Decolorization of synthetic dyes by basidiomycetes isolated from woods of the Atlantic Forest (PE), Brazil

In this study, the biodegradability of synthetic dyes used in the textile industry by Basidiomycetes collected in woods of the Atlantic Forest of Pernambuco, Brazil, was evaluated. Among the fungi, the following species were identified: Caripia montagnei, Datronia caperata, Earliella scabrosa, Fomitopsis feei, Ganoderma stiptatum, Hexagonia hydnoides, Pleurotus ostreatus, Pycnoporus sanguineus and Trametes membranacea. Mycelium discs (2 cm2) of each fungal isolate were transferred to 30 ml of King medium supplemented with 0.05% m/v of each of the following dyes: methyl orange, bromophenol blue (BB), methylene blue, Congo red, phenol red (PR) and methyl green (MG). All these species showed potential to decolorize the tested dyes, with decolorization efficiency varying from 7.26 to 99.2%. H. hydnoides and T. membrancea were the only species able to degrade all dyes. BB was effectively metabolized by P. sanguineus (97.4%), H. hydnoides (95.6%), E. scabrosa (96.6%) and T. membranacea (99.2%). E. scabrosa stood out among the isolates in dyes decolorization, allowing for efficiencies >90% for MG, PR, and BB.     

Molecular characteristics of bovine factor X activated in concentrated sodium citrate solution

Preparations of the zymogen form of bovine factor X were incubated in 25% $\text{w}\text{v}$ sodium citrate at room temperature. The rate of activation of factor X was dependent on the extent of contamination with factor VII, prothrombin, and thrombin. The activated factor X was isolated by DEAE-cellulose chromatography. Analysis of the final product by sedimentation velocity centrifugation coupled with measurements of the rate of boundary spreading, high-speed sedimentation equilibrium, and gel filtration chromatography provided evidence for a single molecular species undergoing reversible association-dissociation with a monomeric molecular weight of 48,000. In the absence of mercaptoethanol a single band was seen by disc electrophoresis and by SDS-acrylamide electrophoresis but after disulfide reduction two components of molecular weights 30,000 and 17,000 were visible. The protein contained large amounts of acidic amino acids but no carbohydrate. The N-terminal amino acids were alanine and isoleucine and 1 mole C-terminal arginine per mole protein was found. These characteristics are very similar to those of factor X activated with Russell's viper venom.When a BaSO₄ eluate of bovine plasma rich in prothrombin was allowed to stand in 25% sodium citrate both thrombin and activated factor X were generated. Chromatography of the isolated activated factor X on Sephadex G-200 as well as disc electrophoresis showed that it behaved identically with the enzyme obtained from purified zymogen and was clearly distinguishable from autoprothrombin c, a glycoprotein possessing qualitatively similar biological activity (Seegers, W. H., Cole, E. R., Harmison, C. R., and Marciniak, E. (1963) Can. J. Biochem. Physiol.41, 1047).     

Feeding ecology and evidence for amino acid synthesis in the periodical cicada (Magicicada)

The periodical cicadas of the genus Magicicada (including M. septendecim, M. cassini, and M. septendecula) have the longest juvenile life span of any insect, living underground for 13 or 17 years and feeding exclusively on root xylem fluids. Due to their inaccessible life cycles very little is known about cicada nutrition, despite the fact that members of Magicicada can achieve a very large biomass in woodland habitats east of the Mississippi and hence constitute a major part of the ecosystem where they occur in high densities. Live cicadas were collected at two sites in early June of 2004, during the emergence of Brood X (both M. septendecim and M. cassini were recovered). We used a combination of stable isotopic measurements (δ¹⁵N and δ¹³C) and multivariate statistical techniques to test for differences in resource acquisition among the cicada species and sexes collected at two locations within the 17-year periodical Brood X range. The amino acid constituents of cicada chitin and organs, plus xylem extracted from a deciduous sapling, were also analyzed. The data show that male and female cicadas have different carbon fractionations, which could reflect differential resource utilization due to oviposition in females. Several essential amino acids for the cicada were absent in xylem. Carbon-isotopic composition of all amino acids in the cicadas was distinctly different from the limited set measured in the xylem. Because of the differences in isotopic composition, we conclude that amino acids were synthesized de novo rather than incorporated directly, most likely produced by endosymbiotic bacteria.     

Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted Cα amino acids

Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C^α amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X = any one of the following residues, A5c, A6c, Tic or Oic and B = any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC₅₀ (<7.5 μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC₅₀ (<7.5 μM) values against selected cancer cell lines.     

Biochemistry of photosynthesis in species of triticum of differing ploidy

Illuminated flag leaves of Triticum monococcum(2X), T. urartu(2X), T. dicoccum(4X), T. dicoccoides(4X), and T. aestivum(6X) were exposed to ¹⁴CO₂ for 10 seconds and subsequently allowed to continue photosynthesis in the ambient air for periods of up to 2 minutes. The relative distribution of ¹⁴C among water-soluble products in the leaves was similar for each species at each sampling time. After the 10-second pulse of ¹⁴CO₂, radioactivity was mainly in phosphate esters with less than 5% in C₄ acids. Subsequently, radioactivity increased in sucrose, glycine, and serine at the expense of that in phosphate esters. By 2 minutes, between 18% and 29% of the ¹⁴C was in glycine plus serine. The results suggest rapid photorespiration in all species and an absence of C₄ photosynthesis.     

A comparison of host-defense peptides in skin secretions of female Xenopus laevis × Xenopus borealis and X. borealis × X. laevis F1 hybrids

Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (X_LB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (X_BL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1was present in low concentration. No peptides were identified in secretions of either X_LB or X_BL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis × X. muelleri F1 hybrids and no novel peptide expression.     

Patterns of nitrogen utilization between the ambrosia beetle Xyleborus dispar and its symbiotic fungus

Some parameters of nitrogen utilization between the ambrosia beetle Xyleborus dispar in mutualistic association with its symbiotic fungus Ambrosiella hartigii, were examined. Qualitative and quantitative analyses of the major nitrogenous excretory products were made on the various life stages of X. dispar. The main nitrogenous product found in excreta and hindguts of beetles, larvae, and pupae, was uric acid (range 7·6–14·8 μg uric acid/beetle). No ninhydrin-positive compounds were located in excreta of the beetles. The concentration of ammonia-nitrogen in the various life stages averaged between 0·70 and 1·13 μg NH₃-N/beetle.Total nitrogen determinations were made on sapwood samples of Malus sylvestris (0·34 ± 0·005% N by dry weight), attacked wood, ‘pre-brood’ (0·31 ± 0·005% N by dry weight), and attacked wood ‘post-brood’ (0·17 ± 0·02% N). Similar determinations of the artificial medium (l-asparagine medium) indicated that a nitrogen requirement of about 0·08–0·1% N by dry weight was necessary before oviposition could occur.Fixation of atmospheric nitrogen by individual X. dispar beetles in vitro was not indicated using the acetylene ethylene reductase method. Proteolytic enzyme activity was not found on examination of diapause beetles, their excreta, larval and pupal excreta, and the ambrosial and mycelial forms of A. hartigii.Comparative concentrations of soluble proteins and free amino acids suggested that fungus in the mycangia was built up from free amino acids of the insects. At the period of emergence, flight, and attack of new hosts, the females were found to have a concentration of soluble proteins more than double that found in the beetles during the remainder of the year. The free amino acids were the lowest values recorded during this period (March–October).     

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae)

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianus Ahl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensis Perret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.     

Morphological Variation among 23 Xiphinema americanum Populations

Morphometrics of 23 United States populations of Xiphinema americanum sensu lato, sharing the characteristics of an offset lip region and conoid tail, were examined and analyzed statistically by canonical discriminant analysis (CDA). Specimens were collected from Arkansas, Georgia, Tennessee, Mississippi, Florida, Oklahoma, California, and North Dakota. Eleven measurements and body ratios obtained from female specimens were used in the analysis. Xiphinema americanum, X. bricolensis, X. californicum, X. citricolum, X, intermedium, X. tarjanense, and X. thornei, and one undescribed species were identified among the 23 populations. Three groups -- X. americanum-group, X. californicum-group, and X. intermedium-group (X. intermedium and X. tarjanense) -- were formed and four populations belonging to four different species were separated consistently from these groups in CDA scatterplots of the 23 populations. Composition of the groups was somewhat related to the geographical origins of the populations in the groups. A population from California had morphometrics intermediate between X. americanum and X. californicum. Separation between the X. americanum-group and X. californicum-group in the CDA scatterplots was not as distinct as that between them and the X. intermedium-group or between any of the three groups and the four single outlying populations.     

Inhibition of reproduction of Xyleborus ferrugineus by ascorbic acid and related chemicals

Effects of various dietary chemicals on the reproduction of the ambrosia beetle, Xyleborus ferrugineus were studied. Ascorbic acid, araboascorbic acid, dehydroascorbic acid, hydroquinone, catechol, cysteine, and α-tocopherol each inhibited progeny production by virgin females. Detailed studies of the effects of ascorbic acid showed that it inhibited progeny production by causing a nutritionally detrimental non-enzymic browning of the dietary casein. Amino acid analyses of such browned and unbrowned casein, after in vitro digestion with pepsin and pancreatin, showed that lesser amounts of certain amino acids were released from the browned material. Effects of the non-enzymic browning were overcome by increasing the casein component of the diet. It was further concluded that araboascorbic acid, dehydroascorbic acid, hydroquinone, and catechol probably inhibit reproduction of X. ferrugineus by the same mechanism as ascorbic acid. No explanation of the inhibitory action of α-tocopherol on X. ferrugineus is offered.     

Structure of single-stranded nucleic acids in the presence of ribosomal protein S1.

We have developed a new method for mounting nucleic acids and nucleic acidprotein complexes for high-resolution electron microscopy, and have used it to characterize the interaction between ribosomal protein S1 and single-stranded nucleic acids. We find that SI unwinds most, but not all of the secondary structure present in MS2 RNA and øX174 viral DNA. The binding of S1 to DNA and RNA is not highly co-operative, and has a stoichiometry of one S1 per 10 to 15 nucleotides. We have not observed any tendency for S1 nucleic acid complexes to form aggregates in either 0·01 m-Na⁺ or 0·1 m-Na⁺. An analogous protein isolated from the 30 S ribosomal subunit of Caulobacter crescentus is indistinguishable from Escherichia coli S1 in these studies. The mono-N-ethylmaleimide derivative of E. coli S1 will bind to both MS2 RNA and øX174 viral DNA with a stoichiometry of one N-ethylmaleimide-S1 per 10 to 15 nucleotides, but will not unwind the secondary structure of either of them.     

Xanthobacter xylophilus sp. nov., a member of the xylotrophic mycobacterial community of low-mineral oligotrophic waters

A novel obligately aerobic heterotrophic bacterium belonging to the genus Xanthobacter, strain Z-0055, was isolated from the bacterial community of dystrophic waters formed by xylotrophic fungi grown on decaying spruce wood. The cells are small (0.4 ±0.7 fum), ovoid, grarrmegative, and nonmotile. The strain reproduces by nonuniform division. Strain Z-0055 is a moderately acidophilic microorganism and grows in a pH range of 4.8–6.8, with an optimum at pH 5.5. The temperature range for growth is 10–28°C, with an optimum at 20°C. The bacterium utilizes salts of organic acids (citrate, oxalate, succinate, and gluconate), as well as xylose and xylan as carbon and energy sources. No growth was detected at NaCl concentrations higher than 1.5 g/l. The DNA G+C base content is 63.6 mol %. According to phylogenetic analysis of the 16S rRNA gene sequences, strain Z-0055 belongs to the cluster containing X. flavus, X. aminoxidans, X. autotrophicus, and X. viscosus. The levels of similarity between the studied strain and these species are almost the same (96.0–97.0%). The genetic and ecophysiological properties of strain Z-0055 support classification of this acid-tolerant inhabitant of oligotrophic waters within the genus Xanthobacter as a new species X. xylophilus sp. nov.     

Phylogenetic Relationships Among Xiphinema and Xiphidorus Nematode Species from Brazil Inferred from 18S rDNA Sequences

Maximum likelihood trees produced from 18S rDNA sequences separated 14 Xiphinema and five Xiphidorus nematode species from Brazil into distinct groups that concurred with their current morphological taxonomic status. Species belonging to the X. americanum group (X. brevicolle, X. diffusum, X. oxycaudatum, and X. peruvianum) formed a single group that was clearly separated from the other Xiphinema species. As with previous taxonomic studies that noted only minor morphological differences between putative X. americanum group species, separation of these species based upon 18S rDNA sequences was inconclusive. Thus it is probable that instead of comprising distinct species, the X. americanum group may in fact represent numerous morphotypes with large inter- and intra- population morphological variability that may be environmentally driven. Within the cluster representing non X. americanum group species, there was little statistical support to clearly separate species. However, three subgroups, comprising (i) the X. setariae/vulgare complex, (ii) X. ifacolum and X. paritaliae, and (iii) X. brasiliense and X. ensiculiferum were well resolved.     

Factor X activator from Vipera lebetina snake venom,molecular characterization and substrate specificity

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6–9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly – factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly – factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg⁵²-Ile⁵³ bond in the heavy chain of human factor X and the Arg²²⁶-Val²²⁷ bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.     

Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel β-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0?~?9.0 and at temperatures below 42 °C. The K _m and V _max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.     

The Metabolome in Finnish Carriers of the MYBPC3-Q1061X Mutation for Hypertrophic Cardiomyopathy

Aims

Mutations in the cardiac myosin-binding protein C gene (MYBPC3) are the most common genetic cause of hypertrophic cardiomyopathy (HCM) worldwide. The molecular mechanisms leading to HCM are poorly understood. We investigated the metabolic profiles of mutation carriers with the HCM-causing MYBPC3-Q1061X mutation with and without left ventricular hypertrophy (LVH) and non-affected relatives, and the association of the metabolome to the echocardiographic parameters.

Methods and Results

34 hypertrophic subjects carrying the MYBPC3-Q1061X mutation, 19 non-hypertrophic mutation carriers and 20 relatives with neither mutation nor hypertrophy were examined using comprehensive echocardiography. Plasma was analyzed for molecular lipids and polar metabolites using two metabolomics platforms. Concentrations of branched chain amino acids, triglycerides and ether phospholipids were increased in mutation carriers with hypertrophy as compared to controls and non-hypertrophic mutation carriers, and correlated with echocardiographic LVH and signs of diastolic and systolic dysfunction in subjects with the MYBPC3-Q1061X mutation.

Conclusions

Our study implicates the potential role of branched chain amino acids, triglycerides and ether phospholipids in HCM, as well as suggests an association of these metabolites with remodeling and dysfunction of the left ventricle.     

Molecular cloning and characterization of PLCB1 (phospholipase C, beta 1) gene from the olive flounder, Paralichthys olivaceus

PLCB1 (phospholipase C, beta 1) cDNA was cloned from olive flounder (Paralichthys olivaceus) cDNA via rapid amplification of cDNA ends (RACE). The cDNA for olive flounder PLCB1 (PoPLCB1) encodes for a polypeptide of 1,244 amino acids in length containing a well-conserved PH domain, catalytic X and Y domains, a C2 domain. From the sequence information of the BAC library, we assembled a contig containing the whole flounder PLCB1 cDNA sequences, and determined the exon/intron structure of the gene spanning > 110,743 bp DNA. PoPLCB1 gDNA sequences demonstrated the new sequence (exon 15), which has only been observed in the fish, is located between the X and Y domain of the PLCB1, and that PoPLCB1 exists as two splice variants-PoPLCB1a (1,244 amino acids) and PoPLCB1b (1,210 amino acids). Phylogenic analysis and sequence comparison of PoPLCB1 with other PLC isozymes showed a close relationship with the PLCB1 isozyme. Tissue-specific mRNA of PoPLCB1 was expressed predominantly in the brain and heart tissues. Between the two splicing variants of PoPLB1 in RT-PCR by tissue, PoPLCB1a showed a major expression pattern in more diverse types of tissues than the PoPLCB1b. PoPLCB1 gene expression was compared with that of the inflammatory cytokines IL-1β and TNF-α in infected spleen and kidney tissues via real-time RT-PCR assays following stimulation with LPS. After the stimulation, the expression of PoPLCB1 increased significantly prior to IL-1B and TNF-α expression. This provided direct evidence suggesting that PoPLCB1 may perform a crucial role in immune responses against pathogens and in inflammation.