Suppressing activity of staurosporine from Streptomyces sp. MJM4426 against rice bacterial blight disease.

AIM: To identify the active compounds from the Streptomyces sp. MJM4426 that can protect rice from bacterial blight disease (BB), and to evaluate the potential of this Streptomyces strains and the compound for biocontrol of rice bacterial blight disease. METHODS AND RESULTS: The ethyl acetate extract of Streptomyces sp. MJM4426 can significantly protect rice leaf explants from the infection of Xanthomonas oryzae pv. oryzaeKACC 10331 (Xoo), the pathogen which cause BB. To identify the active compounds, the ethyl acetate extract of Streptomyces sp. MJM4426 was fractionated through a Sephadex LH-20 column chromatography, and further purified by preparative HPLC guided by the inhibitory activity against BB in rice leaf explants. UPLC-Q-TOF/MS analysis showed the active compound displayed its m/z values at [M+H](+) 467·2086 and [M+FA-H](-) 511·1963, and the molecular formula was estimated as C28 H26 N4 O3 which is identical to commercial standard staurosporine. In this study, the isolated staurosporine dramatically suppressed bacterial blight in rice leaf explants with the lowest concentration at 12·5 µmol l(-1) , however, it exhibited low inhibitory activity against Xoo with the MIC value at 256 µg ml(-1) . In addition, greenhouse study showed both crude extract and purified staurosporine can suppress the bacterial blight at the concentration of 5000 and 200 µg ml(-1) respectively. CONCLUSION: Streptomyces sp. MJM4426 can protect rice leaf explants from the infection of Xoo by producing staurosporine, but not by direct inhibitory activity against Xoo. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that staurosporine can protect rice leaf against bacterial blight disease and showed the potential of Streptomyces sp. MJM4426 as an alternative to chemical bactericide for bacterial blight disease in rice.

Enzymatic hydrolysis of ovomucoid and the functional properties of its hydrolysates.

Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if the protein is cut into small peptides. The objectives of this study were to hydrolyze ovomucoid using various enzyme combinations, and compare the functional properties of the hydrolysates. Purified ovomucoid was dissolved in distilled water (20 mg/mL) and treated with 1% of pepsin, α-chymotrypsin, papain, and alcalase, singly or in combinations. Sodium sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) results of the hydrolysates indicated that pepsin (OMP), alcalase (OMAl), alcalase+trypsin (OMAlTr), and alcalase+papain (OMAlPa) treatments best hydrolyzed the ovomucoid, and the 4 treatments were selected to determine their functional characteristics. Among the 4 enzyme treatments, hydrolysate from OMAlTr showed the highest iron-chelating and antioxidant activities, while OMP showed higher ACE-inhibitory activity, but lower Fe-chelating activity than the other treatments. However, no difference in the copper-chelating activity among the treatments was found. MS/MS analysis identified numerous peptides from the hydrolysates of OMAlPa and OMAlTr, and majority of the peptides produced were <2 kDa. Pepsin treatment (OMP), however, hydrolyzed ovomucoid almost completely and produced only amino acid monomers, di- and tri-peptides. The ACE-inhibitory, antioxidant and iron-chelating activities of the enzyme hydrolysates were not consistent with the number and size of peptides in the hydrolysates, but we do not have information about the quantity of each peptide present in the hydrolysates at this point.

Streptomyces sp. strain PGPA39 alleviates salt stress and promotes growth of 'Micro Tom' tomato plants.

AIMS: To identify an actinobacterial strain that can promote growth and alleviate salinity stress in tomato plants. METHODS AND RESULTS: Actinobacteria were isolated from agricultural soil and screened for ACC deaminase activity, production of indole acetic acid (IAA), solubilization of tricalcium phosphate and sodium chloride (NaCl) salinity tolerance. Among the several strains tested, one strain designated PGPA39 exhibited higher IAA production, and phosphate solubilization in addition to ACC deaminase activity, and tolerance to 1 mol l(-1) NaCl. Strain PGPA39 was identified as a Streptomyces strain based on 16S rDNA sequence and designated Streptomyces sp. strain PGPA39. It promoted the growth of Arabidopsis seedlings in vitro as evidenced by a significant increase in plant biomass and number of lateral roots. Salinity stress-alleviating activity of PGPA39 was evaluated using 'Micro Tom' tomato plants with 180 mmol l(-1) NaCl stress under gnotobiotic condition. A significant increase in plant biomass and chlorophyll content and a reduction in leaf proline content were observed in PGPA39-inoculated tomato plants under salt stress compared with control and salt-stressed noninoculated plants. CONCLUSIONS: Streptomyces sp. strain PGPA39 alleviated salt stress and promoted the growth of tomato plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of Streptomyces sp. strain PGPA39 in alleviating salinity stress in tomato plants and could be utilized for stress alleviation in crop plants under field conditions.

Allometric scaling of marbofloxacin pharmacokinetics: a retrospective analysis.

The association between physiologically dependent pharmacokinetic parameters (CL(B), T1/2beta, Vd(ss)) of marbofloxacin and body weight was studied in eight animal species based on allometric equation Y = aWb, where 'Y' is the pharmacokinetic parameter, 'W' is body weight, 'a' is allometric coefficient (intercept) and 'b' is the exponent that describes relation between pharmacokinetic parameter and body weight. The body clearance of marbofloxacin has shown significant (P < 0.0001) relation with size (Bwt) in various animal species. However, half-life and volume of distribution were not in association with body weight. Although half-life and volume of distribution were not in a good correlation with body weight, statistically significant association between the body clearance and body weight suggests validity of allometric scaling for predicting pharmacokinetic parameters of marbofloxacin in animal species that have not been studied yet. However further study considering large sample size and other parameters influencing pharmacokinetics of marbofloxacin is recommended.

Predicting post-treatment symptom severity for adults receiving psychological therapy in routine care for generalised anxiety disorder: a machine learning approach.

Approximately half of generalised anxiety disorder (GAD) patients do not recover from first-line treatments, and no validated prediction models exist to inform individuals or clinicians of potential treatment benefits. This study aimed to develop and validate an accurate and explainable prediction model of post-treatment GAD symptom severity. Data from adults receiving treatment for GAD in eight Improving Access to Psychological Therapies (IAPT) services (n=15,859) were separated into training, validation and holdout datasets. Thirteen machine learning algorithms were compared using 10-fold cross-validation, against two simple clinically relevant comparison models. The best-performing model was tested on the holdout dataset and model-specific explainability measures identified the most important predictors. A Bayesian Additive Regression Trees model out-performed all comparison models (MSE=16.54 [95 % CI=15.58; 17.51]; MAE=3.19; R²=0.33, including a single predictor linear regression model: MSE=20.70 [95 % CI=19.58; 21.82]; MAE=3.94; R²=0.14). The five most important predictors were: PHQ-9 anhedonia, GAD-7 annoyance/irritability, restlessness and fear items, then the referral-assessment waiting time. The best-performing model accurately predicted post-treatment GAD symptom severity using only pre-treatment data, outperforming comparison models that approximated clinical judgement and remaining within the GAD-7 error of measurement and minimal clinically important differences. This model could inform treatment decision-making and provide desired information to clinicians and patients receiving treatment for GAD.

Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes.

AIM: To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. METHODS AND RESULTS: We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. CONCLUSION: This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process.

Biological control of anthracnose (Colletotrichum gloeosporioides) in yam by Streptomyces sp.MJM5763.

AIM: To find a suitable biocontrol agent for yam anthracnose caused by Colletotrichum gloeosporioides. METHODS AND RESULTS: An actinobacterial strain, MJM5763, showing strong antifungal activity, multiple biocontrol and plant growth-promoting traits was isolated from a yam cultivation field in Yeoju, South Korea. Based on morphological and physiological characteristics and analysis of the 16S rDNA sequence, strain MJM5763 was identified as a novel strain of Streptomyces and was designated as Streptomyces sp. MJM5763. Treatment with MJM5763 and the crude culture filtrate extract (CCFE) was effective in suppressing anthracnose in detached yam leaves in vitro and reduced incidence and severity of anthracnose in yam plants under greenhouse conditions. The CCFE treatment was the most effective of all the treatments and reduced the anthracnose severity by 85-88% and the incidence by 79-81%, 90 days after inoculation with the pathogen. CCFE treatment was also effective under field conditions and showed a reduction of 86 and 75% of anthracnose severity and incidence, respectively. CONCLUSION: Streptomyces sp. strain MJM5763 was effective in biocontrolling anthracnose in yam caused by C. gloeosporioides. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces sp. MJM5763 is a potential alternative to chemical fungicides for reducing yield losses to anthracnose in yam.

Enzymatic hydrolysis of ovomucin and the functional and structural characteristics of peptides in the hydrolysates.

Ovomucin was hydrolyzed using enzymes or by heating under alkaline conditions (pH 12.0), and the functional, structural and compositional characteristics of the peptides in the hydrolysates were determined. Among the treatments, heating at 100 °C for 15 min under alkaline conditions (OM) produced peptides with the highest iron-binding and antioxidant capacities. Ovomucin hydrolyzed with papain (OMPa) or alcalase (OMAl) produced peptides with high ACE-inhibitory activity. The mass spectrometry analysis indicated that most of the peptides from OMPa were <2 kDa, but peptides from OMTr and OM were >2 kDa. OMAl hydrolyzed ovomucin almost completely and no peptides within 700-5000 Da were found in the hydrolasate. The results indicated that the number and size of peptides were closely related to the functionality of the hydrolysates. Considering the time, cost and activities of the hydrolysates, OM was the best treatment for hydrolyzing ovomucin to produce functional peptides.

Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region.

We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.

Polyamine profiles in the urine of patients with leukemia.

Eleven urinary polyamine levels were determined in controls (32 cases) and 43 patients with varying stages of leukemia including initial, relapse and complete remission, using gas chromatography nitrogen-phosphorus detection. Also, to indirectly evaluate the possible involvement of enzymes, precursor to product concentration ratios were compared between controls and patients with each stage of leukemia. As a result, it is confirmed that the ratio of N1-acSpd/N8-acSpd could be used as a diagnostic marker and the level of N1,N12-diacetylspermine could be used for determining disease stage and as a malignancy marker for leukemia. An altered metabolic pathway related to leukemia is also proposed in which N1,N12-diacetylspermine can be produced directly from spermine and N1,N12-diacetylspermine is a major source of N1-acetylspermidine.

Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis.

A gene encoding valine dehydrogenase (Vdh) has been cloned from Streptomyces albus, a salinomycin producer, and expressed in Escherichia coli. The S. albus Vdh is composed of 364 amino acids that showed high homology with several other amino acid dehydrogenases as well as Vdhs from Streptomyces spp. and leucine and phenylalanine dehydrogenases (Ldh and Pdh) from Bacillus spp. A protein of 38 kDa, corresponding to the approximate mass of the predicted S. albus Vdh product (38.4 kDa) exhibiting specific Vdh activity, was observed when the S. albus vdh gene was overexpressed in E. coli under the controlled T7 promoter and was subsequently purified to homogeneity. Among branched- and straight-chain amino acids, L-valine and L-alpha-aminobutyrate were the preferred substrates for the enzyme. Lys-79 and Lys-91 of S. albus Vdh were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydrogenase sequences. To elucidate the functional roles of the lysyl residues, the Lys residues have individually been replaced with Ala by site-directed mutagenesis. Kinetic analyses of the Lys-79 and Lys-91-mutated enzymes revealed that they are involved in the substrate binding site and catalysis, respectively, analogous to the corresponding residues in the homologous Ldh and Pdh.

Phylogenetic diversity of thermophilic actinomycetes and Thermoactinomyces spp. isolated from mushroom composts in Korea based on 16S rRNA gene sequence analysis.

Forty one strains isolated from 21 samples of various mushroom composts in Korea were analyzed by 16S rRNA gene sequencing to investigate the phylogenetic diversity of thermophilic actinomycetes. The 25 strains of thermophilic actinomycete isolates were related to the five genera, Pseudonocardia, Saccharomonospora, Saccharopolyspora, Streptomyces, and Thermobifida, within the order Actinomycetales, and 16 strains were classified into the genus Thermoactinomyces within the family Bacillaceae. Most of 41 isolates were encompassed by two genera, Streptomyces and Thermoactinomyces, that were isolated mainly in composts prepared from waste cotton and hay, respectively. Among them, M104 and M109 were placed in distinct taxonomic positions although these strains formed phylogenetic lineages related to the genus Streptomyces and to the family Streptosporangiaceae, respectively. Therefore, a phenetic and genetic characterization of these strains will be needed to pinpoint their taxonomic position.

Cepacidine A, a novel antifungal antibiotic produced by Pseudomonas cepacia. II. Physico-chemical properties and structure elucidation.

Cepacidine A is a novel glycopeptide with a potent antifungal activity, which is produced by Pseudomonas cepacia AF 2001. Its molecular weight was determined by FAB-MS (m/z 1215). The compound is comprised of glycine (1), serine (2), 2,4-diaminobutyric acid (1), aspartic acid (1), beta-hydroxy tyrosine (1), beta-hydroxy asparagine (1), xylose (1) and 5,7-dihydroxy-3,9-diamino-octadecanoic acid (1). Unfortunately, cepacidine A is a mixture of A1 and A2, either of which is barely distinguishable. Cepacidine A2 includes asparagine (1) instead of beta-hydroxy asparagine (1) of cepacidine A1. The MS data and the NOESY, TOCSY and HMBC spectra show that cepacidine A is a cyclic peptide and xylose is connected to 5,7-dihydroxy-3,9-diaminooctadecanoic acid.

Cepacidine A, a novel antifungal antibiotic produced by Pseudomonas cepacia. I. Taxonomy, production, isolation and biological activity.

Cepacidine A is a potent antifungal antibiotic produced by Pseudomonas cepacia AF 2001. The compound was isolated from the fermentation broth with 1 vol isopropyl alcohol, followed by the collection of the precipitation formed upon concentration of the extract. Purification was effected by chromatography on Diaion HP-20, alumina and reversed phase C18 followed by TLC on silica gel. These techniques afforded the two closely related compounds, cepacidine A1 and cepacidine A2. A mixture of these two compounds called capacidine A, showed high in vitro antifungal activity against the various animal and plant pathogenic fungi. The activity was diminished by the presence of serum. No antibacterial activity was demonstrable.

AL072, a novel anti-Legionella antibiotic produced by Streptomyces sp.

AL072 is a potent anti-Legionella antibiotic produced by Streptomyces strain AL91. The compound was isolated from the fermentation broth with 1 volume of isopropyl alcohol, followed by an ethyl acetate extraction and subsequent concentration under reduced pressure. Purification was performed on an octadecyl silica gel column followed by preparative HPLC. AL072 purified as mentioned above showed extremely specific activity only towards Legionella pneumophila. No antibacterial activity against any other bacteria tested was demonstrable. Its molecular weight was determined by FAB-MS (m/z 648) and the compound was identified as a novel 1,3-diacyl glycerol with the molecular formula C41H76O5. One of the two acyl groups is linoleyl and the other is 3,5-dimethyl octadecanoyl.

Nitrofen-induced congenital malformations of the heart and great vessels in rats: an animal model.

BACKGROUND: Nitrofen (2,4-dichloro-4'-nitrodiphenyl ether), a diapheny ether herbicide, is known to induce in rat fetuses a variety of congenital cardiovascular anomalies, together with diaphragmatic hernia and hydronephrosis. The purpose of the current study was to produce congenital cardiovascular anomalies in rat fetuses by oral nitrofen administration at the indicated doses and days of gestation, and to determine the characteristics of resulting nitrofen-induced cardiovascular anomalies. METHODS: All the observed fetuses were removed from pregnant Sprague-Dawley rats killed on the 21st day of gestation. They were preserved in 10% formalin, and dissection for examination was carried out under a dissecting microscope. RESULTS: The following results were based on dissecting microscopic findings of 482 offspring: (1) The 11th day of gestation was the most sensitive for nitrofen induction of congenital cardiovascular anomalies. The incidence of these was dose related. (2) Ventricular septal defect was the most common single cardiovascular anomaly, representing more than half of all such irregularities. The next most common were aortic arch anomalies and tetralogy of Fallot. (3) Cardiac anomalies derived from infundibular maldevelopment such as tetralogy of Fallot and pulmonary atresia with ventricular septal defect were observed only in the group treated with nitrofen on the 11th gestational day. (4) Aortic arch anomalies were very frequent; the great majority were anomalous right subclavian artery with left aortic arch. CONCLUSION: This animal model is suitable for further embryological investigation of the development of congenital cardiovascular anomalies.

Diagnostic pitfalls of Merkel cell carcinoma and dramatic response to chemotherapy.

Merkel cell carcinoma (MCC) is an unusual malignant tumor that arises from neuroendocrine cells with features of epithelial differentiation. We describe a MCC patient with unusual clinical, histopathological, and immunohistochemical features. Although the microscopic, immunohistochemical, and ultrastructural characteristics of MCC have been well defined, diagnostic difficulties remain, particularly in distinguishing it from lymphoma involving the skin, as suggested by our case. This is an unusual case in which dense lymphoid infiltration masked the true tumor. All the immunohistochemical markers of MCC except neuron-specific enolase (NSE) were negative. The dramatic response to primary chemotherapy was also very noteworthy.

WHOLE BODY NONRIGID CT-PET REGISTRATION USING WEIGHTED DEMONS.

We present a new registration method for whole-body rat computed tomography (CT) image and positron emission tomography (PET) images using a weighted demons algorithm. The CT and PET images are acquired in separate scanners at different times and the inherent differences in the imaging protocols produced significant nonrigid changes between the two acquisitions in addition to heterogeneous image characteristics. In this situation, we utilized both the transmission-PET and the emission-PET images in the deformable registration process emphasizing particular regions of the moving transmission-PET image using the emission-PET image. We validated our results with nine rat image sets using M-Hausdorff distance similarity measure. We demonstrate improved performance compared to standard methods such as Demons and normalized mutual information-based non-rigid FFD registration.

SERIAL NONRIGID VASCULAR REGISTRATION USING WEIGHTED NORMALIZED MUTUAL INFORMATION.

Vascular registration is a challenging problem with many potential applications. However, registering vessels accurately is difficult as they often occupy a small portion of the image and their relative motion/deformation is swamped by the displacements seen in large organs such as the heart and the liver. Our registration method uses a vessel detection algorithm to generate a vesselness image (probability of having a vessel at any given voxel) which is used to construct a weighting factor that is used to modify the intensity metric to give preference to vascular structures while maintaining the larger context. Therefore, our proposing method uses fully data-driven calculated weights and needs no prior knowledge for the weight calculation. We applied our method to the registration of serial MRI lamb images obtained from studies on tissue engineered vascular grafts and demonstrate encouraging performance as compared to non-weighted registration methods.

Catalytic domain of AfsKav modulates both secondary metabolism and morphologic differentiation in Streptomyces avermitilis ATCC 31272.

Genetic characterization of afsK-av (SAV3816) in Streptomyces avermitilis ATCC 31272 was performed to evaluate the role(s) of this eukaryotic-type serine-threonine protein kinase (STPK) in the regulation of morphologic differentiation and secondary metabolism. The afsK-av::neo mutant (SJW4001) was defective in sporulation, melanogenesis, and avermectin production. These phenotypic defects were complemented by introduction of either the intact afsK-av or the 900-nt catalytic domain region. The catalytic domain restored sporulation and melanogenesis to SJW4001 whereas it partially recovered avermectin production. This study reveals that AfsKav is a pleiotropic regulator and demonstrates in vivo that the C-region of AfsKav is not essential for its regulatory role in S. avermitilis differentiations.