Functional and bioinformatics analyses reveal conservation of Cis-regulatory elements between sciaridae and Drosophilidae

The sciarid DNA puff C4 BhC4-1 gene is amplified and transcribed in salivary glands at the end of the larval stage. In transgenic Drosophila, the BhC4-1 promoter drives transcription in prepupal salivary glands and in the ring gland of late embryos. A bioinformatics analysis has identified 162 sequences similar to distinct regions of the BhC4-1 proximal promoter, which are predominantly located either in 5` or 3` regions or introns in the Drosophila melanogaster genome. A significant number of the identified sequences are found in the regulatory regions of Drosophila genes that are expressed in the salivary gland. Functional assays in Drosophila reveal that the BhC4-1 proximal promoter contains both a 129 bp (-186/-58) salivary gland enhancer and a 67 bp (-253/-187) ring gland enhancer that drive tissue specific patterns of developmentally regulated gene expression, irrespective of their orientation.

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Substitution of crude cell wall for neutral detergent fibre in the equations of the Cornell Net Carbohydrate and Protein System that predict carbohydrate fractions: application to sunflower (Helianthus annulus L.)

Prediction of carbohydrate fractions using equations from the Cornell Net Carbohydrate and Protein System (CNCPS) is a valuable tool to assess the nutritional value of forages. In this paper these carbohydrate fractions were predicted using data from three sunflower (Helianthus annuus L.) cultivars, fresh or as silage. The CNCPS equations for fractions B(2) and C include measurement of ash and protein-free neutral detergent fibre (NDF) as one of their components. However, NDF lacks pectin and other non-starch polysaccharides that are found in the cell wall (CW) matrix, so this work compared the use of a crude CW preparation instead of NDF in the CNCPS equations. There were no differences in the estimates of fractions B, and C when CW replaced NDF; however there were differences in fractions A and B2. Some of the CNCPS equations could be simplified when using CW instead of NDF Notably, lignin could be expressed as a proportion of DM, rather than on the basis of ash and protein-free NDF, when predicting CNCPS fraction C. The CNCPS fraction B(1) (starch + pectin) values were lower than pectin determined through wet chemistty. This finding, along with the results obtained by the substitution of CW for NDF in the CNCPS equations, suggests that pectin was not part of fraction B(1) but present in fraction A. We suggest that pectin and other non-starch polysaccharides that are dissolved by the neutral detergent solution be allocated to a specific fraction (B2) and that another fraction (B(3)) be adopted for the digestible cell wall carbohydrates.

European 1: A globally important clonal complex of Mycobacterium bovis

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Diazepam decreases leukocyte-endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

New perspectives on the role of aldosterone excess in cardiovascular disease

1. Evidence from recent experimental and clinical studies suggests that excessive circulating levels of aldosterone can bring about adverse cardiovascular sequelae independent of the effects on blood pressure. Examples of these sequelae are the development of myocardial and vascular fibrosis in uninephrectomized, salt-loaded rats infused with mineralocorticoids and, in humans, an association of aldosterone with left ventricular hypertrophy, impaired diastolic and systolic function, salt and water retention causing aggravation of congestion in patients with established congestive cardiac failure (CCF), reduced vascular compliance and an increased risk of arrhythmias (resulting from intracardiac fibrosis, hypokalaemia, hypomagnesaemia, reduced baroreceptor sensitivity and potentiation of catecholamine effects). 2. These sequelae of aldosterone excess may contribute to the pathogenesis and worsen the prognosis of CCF and hypertension. 3. The heart and blood vessels may be capable of extra-adrenal aldosterone biosynthesis, raising the possibility that aldosterone may have paracrine or autocrine (and not just endocrine) effects on cardiovascular tissues. 4. The high prevalence of CCF, which is associated with secondary aldosteronism, and primary aldosteronism (PAL; recently recognized to be a much more common cause of hypertension than was previously thought) argue for an important role for aldosterone excess as a cause of cardiovascular injury. 5. The recognition of non-blood pressure-dependent adverse sequelae of aldosterone excess raises the question as to whether normotensive individuals with PAL, who have been detected as a result of genetic or biochemical screening among families with inherited forms of PAL, are at excess risk of cardiovascular events. 6. Provided that patients are carefully investigated in order to permit the appropriate selection of specific surgical (laparoscopic adrenalectomy for PAL that lateralizes on adrenal venous sampling) or medical (treatment with aldosterone antagonist medications) management and safety considerations for the use of aldosterone antagonists are kept in mind, the appreciation of a widening role for aldosterone in cardiovascular disease should provide a substantially better outlook for many patients with CCF and hypertension.

Regulation of yeast acetohydroxyacid synthase by valine and ATP

The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC 4.1.3.18). The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity. It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes. One type of subunit contains the catalytic machinery, whereas the other has a regulatory function. Previously, we have shown [Pang and Duggleby (1999) Biochemistry 38, 5222-5231] that yeast AHAS can be reconstituted from its separately purified subunits. The, reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS. High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits. It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5 '-[beta,gamma -imido]triphosphate. The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model.

Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.

Spiroacetals in insects

Spiroacetals, cryptic ketodiols showing a hydroxyl group at both sides of a carbonyl whithin reachable distances are very widespread in nature. A group of 30 different structures, not including stereoisomers, represent volatile, less polar constituents of insect secretions. Five different systems were identified: 1,6-dioxaspirol[4.4]nonanes, 1,6-dioxaspiro[4.5]decanes, 1,6-dioxaspiro[4.6]undecanes, 1,7-dioxaspiro[5.5] undecanes, and 1,7-dioxaspiro[5.6]dodecanes. Some spiroacetals are insect pheromones: (2S,5R)-2-ethyl-1,6-dioxaspiro[4.4]nonane, chalcogran, 1, is a key component of the male produced aggregation pheromone of the spruce bark beetle, Pityogenes cha2cographus. In contrast, (5S,7S)-7-methyl-1,6-dioxaspiro[4.5]decane, 2, conophthorin, acts as a repellent or spacer in several bark beetles. Racemic 1,7-diosaspiro[5.5]undecane, olean, 5, is the female produced sex pheromone of the olive fly, Bactrocera (Dacus) oleae. The most widespread spiroacetal is 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 8. Tt often forms a mixture of (E,E)- and (E,Z)-isomers, the (E,E)-isomer showing (2S,6R,8S)-configuration. In the solitary bee, Andrena wilkella, it serves as an aggregation pheromone. Present knowledge on structures and distribution of volatile spiroacetals is comprehensively compiled. Stereochemical aspects and mass spectrometric fragmentation patterns are discussed in detail to facilitate identifications of hitherto unknown compounds. Synthetic approaches to spiroacetals are classified and reviewed. Last but not least, facts and speculations on the biosynthesis of volatile spiroacetals are presented.

Detection of lateral gene transfer among microbial genomes

An increasingly comprehensive assessment is being developed of the extent and potential significance of lateral gene transfer among microbial genomes. Genomic sequences can be identified as being of putatively lateral origin by their unexpected phyletic distribution, atypical sequence composition, differential presence or absence in closely related genomes, or incongruent phylogenetic trees. These complementary approaches sometimes yield inconsistent results. Not only more data but also quantitative models and simulations are needed urgently.

Long-distance signaling and the control of branching in the rms1 mutant of peal

The ramosus (rms) mutation (rms1) of pea (Pisum sativum) causes increased branching through modification of graft-transmissible signal(s) produced in rootstock and shoot. Additional grafting techniques have led us to propose that the novel signal regulated by Rms1 moves acropetally in shoots and acts as a branching inhibitor. Epicotyl interstock grafts showed that wild-type (WT) epicotyls grafted between rms1 scions and rootstocks can revert mutant scions to a WT non-branching phenotype. Mutant scions grafted together with mutant and WT rootstocks did not branch despite a contiguous mutant root-shoot system. The primary action of Rms1 is, therefore, unlikely to be to block transport of a branching stimulus from root to shoot. Rather, Rms1 may influence a long-distance signal that functions, directly or indirectly, as a branching inhibitor. It can be deduced that this signal moves acropetally in shoots because WT rootstocks inhibit branching in rms1 shoots, and although WT scions do not branch when grafted to mutant rootstocks, they do not inhibit branching in rms1 cotyledonary shoots growing from the same rootstocks. The acropetal direction of transport of the Rms1 signal supports previous evidence that the rms1 lesion is not in an auxin biosynthesis or transport pathway. The different branching phenotypes of WT and rms1 shoots growing from the same rms1 rootstock provides further evidence that the shoot has a major role in the regulation of branching and, moreover, that root-exported cytokinin is not the only graft-transmissible signal regulating branching in intact pea plants.

Microbial interactions with tannins: Nutritional consequences for ruminants

Polyphenolics are widely distributed in the plant kingdom and are often present in the diet of herbivores. The two major groups of plant polyphenolic compounds other than lignin are condensed and hydrolysable tannins. These compounds can have toxic and/or antinutritional effects on the animal. It is well established that tannins complex with dietary proteins can reduce nitrogen supply to the animal, but the ability of gastrointestinal microorganisms to metabolise these compounds and their effects on microbial populations have received little attention. In this paper, we review recent literature on the topic as well as present research from our laboratories on the effect of condensed tannins on rumen microbial ecology and rumen metabolism. Interactions of tannins with dietary components and endogenous protein in the rumen and post-ruminally, and their impact on the nutrition of the animal are considered. (C) 2001 Elsevier Science B.V. All rights reserved.

Exploiting the versatility of human cytochrome P450 enzymes: The promise of blue roses from biotechnology

The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

Antagonism of the two-needle pine stem rust fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum in vitro and in planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degreesC. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wail and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host tell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta -1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.