Terpene glucoside production: Improved biocatalytic processes using glycosyltransferases

Terpenoids are one of the main classes of natural products. In plants, a large fraction of the terpenoids is present as nonvolatile glycosides. The terpene glycosides have attracted much attention as antimicrobials, flavor precursors, and detergents. They are either extracted from plant materials or are synthesized by chemical and biocatalytic methods. Up to now, biotechnological production of terpene glycosides is based on reversed hydrolysis performed by glycosidases. However, this method suffers from low yields as a matter of principle. Recently, the first uridine diphosphate‐glucose:monoterpenol β‐d ‐glucosyltransferase (GT) genes were cloned and characterized from grapevine (Vitis vinifera) and kiwi (Actinidia deliciosa). Heterologous expression in Escherichia coli yielded promiscuous GT enzymes that efficiently glucosylated primary monoterpenols, simple alcohols, and phenols. The GT enzymes differed in substrate preference and activity toward their terpenoid substrates. Biotransformation experiments confirmed the applicability of the novel GTs in biocatalytic processes for the production of these novel compounds. In the near future, terpene glucosides will become commercially available for food, cosmetic, and pharmaceutical industry due to improved biocatalytic processes involving GT enzymes.     

Interaction of complement and leukocytes in severe acute pancreatitis: potential for therapeutic intervention

In acute pancreatitis, local as well as systemic organ complications are mediated by the activation of various inflammatory cascades. The role of complement in this setting is unclear. The aim of the present study was to determine the level of complement activation in experimental pancreatitis, to evaluate the interaction of complement and leukocyte-endothelium activation, and to assess the effects of complement inhibition by soluble complement receptor 1 (sCR1) in this setting. Necrotizing pancreatitis was induced in Wistar rats by the combination of intravenous cerulein and retrograde infusion of glycodeoxycholic acid into the biliopancreatic duct; edematous pancreatitis was induced by intravenous cerulein only. In control animals, a sham operation (midline laparotomy) was performed. Complement activation, leukocyte sequestration, and pancreatic as well as pulmonary injury were assessed in the presence/absence of sCR1. Increased levels of C3a were found in necrotizing but not in edematous pancreatitis. When complement activation in necrotizing pancreatitis was blocked by sCR1, levels of C3a and total hemolytic activity (CH50) were decreased. Leukocyte-endothelial interaction, as assessed by intravital microscopy, and pancreatic as well as pulmonary organ injury (wet-to-dry weight ratio, MPO activity, and histology) were ameliorated by sCR1. As a result of the present study, necrotizing but not edematous pancreatitis is characterized by significant and early complement activation. Based on the interaction of complement and leukocytes, complement inhibition by sCR1 may be a valuable option in the treatment of leukocyte-associated organ injury in severe pancreatitis.     

Antitumor quinols: role of glutathione in modulating quinol-induced apoptosis and identification of putative cellular protein targets

Novel heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) are promising novel anticancer agents. They exhibit in vitro antiproliferative activity against colon, renal, and breast carcinoma cell lines as well as in vivo antitumor activity in colon, renal, and breast tumor xenografts. Elucidation of the mechanism of antitumor action of these compounds is of great importance. We show in this study that the compounds induced apoptosis as demonstrated by caspase 3 and PARP cleavage at doses causing G(2)/M cell cycle arrest. Glutathione was found to play an important role in modulating quinol-mediated cytotoxicity. In HCT 116 cells, treatment with 1 and 2 caused a 2- to 3-fold increase in the total glutathione content, suggestive of a glutathione-mediated antioxidant response. Indeed, buthionine sulfoximine (BSO)-induced glutathione depleted cells were 6-10 times more sensitive to 1 and 2, while glutathione monoethyl ester supplementation decreased the antitumor potencies by 2-3 times. In further studies we determined other cellular proteins which bind to an immobilized quinol analog, and identified several proteins including beta-tubulin, heat shock protein 60, and peroxiredoxin 1 as potential molecular targets of quinols that may contribute to their proapoptotic and antiproliferative effects.     

The xenoestrogen bisphenol A in the Hershberger assay: androgen receptor regulation and morphometrical reactions indicate no major effects

We evaluated androgen-like effects of bisphenol A (BPA) using orchiectomized Wistar rats. Animals were treated p.o. either with vehicle or with 3, 50, 200, 500 mg/kgbw/day BPA (n=13) for 7 days. One group was treated s.c. with 1mg/kgbw/day testosterone propionate (TP). Flutamide (FL) (3mg/kgbw/day, p.o.) was used to antagonize androgen effects of the suprapharmacological dose (500 mg/kgbw/day) of BPA. Androgen-like effects of BPA on prostates and seminal vesicles were assessed by the Hershberger assay, densitometric analysis of androgen receptor (AR) immunoreactivity, cell proliferation-index and a morphometric analysis. Absolute weights of prostates and seminal vesicles were not increased by BPA, whereas the relative weights were increased at higher doses of BPA, most likely due to a decrease in body weight. Staining intensity for AR immunoreactivity was increased at low but not at higher doses of BPA in comparison to the orchiectomized rats. BPA at all doses tested did not cause an increase of the cell proliferation-index. Epithelial height and glandular luminal area were increased by low doses of BPA, whereas higher doses caused a decrease of these parameters. The data provide evidence that BPA does not exert major androgenic effects.     

Growth Deficiencies of Neisseria meningitidis pfs and luxS Mutants Are Not Due to Inactivation of Quorum Sensing

The activated methyl cycle (AMC) is a central metabolic pathway used to generate (and recycle) several important metabolites and enable methylation. Pfs and LuxS are considered integral components of this pathway because they convert S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) and S-ribosylhomocysteine to homocysteine (HCY), respectively. The latter reaction has a second function since it also generates the precursor of the quorum-sensing molecule autoinducer 2 (AI-2). By demonstrating that there was a complete lack of AI-2 production in pfs mutants of the causative agent of meningitis and septicemia, Neisseria meningitidis, we showed that the Pfs reaction is the sole intracellular source of the AI-2 signal. Analysis of lacZ reporters and real-time PCR experiments indicated that pfs is expressed constitutively from a promoter immediately upstream, and careful study of the pfs mutants revealed a growth defect that could not be attributed to a lack of AI-2. Metabolite profiling of the wild type and of a pfs mutant under various growth conditions revealed changes in the concentrations of several AMC metabolites, particularly SRH and SAH and under some conditions also HCY. Similar studies established that an N. meningitidis luxS mutant also has metabolite pool changes and growth defects in line with the function of LuxS downstream of Pfs in the AMC. Thus, the observed growth defect of N. meningitidis pfs and luxS mutants is not due to quorum sensing but is probably due to metabolic imbalance and, in the case of pfs inactivation, is most likely due to toxic accumulation of SAH.     

Hybrid maize breeding with doubled haploids: V. Selection strategies for testcross performance with variable sizes of crosses and S1 families

In hybrid maize (Zea mays L.) breeding, doubled haploids (DH) are increasingly replacing inbreds developed by recurrent selfing. Doubled haploids may be developed directly from S₀ plants in the parental cross or via S₁ families. In both these breeding schemes, we examined 2 two-stage selecting strategies, i.e., considering or ignoring cross and family structure while selection among and within parental crosses and S₁ families. We examined the optimum allocation of resources to maximize the selection gain ΔG and the probability P(q) of identifying the q% best genotypes. Our specific objectives were to (1) determine the optimum number and size of crosses and S₁ families, as well as the optimum number of test environments and (2) identify the superior selection strategy. Selection was based on the evaluation of testcross progenies of (1) DH lines in both stages (DHTC) and (2) S₁ families in the first stage and of DH lines within S₁ families in the second stage (S₁TC-DHTC) with uniform and variable sizes of crosses and S₁ families. We developed and employed simulation programs for selection with variable sizes of crosses and S₁ families within crosses. The breeding schemes and selection strategies showed similar relative efficiency for both optimization criteria ΔG and P (0.1%). As compared with DHTC, S₁TC-DHTC had larger ΔG and P (0.1%), but a higher standard deviation of ΔG. The superiority of S₁TC-DHTC was increased when the selection was done among all DH lines ignoring their cross and family structure and using variable sizes of crosses and S₁ families. In DHTC, the best selection strategy was to ignore cross structures and use uniform size of crosses.     

Structure of Adenovirus Type 21 Knob in Complex with CD46 Reveals Key Differences in Receptor Contacts among Species B Adenoviruses

The complement regulation protein CD46 is the primary attachment receptor for most species B adenoviruses (Ads). However, significant variability exists in sequence and structure among species B Ads in the CD46-binding regions, correlating with differences in affinity. Here, we report a structure-function analysis of the interaction of the species B Ad21 knob with the two N-terminal repeats SCR1 and SCR2 of CD46, CD46-D2. We have determined the structures of the Ad21 knob in its unliganded form as well as in complex with CD46-D2, and we compare the interactions with those observed for the Ad11 knob-CD46-D2 complex. Surface plasmon resonance measurements demonstrate that the affinity of Ad21 knobs for CD46-D2 is 22-fold lower than that of the Ad11 knob. The superposition of the Ad21 and Ad11 knob structures in complex with CD46-D2 reveals a substantially different binding mode, providing an explanation for the weaker binding affinity of the Ad21 knob for its receptor. A critical difference in both complex structures is that a key interaction point, the DG loop, protrudes more in the Ad21 knob than in the Ad11 knob. Therefore, the protruding DG loop does not allow CD46-D2 to approach the core of the Ad21 knob as closely as in the Ad11 knob-CD46-D2 complex. In addition, the engagement of CD46-D2 induces a conformational change in the DG loop in the Ad21 knob but not in the Ad11 knob. Our results contribute to a more profound understanding of the CD46-binding mechanism of species B Ads and have relevance for the design of more efficient gene delivery vectors.The 52 human adenovirus (Ad) serotypes are divided into seven species (species A to G) (20). Species B Ads are of interest, as they cause severe infections of the respiratory tract, urinary tract, and kidney as well as multiorgan system failure and death in immunocompromised patients (2, 23, 24). Species B Ads can be further grouped into subspecies B1 (Ad3, Ad7, Ad16, Ad21, and Ad50) and subspecies B2 (Ad11, Ad14, Ad34, and Ad35). Viruses in the two subspecies differ in their tropisms: while most B1 viruses cause ocular and/or acute respiratory tract infections, the B2 viruses primarily cause persistent infections of the urinary tract as well as eye infections, meningitis, and infections of the gastrointestinal tract (10, 11, 43). The subspecies B1 Ad21, which is the subject of this study, recently caused outbreaks of acute respiratory disease (28).Adenoviruses have a nonenveloped icosahedral capsid with a linear double-stranded DNA (46). The major capsid proteins are the hexon, the penton base, and the fiber. The trimeric fiber protein, which protrudes from each of the 12 capsid vertices, consists of three distinct domains: an N-terminal tail, an elongated shaft, and a globular knob. The knob mediates cellular attachment to the primary receptors CD46 (16, 26, 38), coxsackievirus and adenovirus receptor (CAR) (36), and sialic acid (3). Virus attachment is followed by internalization into the host cell via clathrin-coated endocytosis and macropinocytosis, triggered by αv integrins (17, 27, 45).The species B adenovirus receptor CD46 is a member of a family of proteins that regulate complement activation and are constructed mainly from short consensus repeat (SCR) domains (25). The extracellular portion of CD46 contains four such domains (SCR1 to SCR4), and structural and functional analyses have established that interactions with Ad knobs require only the SCR1 and SCR2 domains (34). The four SCR domains are followed by a 25-amino-acid sequence that is rich in serine, threonine, and proline (the STP region); a single transmembrane segment; and a short cytoplasmic tail. Structural information is currently limited to the N-terminal CD46 domains SCR1 and SCR2. The crystal structure of a fragment comprising these two domains revealed a pronounced kink between the two repeats and some flexibility at the domain interface (7). The protein is expressed on all human cells with the exception of erythrocytes. CD46 acts as a cofactor for factor I, a serine protease that blocks further recruitment of the membrane attack complex by cleaving C3b and C4b (25, 40).CD46 is also a receptor for many other pathogens, including measles virus, human herpesvirus 6, Neisseria gonorrhoeae, Neisseria meningitidis, and group A streptococci (8, 13, 22, 30, 37).The structural analysis of the Ad11 knob in complex with the SCR1-SCR2 fragment of CD46 (CD46-D2) showed that engagement by the knob triggers a conformational change in CD46, producing an elongated, nearly linear conformation that differs substantially from the kinked conformation of the unliganded receptor (34). Furthermore, the structure of this complex provided an explanation for the previously observed critical role of Ad11 knob residue Arg279 in CD46 binding. Earlier mutagenesis studies demonstrated that a mutation of Arg279 to glutamine abolishes binding to CD46-D2 (18). Although the structure of the complex showed that Arg279 does not contact the receptor directly, its side chain lies parallel to that of the CD46-contacting residue Arg280. Stacking interactions between the guanidinium groups of Arg279 and Arg280, resulting in an arginine sandwich, likely play a central role in determining receptor specificity (33), and the mutation of Arg279 is thought to prevent Arg280 from forming contacts with CD46 (18).Although it also uses CD46 as a receptor, the Ad21 knob does not contain an arginine sandwich, as the residue corresponding to Arg279 in Ad11 is a serine. Therefore, the mode of binding of the Ad21 knob to CD46 is likely distinct from that observed for Ad11, consistent with the observation that previously reported mutational studies failed to determine a central binding motif among species B Ads (32, 44). The Ad21 and Ad11 knobs exhibit low sequence identity, especially at the surface loops that mediate binding to CD46 in Ad11. We therefore used a combination of structural and functional studies to establish the mechanism of Ad21 knob binding to CD46-D2. Our structural analysis reveals substantial differences in the numbers and types of contacts between the complexes of Ad11 and Ad21 with CD46-D2 and also in the relative orientations of CD46-D2 and its contacting knobs. Furthermore, our analysis allows the comparison of structural features of the Ad21 and Ad35 knobs, which are closely related in sequence yet also display significantly different CD46-D2-binding properties as well as different tissue tropisms. Thus, our findings result in a significantly enhanced understanding of the interactions between species B Ads and CD46.In addition to their role as pathogens, species B Ads serve as widely used gene delivery vectors, as they can transduce a broad range of possible target cells that are normally poorly permissive to other Ads, such as hematopoietic stem cells, dendritic cells, and malignant tumor cells (19, 41). Therefore, our results should also guide efforts to improve the gene delivery properties of these viruses.     

XTandem Parser: An open‐source library to parse and analyse X!Tandem MS/MS search results

Identification of proteins by MS plays an important role in proteomics. A crucial step concerns the identification of peptides from MS/MS spectra. The X!Tandem Project ( http://www.thegpm.org/tandem ) supplies an open‐source search engine for this purpose. In this study, we present an open‐source Java library called XTandem Parser that parses X!Tandem XML result files into an easily accessible and fully functional object model ( http://xtandem‐parser.googlecode.com ). In addition, a graphical user interface is provided that functions as a usage example and an end‐user visualization tool.