to express a variety of CYPs. The local expression of CYPs in tumors appears to be very important for the management of cancers since CYPs expressed in tumors may be involved in activation and/or inactivation of anticancer drugs. The expression of CYPs in tumors may also convert endogenous substrates to metabolites that facilitate cancer development. In this review, we summarize the association of CYP expression in cancer tissues with carcinogenesis and cancer treatment.

Evidence-based prevention (EBP): approach to lung cancer prevention based on cytochrome 1A1 and cytochrome 2E1 polymorphism.

Now that the human genome has been sequenced, we consider that combinations of 3 to 10 million polymorphic loci scattered throughout the genome contribute to individual differences. Genetic polymorphism analysis is useful for the made-to-order medical treatment of patients and the prevention of disease onset in normal healthy individuals. Individually-tailored disease prevention based on findings such as genetic polymorphism analysis results can be grasped by the concept of evidence-based prevention (EBP). In this paper we outline cytochrome P450 (CYP) 1A1 and 2E1 polymorphism-related differences in metabolic activation of carcinogens in relation to the risk of lung cancer, and explain single nucleotide polymorphism (SNP). We also compare the reports to date on SNP types in CYP1A1 and CYP2E1 in relation to the risk of lung cancer with our study results, examine survival rates of lung cancer patients by CYP1A1 or CYP2E1 genotype and discuss the applicability of SNP research to EBP.

A case-case study comparing the usefulness of serum trace elements (Cu, Zn and Se) and tumor markers (CEA, SCC and SLX) in non-small cell lung cancer patients.

UNLABELLED: Serum copper (Cu), zinc (Zn) the Cu/Zn ratio (Cu/Zn) and selenium (Se) were evaluated in 84 patients with non-small cell lung cancer (NSCLC) before surgery. Serum carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC) and sialyl Lewis X-i antigen (SLX) levels were also determined in the same cases. The cut-off values of Cu, Zn and Se were established to create two categories with equal numbers of patients. We investigated the clinical and prognostic usefulness of assays of serum trace elements (Cu, Zn, Cu/Zn and Se) and compared levels of serum trace elements and serum tumor markers (CEA, SCC and SLX) in NSCLC patients. Furthermore, we evaluated the usefulness of serum trace elements, when compared with tumor markers, in assessing prognosis for NSCLC. IN CONCLUSION: (1) a preoperative increase in Cu/Zn level predicted tumor progression more effectively than changes in Cu or Zn levels; (2) Se levels seemed to vary with age, but there was no relationship between Se level and disease stage; (3) the measurement of Cu/Zn was useful for assessing both prognosis and extent of the disease in NSCLC patients, similar to the measurement of serum tumor markers such as CEA; and (4) the measurement of the Cu/Zn had prognostic significance, but was inferior to disease stage in predicting outcome. Cu and Zn in serum are storable and the determination of Cu/Zn level is so simple and inexpensive that it can be helpful in determining clinical stages and predicting the prognoses of NSCLC patients.

Pleomorphic carcinoma: report of a case with massive pleural effusion and asbestos particles.

Pleomorphic (spindle/giant cell) carcinoma (PC) is one subset of large cell carcinoma. It is well known that PC patients have a poor survival rate. This report describes a 68-year-old man with PC. The patient's tumor had a massive pleural effusion. A left lower lobectomy and partial resection of the left diaphragm, peritoneum, and parietal pleura were performed to remove the tumor. Numerous asbestos particles were found in the left lower lobe. This is the first reported case of PC which may have been caused by asbestos particles. Further investigation is needed into whether asbestos exposure causes PC.