Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis

Aims: Isolation, identification and characterization of a highly efficient isomaltulose producer. Methods and Results: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW). Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. Significance and Impact of Study: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.

Marine isocyanides and related natural products structure, biosynthesis and ecology

This review highlights structural and biosynthetic work on a group of nitrogen-functionalised terpenes that are almost exclusively found in marine invertebrates and the animals that feed on them. The chemical functionality reviewed includes isocyanides, isothiocyanates, formamides, thiocyanates, isocyanates, and dichloroimines. The literature through mid 2003 is reviewed and there are 143 citations.

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9)

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wildtype and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.

Hepatic urea biosynthesis in the euryhaline elasmobranch Carcharhinus leucas

Plasma urea levels and hepatic urea production in the euryhaline bull shark, Carcharhinus leucas, acclimated to freshwater and seawater environments were measured. It was found that plasma urea concentration increased with salinity and that this increase was, in part, the result of a significant increase in hepatic production of urea. This study provides direct evidence that hepatic production of urea plays an important role in the osmoregulatory strategy of C. leucas.

L-arginine attenuates cardiovascular impairment in DOCA-salt hypertensive rats

Nitric oxide (NO) is essential for normal function of the cardiovascular system. This study has determined whether chronic administration of L-arginine, the biological precursor of NO, attenuates the development of structural and functional changes in hearts and blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Uninephrectomized rats treated with DOCA (25 mg every 4th day sc) and 1% NaCl in the drinking water for 4 wk were treated with L-arginine (5% in food, 3.4 +/- 0.3 g.kg body wt(-1).day(-1)). Changes in cardiovascular structure and function were determined by echocardiography, microelectrode studies, histology, and studies in isolated hearts and thoracic aortic rings. DOCA-salt hypertensive rats developed hypertension, left ventricular hypertrophy with increased left ventricular wall thickness and decreased ventricular internal diameter, increased inflammatory cell infiltration, increased ventricular interstitial and perivascular collagen deposition, increased passive diastolic stiffness, prolonged action potential duration, increased oxidative stress, and inability to increase purine efflux in response to an increased workload. L-Arginine markedly attenuated or prevented these changes and also normalized the reduced efficacy of norepinephrine and acetylcholine in isolated thoracic aortic rings of DOCA-salt hypertensive rats. This study suggests that a functional NO deficit in blood vessels and heart due to decreased NO synthase activity or increased release of reactive oxygen species such as superoxide may be a key change initiating many aspects of the cardiovascular impairment observed in DOCA-salt hypertensive rats. These changes can be prevented or attenuated by administration of L-arginine.

Spiroacetal biosynthesis: ()-1,7-dioxaspiro[5.5]undecane in Bactrocera cacuminata and Bactrocera oleae (olive fruit fly)

[GRAPHICS] A biosynthetic scheme rationalizing the formation of (+/-)-1,7-dioxaspiro[5.5]undecane (5) in the fruit fly species Bactrocera cacuminata and Bactrocera oleae (olive fruit fly) is presented. Incorporation studies with deuterium-labeled keto aldehyde (10), 1,5-nonanediol (11), and 1,5,9-nonanetriol (12), and our previous finding that both oxygen atoms of 5 originate from dioxygen, are strongly evidentiary. The racemic condition of the natural spiroacetal 5 is accounted for, and inter alia, it is demonstrated that dihydropyran (18) is not an important intermediate en route to 5.

The influence of dietary sodium chloride, Arginine : Lysine ratio, and methionine source on apparent ileal digestibility of arginine and lysine in acutely heat-stressed broilers

The present study was carried out to determine the ileal digestibility of Arg and Lys in acutely heatstressed broilers using diets varying in Arg:Lys ratio, NaCl concentration, and Met Source. Male broilers were maintained at 22degreesC from 21 to 33 d of age and then at 32degreesC from 33 to 38 d of age. From 28 to 38 d of age, birds were fed a diet with an Arg:Lys ratio of 1.05 and 3 g of supplemental NaCl/kg of diet with or without L-arg free base to increase the Arg:Lys to 1.35, and with or without 3 g/kg of additional NaCl. Methionine was supplied as equimolar amounts of DL-Met or 2-hydroxy-4-(methylthio)-butanoic acid in a 2 x 2 x 2 design. At 38 d of age, digesta were collected from the terminal ileum, and amino acid analyses were conducted on feed and digesta samples and compared with acid-insoluble ash (dietary celite) to calculate the apparent ileal digestibilities of Lys and Arg. Increasing the NaCl concentration and the presence of HMB significantly decreased the digestibility of both Arg and Lys, whereas increasing the Arg:Lys ratio increased the digestibility of only Arg but did increase BW gain (P = 0.08). An interaction between dietary NaCl and Arg:Lys ratio as well as the 3-way interaction suggested that dietary NaCl could affect the apparent ileal digestibility of Arg and Lys at certain Arg:Lys ratios and the response may be influenced by the Met source.

Chemical synthesis and biosynthesis of the cyclotide family of circular proteins

Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed.

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis. (c) 2006 Elsevier Inc. All rights reserved.

The effects of freshwater to seawater transfer on circulating levels of angiotensin II, C-type natriuretic peptide and arginine vasotocin in the euryhaline elasmobranch, Carcharhinus leucas

This study examined the effect of transfer to increased environmental salinity on the circulating levels of angiotensin II (ANG II), C-type natriuretic peptide (CNP), and arginine vasotocin (AVT) in the euryhaline elasmobranch, Carcharhinus letteas. Plasma levels of ANG 11 and CNP were significantly increased in C. leucas chronically acclimated to seawater (SW) in comparison to freshwater (FW) acclimated fish. There was no difference in plasma AVT levels. Acute transfer of FW fish to 75% SW induced an increase in plasma ANG II levels within 12 h, and subsequent transfer from 75 to 100% SW further increased plasma ANG 11 levels at both 24 and 72 h. No change in plasma CNP was observed during acute transfer to increased salinity. However, a significant increase in plasma AVT levels was observed following 96 h in 75% SW and 24 h in 100% SW. In chronically SW acclimated C leucas plasma osmolality, sodium, chloride, and Urea were all significantly higher than FW acclimated fish but there was no difference in haematocrit. Acute transfer of C letteas to 75% SW induced a significant increase in plasma osmolality, sodium and urea concentrations within 96 h of transfer. Subsequent transfer from 75 to 100% SW induced a further increase in these variables within 24 h in addition to a significant increase in plasma chloride above control levels. Haematocrit did not differ between the experimental and control groups throughout the acute study. Circulating levels of ANG 11 were significantly correlated to plasma, sodium, chloride, and urea concentrations during acclimation to SW. Conversely, circulating levels of CNP and AVT did not correlate to plasma osmolytes, however, CNP was significantly correlated to haematocrit during acclimation to seawater. (c) 2005 Elsevier Inc. All rights reserved.

L-arginine improves cardiovascular function in DOCA-salt hypertensive rats

No abstract