Metagenomics,biotechnology with non-culturable microbes

Metagenomics as a new field of research has been developed over the past decade to elucidate the genomes of the non-cultured microbes with the goal to better understand global microbial ecology on the one side, and on the other side it has been driven by the increasing biotechnological demands for novel enzymes and biomolecules. Since it is well accepted that the majority of all microbes has not yet been cultured, the not-yet-cultivated microbes represent a shear unlimited and intriguing resource for the development of novel genes, enzymes and chemical compounds for use in biotechnology. However, with respect to biotechnology, metagenomics faces now two major challenges. Firstly, it has to identify truly novel biocatalysts to fulfil the needs of industrial processes and green chemistry. Secondly, the already available genes and enzymes need to be implemented in production processes to further prove the value of metagenome-derived sequences.     

Molecular characterisation of In51, a class 1 integron containing a novel aminoglycoside adenylyltransferase gene cassette, aadA6, in Pseudomonas aeruginosa

Polymerase chain reaction-amplification and subsequent sequencing of the variable region of a novel integron, In51, from Pseudomonas aeruginosa revealed the presence of a novel aminoglycoside adenylyltransferase gene, aadA6, together with an open reading frame of unknown function, orfD. AADA6 enzyme has only 75% amino acid identity with AADA1 and is able to confer high level resistance to streptomycin and spectinomycin in Escherichia coli.     

Molecular design of synthetic receptors with dynamic, imprinting, and allosteric functions

Sugar recognition in an aqueous system has been achieved using a boronic acid-diol interaction. Combination with an intramolecular amino group has enabled us to read out the binding process as a change in the fluorescence intensity. The novel interaction has been extended to dynamic sugar sensing utilizing an allosteric effect, molecular imprinting, and control of molecular assemblies.     

A novel ferritin gene, SferH-5, reveals heterogeneity of the 26.5-kDa subunit of soybean (Glycine max) seed ferritin

A novel ferritin cDNA, SferH-5, has been cloned from 7-day-old soybean seedlings. Putative SferH-5 has 96% identity with SferH-1 reported previously. All the five amino acid variants distributed in the mature region are not involved in highly conserved residues associated with ferroxidase activity center. We speculate that SferH-5 encodes a novel 26.5-kDa subunit of soybean seed ferritin, which is designated H-5 in this study. Recombinant H-5 was able to assemble, together with co-expressed H-2, as a functional soybean seed ferritin-like complex, H-5/H-2. Our data reveal the potential heterogeneity of the 26.5-kDa subunit of soybean seed ferritin.     

Discovery of novel hydroxy-thiazoles as HIF-alpha prolyl hydroxylase inhibitors: SAR, synthesis, and modeling evaluation

Inhibition of the PHD2 enzyme has been associated with increased red blood cell levels. From a screening hit, a series of novel hydroxyl-thiazoles were developed as potent PHD2 inhibitors.     

New reagents, reactions, and peptidomimetics for drug design.

It has been a major focus in our laboratories to prepare novel reagents and peptidomimetic structures for drug design. We have designed and prepared novel guanidinylation reagents that can be employed in solution or as solid phase reagents. We and others have utilized the reagent 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) for amide bond formation to couple sterically hindered structures. These couplings proceed with remarkably strong resistance to racemization. In the area of peptidomimetics, we have incorporated novel building blocks to create biologically active compounds. These building blocks include thioether and alkylamine bridges, beta-methylated, and beta,beta-dimethylated amino acid residues. These mimetic structures have been incorporated into specific target molecules such as opioids to obtain cyclic peptidomimetics with potent and selective biological activity.     

Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems

We describe the development of a novel PCR assay for the rapid detection of members of the Brucella genus, and the differentiation between six recognized Brucella species. The assay has proven to be highly specific with the additional advantage of being suitable for use with both conventional and real-time PCR.     

Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.     

Synthesis and SAR of selective benzothiophene, benzofuran, and indole-based peroxisome proliferator-activated receptor delta agonists

Recent literature has suggested the benefit of selective PPARdelta agonists for the treatment of atherosclerosis and other disease states associated with the metabolic syndrome. Herein we report the synthesis and structure-activity relationships of a series of novel and selective PPARdelta agonists. Our search began with identification of a novel benzothiophene template which was modified by the addition of various thiazolyl, isoxazolyl, and benzyloxy-benzyl moieties. Further elucidation of the SAR led to the identification of benzofuran and indole based templates. During the course of our research, we discovered three new chemical templates with varying degrees of affinity and potency for PPARdelta versus the PPARalpha and PPARgamma subtypes.     

Design, synthesis, and evaluation of potent and selective benzoyleneurea-based inhibitors of protein geranylgeranyltransferase-I

A series of novel protein geranylgeranyltransferase-I (PGGTase-I) inhibitors based on a benzoyleneurea scaffold has been synthesized. Using a benzoyleneurea scaffold as a mimetic for the central dipeptide (AA), we have developed CAAX peptidomimetic inhibitors that selectively block the activity of PGGTase-I over the closely related enzyme protein farnesyltransferase. In this new class of PGGTase-I inhibitors, compound (6c) with X=L-phenylalanine, displayed the highest inhibition activity against PGGTase-I with an IC50 value of 170 nM. The inhibitors described in this study represent novel and promising leads for the development of potent and selective inhibitors of mammalian PGGTase-I for potential application as antitumor agents.     

Unintentional selection,unanticipated insights: introductions,stocking and the evolutionary ecology of fishes

Natural environmental change has produced countless opportunities for species to disperse into and persist in habitats where they previously did not exist. Introduction and stocking programmes have facilitated similar sorts of colonization opportunities across considerably greater geographical scales and often in much shorter periods of time. Even though the mechanism of colonization differs, the result can be the same: evolutionary change in the colonizing population in response to novel selection pressures. As a consequence, some human‐mediated fish transfers have unintentionally yielded novel research opportunities to study how phenotypes and genes interact with their environment and affect ecological and evolutionary change. The primary purpose here is to explore how work, directly or indirectly involved with human‐mediated transfers, has unintentionally yielded novel research and research opportunities in fish ecology and evolution. Insights have produced new knowledge or altered previously held perceptions on topics such as local adaptation, rate of evolutionary change, phenotypic plasticity, alternative reproductive strategies, population structure and colonization probability. Well‐documented stocking programmes, especially in terms of history, numbers and original population sources, can provide highly fertile ground for generating further insights on the ecology and evolution of fishes and of the factors likely to influence the success of conservation‐based, restoration programmes.     

Design,synthesis, and structure-activity relationship studies of novel triazole agents with strong antifungal activity against Aspergillus fumigatus

The incidence of invasive fungal infections has dramatically increased for several decades. In order to discover novel antifungal agents with broad spectrum and anti-Aspergillus efficacy, a series of novel triazole derivatives containing 1,2,3-benzotriazin-4-one was designed and synthesized. Most of the compounds exhibited stronger in vitro antifungal activities against tested fungi than fluconazole. Moreover, 6m showed comparable antifungal activity against seven pathogenic strains as voriconazole and albaconazole, especially against Aspergillus fumigatus (MIC = 0.25 μg/ml), and displayed moderate antifungal activity against fluconazole-resistant strains of Candida albicans. A clear SAR study indicated that compounds with groups at the 7-position resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.     

The genetics of bird migration: stimulus, timing, and direction

The extent to which genetic factors are directly involved in the control of bird migration and the mode of inheritance involved has been studied systematically over the past 15 years in the Blackcap Sylvia atricapilla by cross-breeding and selective breeding. Results have also been obtained from a few experimental and field studies on Robins Eritfiacus rubecula, Blackbirds Turdus merula and Song Sparrows Melospiza melodia. Cross-breeding of migrants with nonmigrants has resulted in the partial transmission of migratory activity into the F, generation indicating that the urge to migrate is inherited and is based on a multilocus system with a threshold for expression. Migratoriness and sedentariness in obligate partial migrants is probably inherited in a similar way, suggesting that the decision to migrate also has a strong genetic basis. Both traits can be selected to phenotypic uniformity within 3–6 generations indicating an extremely high evolutionary potential. Orientation behaviour can also be transmitted to the offspring of a nonmigratory population by cross-breeding. Cross-breeding individuals with different migratory directions produced offspring with phenotypically intermediate directional preferences, suggesting that the migratory direction is also a predominantly heritable character. In the current development of novel migratory habits in those Central European Blackcaps that now winter in the British Isles, the inheritance of the novel migratory direction may be crucial. Genetic variation in migratory events seems to be sufficient to allow for many microevolutionary processes.     

Target-assisted iterative screening reveals novel interactors for PSD95, Nedd4, Src,Abl and Crk proteins

A new in vitro screening method has been developed and applied to a commercial phage-displayed cDNA library to search for novel protein-protein interactions. PDZ, WW and SH3 domains from PSD95, Nedd4, Src, Abl and Crk proteins were used as targets. 12 novel putative and 2 previously reported interactions were identified in test screens. The novel screening format, dubbed TAIS (target-assisted iterative screening), is discussed as an alternative platform to existing technologies for a pair-wise characterization of protein-protein interactions.     

The Stereostructure of Cotylenol,the Aglycone of Cotylenins Leaf Growth Substances

The stereostructure of cotylenol has been assigned as I on the basis of degradative and spectroscopic evidence. Cotylenol is a novel diterpene with ophiobolane skeleton.     

Energy, Life, and ATP

The mechanism by which ATP is synthesized during oxidative and photophosphorylation has been elucidated by oxygen exchange and other studies: a novel form of catalysis--termed rotary catalysis--is involved.     

Evaluation of a novel aminopeptidase N inhibitor, in vitro, using two assay systems

The activities of the novel aminopeptidase N inhibitor (APNI), beta-Amino-alpha-Hydroxyl-Phenyl butanic acid-Valine (AHPA-Val), were compared with APNI (amastatin). AHPA-Val and amastatin produced competitive inhibition of the hydrolysis of Tyr-Gly in the guinea-pig striatal membrane preparation, with K(i) equal to 14.06 microM and 12.48 microM respectively. Met-enkephalin-induced twitch inhibition of the guinea-pig ileum preparation was enhanced by AHPA-Val and amastatin with pA(1/2) values (the negative logarithm concentration of APNI that decreased the IC(50) of Met-enkephalin by half), of 7.08 and 7.79 respectively. These results suggest that AHPA-Val has good activity as an APNI and that these two assay systems are useful for evaluating the potency of novel APNIs.     

Design and synthesis of a potent,highly selective,orally bioavailable,retinoic acid receptor alpha agonist

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARβ, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARβ (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.     

Toward novel HIV-1 integrase binding inhibitors: molecular modeling, synthesis, and biological studies

The identification of a novel hit compound as integrase binding inhibitor has been accomplished by means of virtual screening techniques. A small family of structurally related molecules has been synthesized and biologically evaluated with one of the compounds showing an IC(50)=12 microM.