Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

Background

The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α₂-antiplasmin (α₂AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α₂AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α₂AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.

Results

Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H₆NQEQVSPLTLLAG₄Y (designated XL1); H₆DQMMLPWAVTLG₄Y (XL2); H₆WQHKIDLPYNGAG₄Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α₂AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α₂AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.

Conclusions

Fusion protein XL5-HSA (DQMMLPWAVTLG₄Y-HSAH₆) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.     

Identifying the neural circuitry of alcohol craving and relapse vulnerability

With no further intervention, relapse rates in detoxified alcoholics are high and usually exceed 80% of all detoxified patients. It has been suggested that stress and exposure to priming doses of alcohol and to alcohol-associated stimuli (cues) contribute to the relapse risk after detoxification. This article focuses on neuronal correlates of cue responses in detoxified alcoholics. Current brain imaging studies indicate that dysfunction of dopaminergic, glutamatergic and opioidergic neurotransmission in the brain reward system (ventral striatum including the nucleus accumbens) can be associated with alcohol craving and functional brain activation in neuronal systems that process attentional relevant stimuli, reward expectancy and experience. Increased functional brain activation elicited by such alcohol-associated cues predicted an increased relapse risk, whereas high brain activity elicited by affectively positive stimuli may represent a protective factor and was correlated with a decreased prospective relapse risk. These findings are discussed with respect to psychotherapeutic and pharmacological treatment options.     

TRPM channels are required for rhythmicity in the ultradian defecation rhythm of <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

Ultradian rhythms, rhythms with a period of less than 24 hours, are a widespread and fundamental aspect of life. The mechanisms underlying the control of such rhythms remain only partially understood. Defecation in C. elegans is a very tightly controlled rhythmic process. Underlying the defecation motor programme is an oscillator which functions in the intestinal cells of the animal. This mechanism includes periodic calcium release and subsequent intercellular calcium waves which in turn regulate the muscle contractions that make up the defecation motor programme. Here we investigate the role of TRPM cation channels in this process.     

A regulatory network modeled from wild-type gene expression data guides functional predictions in Caenorhabditis elegans development

Background

The iJO1366 reconstruction of the metabolic network of Escherichia coli is one of the most complete and accurate metabolic reconstructions available for any organism. Still, because our knowledge of even well-studied model organisms such as this one is incomplete, this network reconstruction contains gaps and possible errors. There are a total of 208 blocked metabolites in iJO1366, representing gaps in the network.

Results

A new model improvement workflow was developed to compare model based phenotypic predictions to experimental data to fill gaps and correct errors. A Keio Collection based dataset of E. coli gene essentiality was obtained from literature data and compared to model predictions. The SMILEY algorithm was then used to predict the most likely missing reactions in the reconstructed network, adding reactions from a KEGG based universal set of metabolic reactions. The feasibility of these putative reactions was determined by comparing updated versions of the model to the experimental dataset, and genes were predicted for the most feasible reactions.

Conclusions

Numerous improvements to the iJO1366 metabolic reconstruction were suggested by these analyses. Experiments were performed to verify several computational predictions, including a new mechanism for growth on myo-inositol. The other predictions made in this study should be experimentally verifiable by similar means. Validating all of the predictions made here represents a substantial but important undertaking.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Predicted structure model of Bungarotoxin from Bungarus fasciatus snake

Snake venoms are cocktails comprising combinations of different proteins, peptides, enzymes and toxins. Snake toxins have diverse characteristics having different molecular configuration, structure and mode of action. Many toxins derived from snake venom have distinct pharmacological activities. Venom from Bungarus fasciatus (commonly known as banded krait) is a species of elapid snake found on the South East Asia and Indian sub-continent, mainly contains neurotoxins. Beta bungartotoxin is the major fraction of Bungarus venom and particularly act pre-synaptically by obstructing neurotransmitter release. This toxin in other snake species functionally forms a heterodimer containing two different subunits (A and B). Dimerization of these two chains is a pre-requisite for the proper functionality of this protein. However, B. fasciatus bungartotoxin contains only B chain and their structural orientation in yet to be resolved. Therefore, it is of interest to describe the predicted structure model of the toxin for functional insights. In this work we analyzed the neurotoxic nature, their alignments, secondary and three dimensional structures, functions, active sites and stability with the help of different bioinformatical tools. A comprehensive analysis of the predicted model provides approaching to the functional interpretation of its molecular action.     

Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium

A combination of stable isotopes (¹⁵N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N₂ production along a transect from the Omani coast to the central–northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world''s oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (<48 h) anoxic incubations with ¹⁵N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-¹⁵N-labeled N₂ from ¹⁵NO₂⁻, a signal often taken as the lone evidence for denitrification in the past. Although the central–NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.     

Anaerobic ammonia oxidation in a fertilized paddy soil

Evidence for anaerobic ammonium oxidation in a paddy field was obtained in Southern China using an isotope-pairing technique, quantitative PCR assays and 16S rRNA gene clone libraries, along with nutrient profiles of soil cores. A paddy field with a high load of slurry manure as fertilizer was selected for this study and was shown to contain a high amount of ammonium (6.2–178.8 mg kg⁻¹). The anaerobic oxidation of ammonium (anammox) rates in this paddy soil ranged between 0.5 and 2.9 nmolN per gram of soil per hour in different depths of the soil core, and the specific cellular anammox activity observed in batch tests ranged from 2.9 to 21 fmol per cell per day. Anammox contributed 4–37% to soil N2 production, the remainder being due to denitrification. The 16S rRNA gene sequences of surface soil were closely related to the anammox bacteria ‘Kuenenia'', ‘Anammoxoglobus'' and ‘Jettenia''. Most of the anammox 16S rRNA genes retrieved from the deeper soil were affiliated to ‘Brocadia''. The retrieval of mainly bacterial amoA sequences in the upper part of the paddy soil indicated that nitrifying bacteria may be the major source of nitrite for anammox bacteria in the cultivated horizon. In the deeper oxygen-limited parts, only archaeal amoA sequences were found, indicating that archaea may produce nitrite in this part of the soil. It is estimated that a total loss of 76 g N m⁻² per year is linked to anammox in the paddy field.     

<i>In vitro</i> evaluation of antifungal activity of Agave (<i>Agave scabra</i>, Salm Dyck) extracts against post-harvest mushrooms

The agricultural sector, and particularly the horticultural production, has a singular importance in agriculture, considering that it ranks second on agricultural products, nationally and worldwide. Fungal diseases are one of the major causes of vegetable loss during storage, reducing their nutritional value, quality and sale price. Vegetables are usually exposed to diverse treatments with chemical products before storage; as a result, fungal populations develop an increased resistance over time becoming more difficult to control. Because of this, research efforts toward finding more suitable chemicals to control fungal diseases are needed. Natural extracts may be an alternative solve this problem. In the present investigation the fungicidal activity of aqueous and ethanol extracts of Agave scabra was evaluated on the growth of Botrytis cinerea, Mucor sp., Aspergillus niger, Fusarium sp. and Penicillium sp., whose strains were isolated from potato and tomato. To assess their effects, the agar-dilution and agar-well techniques were performed. The ethanol extract was more effective against Botrytis cinerea and Mucor sp. when the agar-well method was used. However, when using the agar-dilution method the ethanol extract of Agave scabra inhibited the growth of Botrytis cinerea, Mucor sp. and Penicillium sp.