A case of inflammatory fibroid polyp causing small-bowel intussusception in which retrograde double-balloon enteroscopy was useful for the preoperative diagnosis.

We showed a newly developed method, retrograde double-balloon enteroscopy, to be useful for preoperative diagnosis in a case of inflammatory fibroid polyp accompanied by small-bowel intussusception. A 64-year-old woman was admitted to our hospital with small-bowel intussusception. Results of radiographic and ultrasonographic examination were suggestive of a small-bowel mass. Retrograde double-balloon enteroscopy was performed in an attempt to make a preoperative diagnosis. Endoscopic observation, in combination with histological findings derived from endoscopic biopsy, was suggestive of an inflammatory fibroid polyp. The patient then underwent laparotomy with minimal incision, which revealed a polypoid mass leading to a jejunojejunal intussusception, without bowel necrosis, and a partial small-bowel resection was performed. The pathological diagnosis was an inflammatory fibroid polyp.

Role of T cells in development of chronic pancreatitis in male Wistar Bonn/Kobori rats: effects of tacrolimus.

We assessed T cell association with acinar cell apoptosis and a preventive effect of tacrolimus, a T cell suppressant, on the development of chronic pancreatitis in male Wistar Bonn/Kobori rats. At 15 wk, cellular infiltrates composed of F4/80-positive cells (monocytes/macrophages), CD4-positive cells, and CD8-positive cells were extensive in the interlobular connective tissue and parenchyma. In particular, CD8-positive cells invaded pancreatic lobules and formed close associations with acinar cells, some of which demonstrated features of apoptosis. At 20 wk, CD8-positive cells were still abundant in the fibrotic tissue formed with loss of acinar cells. Repeated subcutaneous injection of 0.1 mg x kg(-1) x day(-1) but not 0.025 mg x kg(-1) x day(-1) of tacrolimus for 10 wk completely prevented the occurrence of acinar cell apoptosis, infiltration of CD4- and CD8-positive cells, and development of pancreatitis at the age of 20 wk, but these maneuvers did not recover the decreased plasma corticosterone levels, which may be responsible for the development of disease. We demonstrated that T cells, possibly CD8-positive cells, are involved in inducing apoptosis of acinar cells, raising the possibility that tacrolimus might find clinical application in the treatment of autoimmune chronic pancreatitis.

Critical timing of mitochondrial K(ATP) channel opening for enhancement of myocardial tolerance against infarction.

OBJECTIVE: The present study was designed to assess the relationship between the timing of a mitoK(ATP) channel opener, diazoxide, and its infarct size-limiting effect. METHODS: In isolated rabbit hearts, infarction was induced by 30 min of global ischemia and 2 h of reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of the left ventricle (%IS/LV). Diazoxide, a mitoK(ATP) channel selective opener, and/or 5-hydroxydecanoate (5-HD), a mitoK(ATP) channel blocker, were infused before or after the onset of ischemia. When these agents were infused during the ischemic period, they were dissolved in a hypoxic buffer at concentrations 10-fold higher than those in the pre-ischemic period, and the infusion rate was set at 2% of the pre-ischemic coronary flow. RESULTS: In untreated controls, %IS/LV was 53.2+/-4.1 (SE). Pretreatment with diazoxide (100 microM) with a 10-min washout period reduced %IS/LV to 7.8+/-2.4 and this protection was abolished by co-infusion of 5-HD (50 microM). Pre-ischemic infusion of diazoxide without a washout period reduced %IS/LV to 7.3+/-1.4, and infusion of diazoxide from 10 min after the onset of ischemia also limited %IS/LV to 14.9+/-4.6. However, diazoxide infusion from 25 min after the onset of ischemia failed to reduce infarct size (%IS/LV = 54.5+/-7.2). Furthermore, pretreatment with 5-HD (50 microM) also completely abolished the protection afforded by early post-ischemic diazoxide infusion (%IS/LV = 48.3+/-6.5). Neither infusion of 5-HD nor the anoxic vehicle alone during ischemia modified %IS/LV. CONCLUSION: These findings suggest that opening of mitoK(ATP) channels before ischemia and during early ischemia, but not that upon reperfusion, is important for enhancement of myocardial tolerance against infarction.

Studies on intake of heavy metals by Bradybaena similaris, land snails, by XAFS measurement.

We have applied XAFS in order to determine both the chemical form and the place where heavy metals are stored in cultivated land snails. From Cu and Zn XANES spectra, the shells showed similar patterns as those of soft tissues and not like carbonates. This indicates that heavy metals are not completely taken into carbonate structures but are present within organic components in the shells. In addition, Cu XANES spectra of the samples showed low absorption edge-energy in the order of hepatopancreas, mantle, body, and shell. By comparing samples with standard reagents, each of which has only S- or O-ligand, it was found that the metals in hepatopancreas exist mostly as S-bound chemical components. To quantify the relative abundance of S-bound chemical component, partial least-squares (PLS) regression was applied. The PLS result indicated that for Cu, S-bound compound was higher in the order of hepatopancreas > mantle > body >shell.

Purification and characterization of the recombinant Thermus sp. strain T2 alpha-galactosidase expressed in Escherichia coli.

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable alpha-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514). The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h. The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

Antenatal three-dimensional sonographic features of Pierre-Robin syndrome. Case report.

A case of Pierre-Robin syndrome with polyhydramnios is described. Three-dimensional sonography clearly showed fetal micrognathia and hypoplastic ear antenatally. The benefits and advantages of the use of three-dimensional sonography to diagnose Pierre-Robin syndrome in utero are discussed.

Late second-trimester nonstress test characteristics in preterm delivery before 32 weeks of gestation.

OBJECTIVE: To clarify electronic fetal heart rate (FHR) monitoring characteristics in pregnancies with preterm delivery before 32 weeks of gestation, using the late second-trimester nonstress test. METHODS: Among 953 children born from 1993 to 1996, we identified 100 singleton infants born before 32 weeks of gestation in whom second-trimester (24-27 weeks of gestation) electronic fetal monitoring (EFM) records were obtained. Individual components of the FHR patterns [baseline rate, baseline FHR variability, presence of acceleration (at least 10 beats/min for at least 10 s) and periodic or episodic deceleration (at least 25 beats/min for at least 15 s)] and birth characteristics were compared between pregnancy with or without second-trimester decelerations. RESULTS: Among 100 infants, 65 had and 35 did not have second-trimester decelerations. There were no significant differences in gestational age at birth, birth weight, cord arterial blood pH, Apgar score and meconium staining between pregnancies with second-trimester decelerations and those without second-trimester decelerations. There were no significant differences in baseline rate and baseline variability between pregnancies with or without second-trimester decelerations. The number of accelerations in pregnancies with second-trimester decelerations was significantly more frequent than that in pregnancies without second-trimester decelerations (p < 0.001). There was a significant increase in the occurrence of premature rupture of the membranes (PROM; 60.0%) in pregnancies with second-trimester decelerations, when compared with events (37.1%) related to pregnancies without second-trimester decelerations (p < 0.05). There were no significant differences in the onset of breech presentation, cervical incompetency, preeclampsia and abnormal FHR pattern at birth between pregnancies with second-trimester decelerations and those without second-trimester decelerations. Pregnancies with PROM after second-trimester EFM were significantly more likely to have second-trimester decelerations than those without PROM (75.0 vs. 54.2%, p < 0.05). CONCLUSION: Periodic or episodic decelerations during late second-trimester EFM were associated with an increased risk of the occurrence of PROM in pregnancies with preterm delivery before 32 weeks of gestation.

Construction of the dual-functional RNA ligand against HCV NS3 protease and helicase.

Non-structural protein 3 (NS3) of hepatitis C virus (HCV) contains two distinct activities, protease and helicase which are essential for the HCV replication. In the previous study, we succeeded to obtain RNA aptamers, G9-I, G9-II and G9-III specific for the NS3 protease domain (delta NS3) by in vitro selection (1). As the result of mutational analysis in G9-I, we could obtain the minimum length of RNA structure, delta NEO-III maintaining the full inhibitional activity as shown in G9-I. Furthermore, we created a bi-functional novel RNA ligand, NEO-III-14U which was constructed by connecting delta NEO-III with (U)14 at the 3' terminal. NEO-III-14U was able to inhibit the unwinding of duplex DNA catalyzed by the Full-NS3 helicase activity as well as the protease activity in vitro. Consequently, we could obtain the dual-functional RNA ligand which could inhibit both NS3 protease and helicase activities essential for the HCV proliferation.

Three-dimensional sonographic assessment of fetal behavior in the early second trimester of pregnancy.

OBJECTIVE: To evaluate the fetal behavior pattern in the early second trimester of pregnancy by use of specially developed abdominal dynamic three-dimensional sonography. METHODS: Dynamic three-dimensional sonographic examinations were performed on 11 healthy pregnant women at 14 to 18 weeks of gestation. This imaging system provided continuous three-dimensional sonographic images every 1 to 2 seconds. Fetal movements were recorded continuously for 60 minutes in each fetus. The rate of occurrence of head, mouth, arm, trunk, and leg movements was evaluated. All fetal behavioral patterns were observed during the period studied. RESULTS: The active phase (time with fetal movements) was 59.4%, and the resting phase was 40.6%. The most active fetal behavior pattern was an arm movement, whereas the least was a mouth movement. Moreover, each fetal movement was synchronized and harmonized with other fetal movements (a few movement patterns were found to be generated simultaneously). CONCLUSIONS: Dynamic three-dimensional sonography provides a novel means for evaluation of fetal behavior in the early second trimester of pregnancy. These results suggest that dynamic three-dimensional sonography may be an important modality in future early fetal behavior research and in evaluation of early fetal well-being.

Recognition of tRNA by aminoacyl-tRNA synthetase from hyperthermophilic archaea, Aeropyrum pernix K1.

To study the recognition sites of tRNA for archaeal aminoacyl-tRNA synthetase, several aminoacyl-tRNA synthetase genes from hyperthermophilic archaeon, Aeropyrum pernix K1 were cloned and expressed. All the expressed enzymes showed extreme thermostability. Expressed threonyl-tRNA synthetase threonylated not only archaeal (A. pernix and Haloferax volcanii) threonine tRNAs but also Escherichia coli threonine tRNA. However, threonyl-tRNA synthetase from H. volcanii did not threonylate E. coli threonine tRNA.

Novel sugar-binding specificity of the type XIII xylan-binding domain of a family F/10 xylanase from Streptomyces olivaceoviridis E-86.

The type XIII xylan-binding domain (XBD) of a family F/10 xylanase (FXYN) from Streptomyces olivaceoviridis E-86 was found to be structurally similar to the ricin B chain which recognizes the non-reducing end of galactose and specifically binds to galactose containing sugars. The crystal structure of XBD [Fujimoto, Z. et al. (2000) J. Mol. Biol. 300, 575-585] indicated that the whole structure of XBD is very similar to the ricin B chain and the amino acids which form the galactose-binding sites are highly conserved between the XBD and the ricin B chain. However, our investigation of the binding abilities of wt FXYN and its truncated mutants towards xylan demonstrated that the XBD bound xylose-based polysaccharides. Moreover, it was found that the sugar-binding unit of the XBD was a trimer, which was demonstrated in a releasing assay using sugar ranging in size from xylose to xyloheptaose. These results indicated that the binding specificity of the XBD was different from those of the same family lectins such as the ricin B chain. Somewhat surprisingly, it was found that lactose could release the XBD from insoluble xylan to a level half of that observed for xylobiose, indicating that the XBD also possessed the same galactose recognition site as the ricin B chain. It appears that the sugar-binding pocket of the XBD has evolved from the ancient ricin super family lectins to bind additional sugar targets, resulting in the differences observed in the sugar-binding specificities between the lectin group (containing the ricin B chain) and the enzyme group.

Three-dimensional sonographic features of fetal central nervous system anomaly.

OBJECTIVE: To visualize an intracranial structure of the fetal central nervous system (CNS) anomaly using transabdominal three-dimensional (3D) sonography. METHODS: A total of 12 cases with fetal CNS anomalies (one unilateral ventriculomegaly; three hydrocephalus; three anencephaly; three holoprosencephaly; one Dandy-Walker cyst; and one enlarged cisterna magna) from 17 to 37 weeks of gestation were studied with transabdominal 3D sonography (3.5 MHz). RESULTS: In unilateral ventriculomegaly, insight view of dilated lateral ventricle, especially dilated atrium was depicted. In hydrocephalus, severely dilated bilateral ventricles and thin brain mantle were very clearly shown. In anencephalus, an absence of the brain and defect of the vault of the skull was clearly noted. In holoprosencephaly, absent interhemispheric fissure, common ventricle, and the extent of thalamic fusion were evident. In Dandy-Walker cyst, cerebellar hemisphere was clearly depicted due to the agenesis of cerebellar vermis. In enlarged cisterna magna, posterior intracranial view of the fetus showed a large space of cisterna magna. Although the diagnosis of each CNS anomaly was made using conventional two-dimensional sonography, 3D sonography proved most helpful delineating the exact nature and anatomic level of the anomaly. CONCLUSIONS: These results suggest that 3D sonography provides a novel means of visualizing fetal CNS anomalies in utero. However, it should be noted that our 3D sonography cannot depict intracranial brain structures in normal fetuses or some CNS anomaly such as intracranial tumor.

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain.

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.

Syntheses of 4-methylumbelliferyl-beta-D-xylobioside and 5-bromo-3-indolyl-beta-D-xylobioside for sensitive detection of xylanase activity on agar plates.

4-Methylumbelliferyl-beta-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-beta-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate.

Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86.

A beta-xylanase (GXYN) was purified from the culture filtrate of Streptomyces olivaceoviridis E-86 by successive chromatography on DE-52, CM-Sepharose and Superose 12. The molecular mass of the xylanase was estimated to be 23 kDa, indicating that the enzyme consists of a catalytic domain only. The enzyme displayed an optimum pH of 6, a temperature optimum of 60 degrees C, a pH stability range from 2 to 11 and thermal stability up to 40 degrees C. The N-terminal amino acid sequence of GXYN was A-T-V-I-T-T-N-Q-T-G-T-N-N-G-I-Y-Y-S-F-W-, and sharing a high degree of similarity with the N-terminal sequence of xylanases B and C from Streptomyces lividans, indicating GXYN belongs to family G/11 of glycoside hydrolases. GXYN was inferior to xylanase B from Streptomyces lividans in the hydrolysis of insoluble xylan because of its lack of a xylan binding domain.

Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901.

alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. alpha-L-Arabinofuranosidase I hydrolysed all of the alpha-linkage types that normally occur between two alpha-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: alpha-(1-->2) alpha-(1-->3) alpha-(1-->5). This enzyme cleaved the (1-->3) linkages of the arabinosyl side-chains of methyl 3, 5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside in preference to the (1-->5) linkages. alpha-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, alpha-L-Arabinofuranosidase II hydrolysed only (1-->5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1-->5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of alpha-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of alpha-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1, 5-alpha-L-arabinofuranosidase, it represents a novel type of enzyme.

Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi.

To facilitate an understanding of structure-function relationships, chimeric xylanases were constructed by module shuffling between the catalytic domains of the FXYN from Streptomyces olivaceoviridis E-86 and the Cex from Cellulomonas fimi. In the family F/10 xylanases, the modules M4 and M5 relate to substrate binding so that modules M4 and M5 of the FXYN were replaced with those of the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 and FCF-C4,5 were constructed. The k(cat) value of FCF-C5 for p-nitrophenyl-beta-D-cellobioside was similar to that of the FXYN (2.2 s(-1)); however, the k(cat) value of FCF-C4 for p-nitrophenyl-beta-D-cellobioside was significantly higher (7.0 s(-1)). The loss of the hydrogen bond between E46 and S22 or the presence of the I49W mutation would be expected to change the position of Q88, which plays a pivotal role in discriminating between glucose and xylose, resulting in the increased k(cat) value observed for FCF-C4 acting on p-nitrophenyl-beta-D-cellobioside since module M4 directly interacts with Q88. To investigate the synergistic effects of the different modules, module M10 of the FCF-C4 chimera was replaced with that of the Cex. The effects of replacement of module M4 and M10 were almost additive with regard to the K:(m) and k(cat) values.

Endothelium-dependent coronary response is impaired in the myocardium at an early phase of post-infarct remodeling.

The aim of the present study was to characterize endothelium-dependent and -independent coronary functions in remodeling hearts after infarction. First, echocardiography showed that the left ventricular diastolic dimension and thickness of the non-ischemic region were increased by 25% and 20%, respectively, at 2 weeks after coronary ligation in the rabbit heart. In the second series of experiments, 2 weeks after coronary ligation or a sham operation, the heart was isolated and perfused with modified Krebs-Henseleit buffer at 75 mmHg, and effluent from the pulmonary artery was measured as total coronary flow (CF). Regional CF analysis by microspheres indicated that flow to the infarcted region as a percentage of total CF is negligibly small. There was no significant difference between CF responses to sodium nitroprusside (10(-9)-10(-5) mole/l) in the sham-operated and remodeling hearts. However, the increase in CF after acetylcholine (ACh: 10(-8)-10(-5) mole/l) injection was significantly reduced by approximately 50% in the remodeling hearts compared to that in the sham-operated hearts. Furthermore, the percent increase in CF by ACh (10(-5) mole/l) was inversely correlated with weight of the remodeling myocardium (r = -0.630, p < 0.05). These results suggest that endothelium-dependent vasodilatory function is impaired in the myocardium at the early stage of post-infarct remodeling and that this endothelial dysfunction is closely related to the degree of hypertrophy of the remodeling myocardium.

Threonyl-tRNA synthetase of archaea: importance of the discriminator base in the aminoacylation of threonine tRNA.

To investigate the contribution of the discriminator base of archaeal tRNA(Thr) in aminoacylation by threonyl-tRNA synthetase (ThrRS), cross-species aminoacylation between Escherichia coli and Haloferax volcanii, halophilic archaea, was studied. It was found that E. coli ThrRS threonylated the H. volcanii tRNA(Thr) but that E. coli threonine tRNA was not aminoacylated by H. volcanii ThrRS. Results of a threonylation experiment using in vitro mutants of E. coli threonine tRNA showed that only the mutant tRNA(Thr) having U73 was threonylated by H. volcanii ThrRS. These findings indicate that the discriminator base U73 of H. volcanii tRNA(Thr) is a strong determinant for the recognition by ThrRS.