New thermostable endoglucanase from Spirochaeta thermophila and its mutants with altered substrate preferences.

Endoglucanases are key elements in several industrial applications, such as cellulosic biomass hydrolysis, cellulose fiber modification for the production paper and composite materials, and in nanocellulose production. In all of these applications, the desired function of the endoglucanase is to create nicks in the amorphous regions of the cellulose. However, endoglucanase can be diverted from its activity on the fibers by other substrates-soluble oligosaccharides.

Chemo-enzymatic three-step conversion of glucose to kojic acid.

Kojic acid is an important biomolecule, currently produced by fermentation and having a wide range of potential applications. A faster and more direct chemical route could open the door for its large-scale production and wider utilization in biorefineries. Here we describe an efficient method for the preparation of kojic acid from d-glucose via glucosone by a three-step chemo-enzymatic route.

Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display.

Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5 and to construct new artificial TNF-binding proteins. We obtained a Fn3 gene combinatorial library and screened it using the bacterial display method.

Enzymatic Processes to Unlock the Lignin Value.

Main hurdles of lignin valorization are its diverse chemical composition, recalcitrance, and poor solubility due to high-molecular weight and branched structure. Controlled fragmentation of lignin could lead to its use in higher value products such as binders, coatings, fillers, etc. Oxidative enzymes (i.

SNP detection using trityl mass tags.

A new method suitable for single nucleotide polymorphism (SNP) detection using differential oligonucleotide probe extension has been developed. Sulfur-linked laser-cleavable trityl labels are implemented in this protocol. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption-ionization of the cleavable labels.

Role of protein kinase C in Ca(2+) homeostasis disorders in cultured rat neurons during hyperstimulation of glutamate receptors.

The primary culture of rat cerebellar neurons was used to study protein kinase C activity, intracellular variations in calcium concentration ([Ca(2+)]i), changes in the mitochondrial potential, and neuronal death during hyperstimulation of glutamate receptors and after 24-h incubation with phorbol ester. Prolonged exposure of neurons to glutamate (100 microM, 45 min) was followed by the development of delayed calcium dysregulation. Protein kinase C activity depended on the time of cell incubation with glutamate.

Novel mass tags for single nucleotide polymorphism detection.

A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption/ionization of the cleavable labels. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study.

Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo.

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs).

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli.

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes.

Interaction of "readthrough" acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behavior.

Behavioral reactions to stress are altered in numerous psychiatric and neurodegenerative syndromes, but the corresponding molecular processes and signal transduction pathways are yet unknown. Here, we report that, in mice, the stress-induced splice variant of acetylcholinesterase, AChE-R, interacts intraneuronally with the scaffold protein RACK1 and through it, with its target, protein kinase CbetaII (PKCbetaII), which is known to be involved in fear conditioning. In stress-responsive brain regions of normal FVBN mice, the mild stress of i.

Complex regulation of acetylcholinesterase gene expression in human brain tumors.

To study the regulation of acetylcholinesterase (AChE) gene expression in human brain tumors, 3' splice variants of AChE mRNA and potentially relevant transcription factor mRNAs were labeled in primary astrocytomas and melanomas. AChE-S and AChE-R mRNA, as well as Runx1/AML1 mRNA accumulated in astrocytomas in correlation with tumor aggressiveness, but neither HNF3beta nor c-fos mRNA was observed in melanoma and astrocytomas. Immunohistochemistry demonstrated nuclear Runx1/AML1 and cellular AChE-S and AChE-R in melanomas, however, only AChE-S, and not the secreted AChE-R variant, was retained in astrocyte tumor cells.

The structure, function and application of the hammerhead ribozyme.

The hammerhead ribozyme is one of the smallest ribozymes known and catalyses the site-specific hydrolysis of a phosphodiester bond. This small ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression.

Probing accessible sites for ribozymes on human acetylcholinesterase RNA.

In order to design ribozymes for the efficient cleavage of a human acetylcholinesterase (AChE) in vitro transcript, a completely randomized decadeoxyribonucleotide (dN10) was used in conjunction with RNase H to identify suitable sites for annealing. Based on the observed cleavage pattern, ribozymes were designed to cleave the transcript at these positions. Five ribozymes so designed proved to be efficient in the transcript cleavage (k(react)/Km ranged from 0.

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene.

[New site-specific endonucleases from strains of Bacillus].

In a search for new restriction endonucleases type II, among forty bacterial strains of the Bacillus genus two strains producing site-specific endonucleases have been found. Endonucleases BbvAIII and BspFI, isolated from B. brevis BLM B-677 and B.

Reagent for introducing pyrene residues in oligonucleotides.

A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.

[Artificial DNA splicing using directed ligation].

An approach to the directed genetic recombination in vitro has been devised, which allows for joining, in a predetermined chemical-enzymatic way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA Splicing by Directed Ligation, SDL). The approach makes use of amplification, by several polymerase chain reactions (PCR), of the chosen DNA segments. The corresponding primers contain recognition sites of the class IIS restriction endonucleases, yielding protruding ends of unique primary structures.

A modified approach to identification of the sickle cell anemia mutation by means of allele-specific polymerase chain reaction.

The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue.

Method of artificial DNA splicing by directed ligation (SDL).

An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary.

Synthesis and modification of genes through artificial splicing by directed ligation (ASDL).

An approach to the directed genetic recombination in vitro mediated by synthetic oligodeoxynucleotides and polymerase chain reaction (PCR) is devised, which allows the joining, in a predetermined chemical-enzymatic way, of a series of DNA segments to give a precisely spliced polynucleotide sequence (Artificial Splicing by Directed Ligation, ASDL). The approach can thus lead to the totally processed eukaryotic genes using genomic DNA, with no mRNA needed. This approach has been used for the synthesis of artificial genes of interleukin-1 alpha and, in combination with PCR on the mRNA-cDNA duplex as template, of interleukin-1 receptor antagonist and their analogues, as well as for the modified genes.