Discovery and biochemical characterization of a mannose phosphorylase catalyzing the synthesis of novel β-1,3-mannosides

BackgroundMannoside phosphorylases are frequently found in bacteria and play an important role in carbohydrate processing. These enzymes catalyze the reversible conversion of β-1,2- or β-1,4-mannosides to mannose and mannose-1-phosphate in the presence of inorganic phosphate.MethodsThe biochemical parameters of this recombinantly expressed novel mannose phosphorylase were obtained. Furthermore purified reaction products were subjected to ESI- and MALDI-TOF mass spectrometry and detailed NMR analysis to verify this novel type of β-1,3-mannose linkage.ResultsWe describe the first example of a phosphorylase specifically targeting β-1,3-mannoside linkages. In addition to mannose, this phosphorylase originating from the bacterium Zobellia galactanivorans could add β-1,3-linked mannose to various other monosaccharides and anomerically modified 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars).ConclusionsAn unique bacterial phosphorylase specifically targeting β-1,3-mannoside linkages was discovered.General significanceFunctional extension of glycoside hydrolase family 130.     

Do sperm contribute to the buffering capacity of steelhead trout (Oncorhynchus mykiss) semen?

The motility of salmonid sperm is pH-sensitive and the buffering capacity of the seminal plasma is low. The objective of the present study was to determine the extent to which sperm contribute to the buffering capacity of whole steelhead (Oncorhynchus mykiss) semen. To determine the buffering capacity, semen and seminal plasma samples were titrated with HCl and pH measurements taken at 1-2 min. The buffering capacity of semen was not different from that of seminal plasma over the pH range 7.5 to 8.5 and was approximately 15% to 20% less over the range 6.0 to 7.0. Comparable results were obtained for the semen and seminal plasma of the chinook salmon (Oncorhynchus tshawytscha). To assess whether the intracellular environment could influence the buffering capacity, the effects of cell disruption with n-butanol and Triton X-100 (TX-100) were determined. Over the pH range 7.5 to 8.5, the presence of n-butanol or TX-100 resulted in a doubling of the buffering capacity of the semen; TX-100, but not n-butanol, increased semen buffering capacity over the pH range 6.0 to 7.0. To determine whether the sperm's intracellular compartment might contribute to the buffering capacity over a longer duration, semen and seminal plasma samples were acidified with HCl and the pH measured over several hours. These data suggest that intact sperm contribute no more than about 25% to the buffering capacity of whole semen. The buffering capacity of steelhead semen and seminal plasma were comparably and modestly temperature sensitive. The results suggest that the sperm may contribute to the buffering capacity of the semen over a physiological pH range, however, if so, the effect is relatively small.     

Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C₁₈ precursors.     

Identification and characterization of RAPD–SCAR markers linked to glyphosate-susceptible and -resistant biotypes of Eleusine indica (L.) Gaertn

Eleusine indica is one of the most common weed species found in agricultural land worldwide. Although herbicide-glyphosate provides good control of the weed, its frequent uses has led to abundant reported cases of resistance. Hence, the development of genetic markers for quick detection of glyphosate-resistance in E. indica population is imperative for the control and management of the weed. In this study, a total of 14 specific random amplified polymorphic DNA (RAPD) markers were identified and two of the markers, namely S4R727 and S26R₆976 were further sequence characterized. Sequence alignment revealed that marker S4R727 showing a 12-bp nucleotides deletion in resistant biotypes, while marker S26R₆976 contained a 167-bp nucleotides insertion in the resistant biotypes. Based on these sequence differences, three pairs of new sequence characterized amplified region (SCAR) primers were developed. The specificity of these primer pairs were further validated with genomic DNA extracted from ten individual plants of one glyphosate-susceptible and five glyphosate-resistant (R2, R4, R6, R8 and R11) populations. The resulting RAPD–SCAR markers provided the basis for assessing genetic diversity between glyphosate-susceptible and -resistant E. indica biotypes, as well for the identification of genetic locus link to glyphosate-resistance event in the species.     

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47?kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84?°C and an activity half-life at 75?°C of 112?min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis.     

Plasticity of boldness in rainbow trout,Oncorhynchus mykiss: do hunger and predation influence risk-taking behaviour?

Boldness, a measure of an individual's propensity for taking risks, is an important determinant of fitness but is not necessarily a fixed trait. Dependent upon an individual's state, and given certain contexts or challenges, individuals may be able to alter their inclination to be bold or shy in response. Furthermore, the degree to which individuals can modulate their behaviour has been linked with physiological responses to stress. Here we attempted to determine whether bold and shy rainbow trout, Oncorhynchus mykiss, can exhibit behavioural plasticity in response to changes in state (nutritional availability) and context (predation threat). Individual trout were initially assessed for boldness using a standard novel object paradigm; subsequently, each day for one week fish experienced either predictable, unpredictable, or no simulated predator threat in combination with a high (2% body weight) or low (0.15%) food ration, before being reassessed for boldness. Bold trout were generally more plastic, altering levels of neophobia and activity relevant to the challenge, whereas shy trout were more fixed and remained shy. Increased predation risk generally resulted in an increase in the expression of three candidate genes linked to boldness, appetite regulation and physiological stress responses - ependymin, corticotrophin releasing factor and GABA(A) - but did not produce a significant increase in plasma cortisol. The results suggest a divergence in the ability of bold and shy trout to alter their behavioural profiles in response to internal and exogenous factors, and have important implications for our understanding of the maintenance of different behavioural phenotypes in natural populations.     

Identification and characterization of a QTL on chromosome 2 for cytosolic glutamine synthetase content and panicle number in rice

A quantitative trait locus (QTL) associated with the protein content of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in senescing leaves, panicle number, and panicle weight was characterized in rice (Oryza sativa L.). A near-isogenic line (NIL), C-22, developed by marker-assisted selection was grown under different nitrogen levels in the greenhouse and in a paddy field. Chromosome 2 of C-22 had an approximately 50-cM segment substituted from the Kasalath (indica) chromosome in a Koshihikari (japonica) genetic background. C-22 showed a 12–37% lower content of GS1 protein in leaf blades than Koshihikari, which was in good agreement with a QTL region positively affected by the japonica chromosome. At an early vegetative stage, C-22 had more active tillers than Koshihikari in the greenhouse. At the reproductive stage, both panicle number and total panicle weight of C-22 were significantly higher than those of Koshihikari, particularly when the plants were grown under a low-nitrogen condition. These traits of C-22 were further confirmed in a paddy field. Thus, tiller development was positively affected by the Kasalath chromosome at an early vegetative stage, which resulted in an increased panicle number and panicle weight at the mature stage in C-22. These data indicate that the target QTL (Pnn1; panicle number 1) is important in the development of tillers and panicles in rice. Linkage analyses for panicle number and ratio of developing tiller formation in the second axil (RDT) revealed that Pnn1 was delimited at the 6.7-cM region.     

Application of RAPD markers for characterization of γ-ray-induced rose mutants and assessment of genetic diversity

Six parent and their 12 gamma ray-induced somatic flower colour mutants of garden rose were characterized to discriminate the mutants from their respective parents and understanding the genetic diversity using Random amplification of polymorphic DNA (RAPD) markers. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. The other 7 primers gave highly polymorphic banding patterns among the radiomutants. All the cultivars were identified by using only 7 primers. Moreover, individual mutants were also distinguished by unique RAPD marker bands. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. A dendrogram was constructed based on the similarity matrix using the Neighbor Joining Tree method showed three main clusters. The present RAPD analysis can be used not only for estimating genetic diversity present in gamma ray-induced mutants but also for correct identification of mutant/new varieties for their legal protection under plant variety rights.     

Discovery of O6-benzyl glaziovianin A,a potent cytotoxic substance and a potent inhibitor of α,β-tubulin polymerization

We have discovered O⁶-benzyl glaziovianin A, which showed stronger inhibition of microtubule polymerization (IC₅₀ = 2.1 μM) than known α,β-tubulin inhibitors, such as colchicine and glaziovianin A. Also, we performed competition binding experiments of O⁶-benzyl glaziovianin A and revealed that O⁶-benzyl glaziovianin A binds to the colchicine binding site with high affinity. It is interesting that glaziovianin A derivatives change their mode of action in benzylation at the O⁶ (α,β-tubulin inhibitor) or O⁷ (γ-tubulin-specific inhibitor) position.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Expression and characterization of recombinant NH2-terminal cell binding fragment of vitronectin in E. coli

A recombinant peptide fragment of vitronectin (rVN143), that includes the Arg-Gly-Asp (RGD) cell recognition site, was expressed in Escherichia coli using a prokaryotic expression system. The addition of recombinant rVN143 peptide enhances cell adhesion and proliferation similar (approximately 70%) to those of native VN.     

The UvrY response regulator of the BarA–UvrY two-component system contributes to Yersinia ruckeri infection of rainbow trout (Oncorhynchus mykiss)

To identify virulence-associated genes of a fish pathogen Yersinia ruckeri, we screened a total of 1056 mini-Tn5-Km2 signature-tagged mutants in rainbow trout by immersion challenge. Of 1056, 25 mutants were found survival-defective as they could not be re-isolated from fish kidney 7 days after infection. Mutated gene in F2-4 mutant, one of the 25 mutants, was homologous to uvrY that encodes UvrY response regulator of BarA–UvrY two-component system (TCS). Mutant F2-4 was significantly more sensitive (P < 0.05) to H₂O₂-mediated killing and was less able to infect Epithelioma papulosum cyprini cells. However, UvrY mutation did not affect survival of F2-4 mutant in the presence of non-immune fish serum and its ability to grow under iron starvation. In a time-course co-infection, mutant F2-4 had lower bacterial loads on day 1 itself, and by day 5 there was nearly a 1,000-fold difference in infection levels of the parent and mutant strains. The barA homolog of Y. ruckeri was PCR-amplified and sequence analyses identified four domains that were characteristic of hybrid histidine kinases. To conclude, the BarA–UvrY TCS contributes to the pathogenesis of Y. ruckeri in its natural host rainbow trout, possibly by regulating invasion of epithelial cells and sensitivity to oxidative stress induced by immune cells.     

Cystathionine β-Synthase in the Brain of the Trout Oncorhynchus mykiss after Unilateral Eye Damage and in Conditions of in vitro Cultivation

Russian Journal of Developmental Biology - Expression of cystathionine β-synthase (CBS) in the brain of adult trout under normal conditions and 1 week after an eye injury was assessed using...     

High-level expression of a polypeptides encoded by a large segment of the envelope gene of HIV-1 in E. coli by directed evolution

A random mutagenesis library of gp120-801 (a large segment of the envelope protein gene of HIV-1) was constructed using error-prone PCR and DNA shuffling methods, and one mutant of gp120-801 was selected from this library using a green fluorescent protein (GFP) fusion vector. After being cloned into pEX31b and expressed in E. coli, the expressed fusion protein reached to 15% of total bacterial proteins for the mutant but was just 1–2% for the wild type.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.