bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Essential Lysyl Residue (Lys208) in the Substrate‐Binding Site of Porcine FAD‐Containing Monooxygenase
Authors: Wu Ru‐Feng , Ichikawa Yoshiyuki 
Published: July 2010

The substrate (amine)‐binding site of porcine FAD‐containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5′‐phosphate (pyridoxal‐P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal‐P. Upon reduction of pyridoxal‐P‐treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal‐Pappeared at 325 nm. The amino acid residue compositions of the native and pyridoxal‐P‐treated FMOs indicated that the lysyl residues were modified by pyridoxal‐P. The about 74% inactivation of the enzymic activity on covalent pyridoxal‐Ptreatment of the FMO was nearly completely prevented in the presence of the substrate,N,N‐dimethylaniline. The FMO covalently modified with pyridoxal‐Pin the presence or absence ofN,N‐dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse‐phase high‐performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal‐Pand was detected at 325 nm in the absence ofN,N‐dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186–208.These results suggest that Lys208 plays an important role in the substrate (amine)‐binding site of FMO.
Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor
Authors: Takagi Junichi , Aoyama Toshihiro , Ueki Shoko , Ohba Hirotsugu , Saito Yuji , Lorand Laszlo 
Published: July 2010

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100‐kDa propeptide, called the propolypeptide of von Willebrand factor (pp‐vWF), is generated during biosynthesis. We found that pp‐vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311–123161. As such, it could form cross‐linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp‐vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861–867].In this work, we identified the Gln residues in pp‐vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme‐reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti‐dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp‐vWF, only four could be modified in the factor‐XIIIa‐catalyzed reaction.
Mutational Analysis of Mau Genes Involved in Methylamine Metabolism in Paracoccus Denitrificans
Authors: Van Der Palen Carol J. N. M. , Slotboom Dirk‐Jan , Jongejan Laurian , Reijnders Willem N. M. , Harms Nellie , Duine Johannis A. , Van Spanning Rob J. M. 
Published: July 2010

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans.MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme‐binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin‐like proteins and contain four and two motifs, respectively, characteristic for [4Fe‐4S] clusters.Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye‐linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the β subunit of MADH, but the α subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the α and β subunits of MADH as well as amicyanin. However, no dye‐linked MADH activity was found in this mutant strain. In addition, as the wild‐type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the β subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH.The mau gene cluster mauRFBEDACJGMN was cloned on the broad‐host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.
3‐Deoxy‐Octulosonic‐Acid‐Containing Hexasaccharide Fragment of Unusual Core Type Isolated from Hafnia alvei 2 Lipopolysaccharide
Authors: Ravenscroft Neil , Gamian Andrzej , Romanowska Elżbieta 
Published: July 2010

The hexasaccharide containing 3‐deoxy‐octulosonic acid (Kdo) was isolated from the carbohydrate material obtained after the mild acid hydrolysis of lipopolysaccharide of Hafnia alvei strain 2. The hexasaccharide was purified by gel filtration on Bio‐Gel P‐4 and P‐2 columns. On the basis of sugar and methylation analyses, one‐ and two‐dimensional NMR spectroscopic methods, the hexasaccharide was identified as
image
The tetrasaccharide linked to position 7 of Kdo is also part of the sialic‐acid‐containing O‐specific unit. Di‐substitution of Kdo at positions 7 and 8 has not been previously reported.
Studies on a Stabilisation of Ubisemiquinone by Escherichia coli Quinol Oxidase, Cytochrome bo
Authors: Ingledew W. John , Ohnishi Tomoko , Salerno John C. 
Published: July 2010

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome‐c ‐binding site and its attendant CuA prosthetic group with a quinone‐binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one‐electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5–10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two‐electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two‐electron couple is –60 mV/pH, indicative of a 2H+/2e‐ reaction.The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q˙‐ species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q˙‐ radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q˙‐bo (QH˙bo).
Binding of Thyroxine to Pig Transthyretin, its cDNA Structure, and Other Properties
Authors: Duan Wei , Richardson Samantha J. , Köhrle Josef , Chang Linus , Southwell Bridget R. , Harms Paul J. , Brack Charlotte M. , Pettersson Tom M. , Schreiber Gerhard 
Published: July 2010

Thyroxine binding to proteins in pig plasma during electrophoresis was observed in the albumin, but not in the prealbumin and post‐albumin regions.Transthyretin could be identified in medium from in vitro pig choroid plexus incubations by size and number of subunits and a very high rate of synthesis and secretion. Its electrophoretic mobility was intermediate between that of thyroxine‐binding globulin and albumin. It bound thyroxine, retinol‐binding protein, anti‐(rat transthyretin) antibodies and behaved similarly to transthyretins from other vertebrate species when plasma was extracted with phenol. Inhibition experiments with the synthetic flavonoid F 21388, analysing the binding of thyroxine, suggested that transthyretin is not a major thyroxine carrier in the bloodstream of pigs.Cloning and sequencing of transthyretin cDNA from both choroid plexus and liver showed that the same transthyretin mRNA is expressed in pig choroid plexus and liver. The amino acid sequence derived from the nucleotide sequence revealed that pig transthyretin differs from the transthyretins of all other studied vertebrate species by an unusual C‐terminal extension consisting of the amino acids glycine, alanine and leucine. This extension results from the mutation of a stop codon into a codon for glycine. The unusual C‐terminal extensions do not seem to interfere with the access of thyroxine to its binding site in the central channel of transthyretin.
Transitions of gene expression induced by short‐term blue light
Authors: Lehmann P. , Nöthen J. , Schmidt von Braun S. , Bohnsack M. T. , Mirus O. , Schleiff E. 
Published: July 2010

AbstractBlue light modulates many processes in plants and plant cells. It influences global and long‐term responses, such as seedling development and phototropism, and induces short‐term adaptations like stomatal opening and chloroplast movement. Three genes were identified as important for the latter process, namely PHOT1, PHOT2 and CHUP1. The former two phototropin blue light receptors act in perception of the blue light signal. The protein CHUP1 is localised to the outer envelope membrane of chloroplasts and is involved in chloroplast movement. To explore whether short‐term reactions required for chloroplast movement are under transcriptional control, we analysed the transcriptome in wild‐type Arabidopsis thaliana, phot1, phot2 and chup1 with different blue light treatments for 5 or 30 min. Blue light‐induced changes in transcription depended on illumination time and intensity. Illumination with 100 μmol·m−2·s−1 blue light induced down‐regulation of several genes and might point to cascades that could be important for sensing low levels of blue light. Analysis of the transcriptome of the mutants in response to the different light regimes suggests that the transcriptional response to blue light in the wild‐type can be attributed to phot1 rather than phot2, suggesting that blue light‐induced alteration of expression is a function of phot1. In contrast, the blue light response at the transcriptional level of chup1 plants was unique, and confirmed the higher light sensitivity of this mutant.
OCT4 Expression in Outgrowth Colonies Derived from Porcine Inner Cell Masses and Epiblasts
Authors: Wolf XA , Rasmussen MA , Schauser K , Jensen AT , Schmidt M , Hyttel P 
Published: July 2010

ContentsThe present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM‐ and epiblast‐derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)‐like morphology. A total of 104 zona pellucida‐enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC‐like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC‐like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC‐like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT‐PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC‐like morphology. In conclusion, we have established a robust system for derivation of ESC‐like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC‐like morphology although this relationship is lost during early passages.
Effect of mSOF and G1.1/G2.2 Media on the Developmental Competence of SCNT‐Derived Bovine Embryos
Authors: Wang YS , Tang S , An ZX , Li WZ , Liu J , Quan FS , Hua S , Zhang Y 
Published: July 2010

ContentsThe objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.
CLINICAL CHARACTERISTICS AND TREATMENT FOR PATIENTS PRESENTING WITH BLEEDING DUODENAL VARICES
Authors: Kakizaki Satoru , Toyoda Mitsuo , Ichikawa Takeshi , Sato Ken , Takagi Hitoshi , Arai Hirotaka , Sohara Naondo , Iizuka Haruhisa , Onozato Yasuhiro , Mori Masatomo 
Published: July 2010

Background and Aim:  Bleeding from ectopic varices, including duodenal varices, is uncommon, but it can be difficult to manage. The clinical data of patients diagnosed and treated for duodenal varices were reviewed to investigate the strategy for treatment.Methods:  The present study reviewed the clinical data of 10 patients with duodenal varices (mean age, 58.2 ± 15.6 years) at our associated institutes during the period between January 1996 and December 2008.Results:  Nine patients had duodenal varices located in the second portion, whereas in one case they were located in the duodenal bulbus. The underlying diseases included liver cirrhosis in eight patients, and extrahepatic portal vein obstruction in two patients. The lesions were identified with bleeding from varices in eight of 10 patients. Initial hemostasis was achieved in all eight patients. However, among four patients treated endoscopically only, two patients died from rebleeding from varices and two died from hepatic failure resulting from variceal bleeding. Additional interventional radiology (IVR) was used in three patients and additional surgery was carried out in one case. One patient who was treated with balloon‐occluded retrograde transvenous obliteration rebled during IVR and died from bleeding. Two patients who underwent double balloon‐occluded embolotherapy and one case who had surgery achieved good clinical outcomes.Conclusions:  Although endoscopic treatment is useful for initial hemostasis of hemorrhagic duodenal varices, the patients who underwent additional IVR after endoscopic treatment achieved good outcomes.
STRICTURE AFTER ENDOSCOPIC SUBMUCOSAL DISSECTION FOR EARLY GASTRIC CANCERS AND ADENOMAS
Authors: Iizuka Haruhisa , Kakizaki Satoru , Sohara Naondo , Onozato Yasuhiro , Ishihara Hiroshi , Okamura Shinichi , Itoh Hideaki , Mori Masatomo 
Published: July 2010

Background and Aim:  Stricture is a complication that may occur after endoscopic submucosal dissection (ESD) of gastric neoplasms. The goal of the present study was to investigate the incidence, risk factors and management of gastric stricture after ESD.Methods:  The medical records of 308 patients who underwent ESD for gastric neoplasms were reviewed. Stricture is defined as having symptoms caused by an obstruction through which a 1‐cm diameter endoscopic fiber cannot be passed.Results:  Stricture was identified in six of 308 patients (1.9%). Three of the six lesions were located in the prepylorus, two cases in the antrum and one in the cardia. The mean longitudinal distance and the mean area of the resected specimens in the six cases with stricture (7.8 ± 2.0 cm, 34.0 ± 15.8 cm2, respectively) was significantly larger than in those without stricture (4.5 ± 1.4 cm, 12.7 ± 8.3 cm2, respectively, P < 0.01). The ratio of the resected circumference/whole circumference was 83.3 ± 7.5% in those with stricture in comparison to 25.4 ± 16.3% in those without stricture (P < 0.01). All six patients underwent endoscopic balloon dilations, and obtained relief from stricture. However, one patient experienced a gastric perforation and recovered following conservative therapy.Conclusion:  Sub‐circumferential resection over 75% of the circumference by ESD in the prepylorus, antrum and cardia is a risk factor for the occurrence of stricture. Early intervention might be considered for this high‐risk group to avoid a perforation during balloon dilation.
New attempt using ablative curettage technique for managing benign premenopausal uterine bleeding
Authors: Thabet Saeed Mohamed Ahmad 
Published: July 2010

AbstractAim:  To assess the efficacy of a newly suggested ablative curettage technique in managing premenopausal dysfunctional uterine bleeding.Methods:  A prospective controlled operative study and clinical trial was carried out at the Department of Obstetrics and Gynecology, Kasr El‐Aini Hospital, Cairo University, Egypt. One hundred cases of premenopausal bleeding were divided into 50 controls and 50 studied cases. Routine diagnostic and follow‐up techniques for managing premenopausal bleeding were carried out. Two operative techniques were compared: overcurettage, using Sims sharp curette, and the newly suggested dilatation and ablative curettage, using a Thabet double‐bladed sharp curette. Histopathology of curetted material and the endomyometrium after ablation in the failed cases that needed hysterectomy was also included.Results:  Successful endometrial ablation and control of the premenopausal bleeding were recorded in 66% of overcurettage cases and in 90% of cases treated with ablative curettage. The latter had significantly fewer operative and postoperative complications. These advantages were also significantly reduced in both techniques compared with other ablative techniques and hysterectomy. Complete ablation could be confirmed histopathologically in the ablated endometrium and in the endomyometrium of the hysterectomy specimens.Conclusion:  Ablative curettage is an efficient technique in managing premenopausal dysfunctional uterine bleeding, especially in developing countries where there are limited financial resources.
Group A streptococcal toxic shock syndrome with extremely aggressive course in the third trimester
Authors: Sugiyama Takashi , Kobayashi Takumi , Nagao Kenji , Hatada Tsuyoshi , Wada Hideo , Sagawa Norimasa 
Published: July 2010

AbstractGroup‐A‐streptococcus‐(GAS)‐induced toxic shock syndrome (TSS) is uncommon, but carries a high risk of maternal mortality during pregnancy. The onset of gravidic GAS‐TSS has been reported mostly during the puerperium.A 16‐year‐old woman, who was at 37 weeks of gestation, and without obstetrical care during the last 30 weeks, was referred to our hospital. She complained of fever for one day with headache and abdominal pain after the fever developed. On admission, her consciousness was drowsy, intrauterine fetal death was recognized, and she rapidly developed shock status with coma and hypotension, hemolysis, disseminated intravascular coagulation (DIC), and multi‐organ failure. Although we had not obtained the results of a bacterial culture, we suspected sepsis with DIC with homolysis and multi‐organ failure resulting from an infection. The patient was treated with antibiotics and intubation because of respiratory insufficiency. Twelve hours after admission to the intensive care unit in our hospital, she died. Cultures from blood, subcutaneous tissue, vaginal discharge, and pharynx all revealed GAS bacteria, and therefore she was diagnosed as having GAS‐TSS.GAS‐TSS in pregnancy is rare. However, once the infection occurs in a pregnant woman, it rapidly develops into sepsis with multi‐organ failure. Therefore, early recognition and intensive treatment for GAS during pregnancy is recommended in women with high fever, muscular pain, hemolysis and DIC during pregnancy.