A common allosteric mechanism regulates homeostatic inactivation of auxin and gibberellin.

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

Efficacy of non-rigid registration technique for misregistration in 3D-CTA fusion imaging.

PURPOSE: To assess whether fusion 3D-CTA images can be corrected using non-rigid registration (NRR) for gastroenterology imaging. METHODS: This study included 55 patients before gastroenterology surgery who underwent preoperative 3D-CTA prior to gastroenterological surgery. We recorded the coordinate of measurement points on the arterial vessels (X, Y, and Z) in each portal phase, original image of the arterial phase, and arterial phase with NRR. The distance of misregistration between the two points was calculated with the coordinate of the original image with NRR and that of the portal phase as true value. RESULTS: The distance of misregistration between the two points in the original arterial and portal phase images was significantly higher than that in the arterial phase image with NRR on all directions (p < 0.01). CONCLUSIONS: This study showed that NRR may correct misregistration on fusion 3D-CTA imaging. Hence, it can visualize correctly the anatomy of the vessel.

Prediction of difficult laparoscopic cholecystectomy for acute cholecystitis.

BACKGROUND: No report has described the predictive factor of surgical difficulty for laparoscopic cholecystectomy (LC) by preoperative computed tomography (CT) findings. This study aimed to investigate whether dynamic CT findings can predict the difficulty of LC for acute cholecystitis. MATERIALS AND METHODS: Fifty-seven patients who underwent emergency LC and dynamic CT preoperatively were enrolled. Difficult LC (DLC) was defined as any patient with an operative time ≥3 h, bleeding volume ≥300 mL, common bile duct injury, partial cholecystectomy, the need for a second surgeon, and/or conversion to open surgery. Patients were assigned to either the DLC (+) or DLC (-) group. We determined the CT attenuation ratio of the arterial phase (ARAP) to represent the degree of transient focal enhancement of the liver adjacent to the gallbladder. The ARAP cutoff value for a DLC predictor was determined using receiver operating characteristic curve analysis. Patients' characteristics and CT findings, including the ARAP, were compared between the groups. The Fisher exact test for categorical variables and the Mann-Whitney U test for continuous variables with Bonferroni correction were used to evaluate the significance of differences. RESULTS: Fifteen patients were assigned to the DLC (+) group. The ARAP was significantly higher in the DLC (+) group than in the DLC (-) group (P = 0.006). The ARAP cutoff value was 1.55. Regarding the CT findings, an ARAP ≥1.55 (P = 0.005) was significantly correlated with DLC. CONCLUSIONS: Among dynamic CT findings, an increased ARAP is a predictive factor for DLC.

Use of dynamic CT attenuation value for diagnosis of acute gangrenous cholecystitis.

OBJECTIVE: To determine if the increase in transient focal enhancement of the liver adjacent to the gallbladder seen on dynamic computed tomography (CT) is greater in gangrenous cholecystitis than in nongangrenous cholecystitis by determining the CT attenuation value. METHODS: We retrospectively reviewed the medical records of 57 patients who underwent emergency cholecystectomy for acute cholecystitis and preoperative dynamic CT scans between March 2011 and April 2016. Based on the pathology findings, patients were assigned to a gangrenous group or a nongangrenous group. The CT attenuation ratio (AR) was defined as the ratio of the maximum CT attenuation value in the region of interest in segment 5 (liver parenchyma adjacent to the gallbladder) and that in the control region of interest in segment 8. The patient characteristics and CT findings were compared between the groups. The appropriate AR of the arterial phase (ARAP) cutoff value for a diagnosis of gangrenous cholecystitis was determined using receiver operating characteristic curve analysis. RESULTS: The ARAP was significantly higher in the gangrenous group than in the nongangrenous group (P < .001); the area under the receiver operating characteristic curve was 0.849. The ARAP cutoff value of 1.46 had a sensitivity and specificity of 84.6% and 83.9%, respectively (odds ratio, 21.17; 95% confidence interval, 4.90-118.91), and ARAP ≥1.46 was significantly correlated with a diagnosis of gangrenous cholecystitis (P < .001). CONCLUSIONS: The increase in transient focal enhancement of the liver adjacent to the gallbladder during the arterial phase of dynamic CT was greater in gangrenous cholecystitis than in nongangrenous cholecystitis.

Effect of liquid whey feeding on fecal microbiota of mature and growing pigs.

The effect of liquid whey feeding on fecal bacteria and their metabolites was assessed in five pregnant sows and 66 growing pigs. Sows were fed a control diet for 4 weeks (control period) followed by the same diet but with whey feeding (5 L/day/pig) for 4 weeks (whey period). One group of growing pigs was given 267 L of whey per pig (whey group), while the other group was not (control group). In both cases, liquid whey was given separately from control diet. Sows in the whey period had feces showing lower pH, lower ammonia concentration, and larger population sizes of total bacteria, lactobacilli and bifidobacteria. The bacterial gene library analysis indicated that Mitsuokella and Megasphaera were more frequently detected, while Clostridium disporicum were detected less frequently in the whey period. Feces from whey-fed growing pigs showed lower pH than that from control pigs in the early stage of growing. Also, larger populations of total bacteria, lactobacilli and bifidobacteria were recorded in the whey group. From the bacterial gene library analysis, the detection frequency of Lactobacillus reuteri tended to be higher in the whey group. These results indicate that whey feeding influences the hindgut microbiota of pigs, possibly leading to a fermentation shift that is favorable for animal health.

A precise and useful method for coordinating computed tomography and jaw movement frames of reference.

PURPOSE: The aim of this study was to develop a means of coordinating helical computed tomography (CT)- based morphological data in 3 dimensions (3-D) with that pertaining to jaw movement as recorded by a device that measures jaw movement in six-degrees-of-freedom (6-DOF), thus producing multi-point movement analysis of the condyle. METHODS: The study sample was two volunteers. One of the subjects had erosive bony changes in both condyles, while the other had healthy condyles. We employed a customized facebow, which enabled us to coordinate jaw movement data and morphological volume data from CT. Total uncertainty of the coordination was computed, according to International Organization for Standardization (ISO). In order to demonstrate the effects of multi-point analysis for complex condylar movement, we tried to visualize the trajectory of the working condyle in lateral excursion. RESULTS: The overall uncertainty at a condylar center chosen as an example to illustrate the method was 0.38 mm, 0.19 mm, and 0.50 mm in antero-posterior, lateromedial, and supero-inferior directions, respectively, in terms of 95% coverage as defined by the ISO. CONCLUSION: We developed facebow-based X-ray markers with high clinical operability, which could correlate the helical CT's coordinate system with our 6-DOF jaw movement measuring system for precise analysis of 3-D condylar movements. In motion analysis of rotational condyle, even a small amount of measurement error cannot necessarily be neglected. Then, a multi-point approach such as that realized by our system presents the best option.

Investigation of substrate binding and product stereochemistry issues in two linoleate 9-lipoxygenases.

Herein we characterize the Arabidopsis thaliana AtLOX1 and tomato (Solanum lycopersicum) LOXA proteins as linoleate 9S-lipoxygenases (9-LOX), and use the enzymes to test a model that predicts a relationship between substrate binding orientation and product stereochemistry. The cDNAs were heterologously expressed in E. coli and the proteins partially purified by nickel affinity chromatography using a N-terminal (His)(6)-tag. Both enzymes oxygenated linoleic acid almost exclusively to the 9S-hydroperoxide with turnover numbers of 300-400/s. AtLOX1 showed a broad range of activity over the range pH 5-9 (optimal at pH 6); tomato LOXA also showed optimal activity around pH 5-7 dropping off more sharply at pH 9. Site-directed mutagenesis of a conserved active site Ala (Ala562 in AtLOX1, Ala 564 in tomato LOXA, and typically conserved as Ala in S-LOX and Gly in R-LOX), revealed that substitution with Gly led to the production of a mixture of 9S- and 13R-hydroperoxyoctadecadienoic acids from linoleic acid. To follow up on earlier reports of 9-LOX metabolism of anandamide (van Zadelhoff et al. Biochem. Biophys. Res. Commun. 248:33-38, 1998), we also tested this substrate with the mutants, which produced predictable shifts in product profile, including a shift from the prominent 11S-hydroperoxy derivative of wild-type to include the 15R-hydroperoxide. These results conform to a model that predicts a head-first substrate binding orientation for 9S-LOX. We also found that linoleoyl-phosphatidylcholine is not a 9S-LOX substrate, which is consistent with this conclusion.

Large-scale identification of protein-protein interaction of Escherichia coli K-12.

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology.

The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain.

The SCO2299 gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N-terminal region shows high amino acid sequence similarity to RNase HI, whereas its C-terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature-sensitive growth defect of an Escherichia coli RNase H-deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA.DNA hybrid in vitro. The N-terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C-terminal domain showed no CobC-like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N-terminal and C-terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.

Application of capillary electrophoresis-mass spectrometry to synthetic in vitro glycolysis studies.

We propose an approach designed to reconstitute a metabolic pathway composed of multistep biochemical reactions, rather than to dissect the individual reactions that make up the pathway. A synthetic in vitro glycolysis was reconstructed from ten purified Escherichia coli (E. coli) enzymes to obtain a better understanding of the regulation of sequential enzymatic reactions. The key to the success of this approach is the ability to perform direct and simultaneous determination of the diverse metabolic intermediates in the pathway by capillary electrophoresis-mass spectrometry. We observed that the pathway is regulated by a delicate balance between the changing metabolite concentrations and behaves like a natural biological oscillating network that has hitherto not been reported for E. coli glycolysis. The end-product, pyruvate, was periodically synthesized from glucose at an overall efficiency of 30%, corresponding to an average of 90% conversion efficiency for each of the ten steps involved. This approach is likely useful for the synthesis of natural products requiring complex sequential biocatalytic reactions.

Identification of a jasmonate-regulated allene oxide synthase that metabolizes 9-hydroperoxides of linoleic and linolenic acids.

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants. Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed 9- and 13-hydroperoxides of linoleic and linolenic acid to alpha-ketol, gamma-ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3 was approximately 10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling. These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.