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At least 865 records · Page 48Linked to original sources
PubMed2000

A functional role for complex gangliosides: motor deficits in GM2/GD2 synthase knockout mice.

Although gangliosides are abundant molecular determinants on all vertebrate nerve cells (comprising approximately 1.5% of brain dry weight) their functions have remained obscure. We report that mice engineered to lack a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase), and which express only the simple ganglioside molecular species GM3 and GD3, develop significant and progressive behavioral neuropathies, including deficits in reflexes, strength, coordination, and balance. Quantitative indices of motor abilities, applied at 8 and 12 months of age, also revealed progressive gait disorders in complex ganglioside knockout mice compared to controls, including reduced stride length, stride width, and increased hindpaw print length as well as a marked reduction in rearing. Compared to controls, null mutant mice tended to walk in small labored movements. Twelve-month-old complex ganglioside knockout mice also displayed significant incidence of tremor and catalepsy. These comprehensive neurobehavioral studies establish an essential role for complex gangliosides in the maintenance of normal neural physiology in mice, consistent with a role in maintaining axons and myelin (Sheikh, K. A. , J. Sun, Y. Liu, H. Kawai, T. O. Crawford, R. L. Proia, J. W. Griffin, and R. L. Schnaar. 1999. Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc. Natl. Acad. Sci. USA 96: 7532-7537), and may provide insights into the mechanisms underlying certain neural degenerative diseases.

Animals↗
PubMed1999

High-level expression of Rhizopus niveus lipase in the yeast Saccharomyces cerevisiae and structural properties of the expressed enzyme.

Rhizopus niveus lipase (RNL) has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain). To improve this enzyme's properties by protein engineering, we have developed a new expression system for the production of recombinant lipase in the yeast Saccharomyces cerevisiae. For the present study, we developed a more efficient expression system using the strain ND-12B and the multicopy-type plasmid pJDB219. We purified two types of recombinant lipases, each to a single peak by gel-filtration HPLC, although they were found to be heterogeneous by SDS-PAGE. Analysis of reversed-phase HPLC, N-terminal amino acid sequence, and sugar content showed that the difference between the two types of lipases was due mainly to their sugar content (high or low mannose type). Moreover, there were two species within each type of lipase. One kind was processed to the A-chain and B-chain as in the native lipase, while the other remained unprocessed. Although these yeast-purified lipases contained several posttranslational modifications and different glycosylations, their secondary structures were the same as those of the native lipase as measured by circular dichroism spectra and determination of disulfide bonding. This suggests that protein folding of the recombinant lipase occurred correctly in yeast.

Amino Acid Sequence↗
PubMed1998

Phytase: sources, preparation and exploitation.

This review deals with phytase (myo-inositol hexakisphosphate phosphohydrolase) and covers microbiological sources, phytase occurrence in plants and animals, its purification, physico-chemical and molecular properties. Protein engineering of phytase and potential enzyme applications are discussed.

6-Phytase↗
PubMed2003

Evolutionary landscapes for the acquisition of new ligand recognition by RNA aptamers.

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro--unconstrained by confounding biological factors--is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences--defined as the set's functional tolerance for point mutations--and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.

Base Sequence↗
PubMed2003

Chitinase-mediated inhibitory activity of Brassica transgenic on growth of Alternaria brassicae.

Chitinase, capable of degrading the cell walls of invading phytopathogenic fungi, plays an important role in plant defense response, particularly when this enzyme is overexpressed through genetic engineering. In the present study, Brassica plant (Brassica juncea L.) was transformed with chitinase gene tagged with an overexpressing promoter 35 S CaMV. The putative transgenics were assayed for their inhibitory activity against Alternaria brassicae, the inducer of Alternaria leaf spot of Brassica both in vitro and under polyhouse conditions. In in vitro fungal growth inhibition assays, chitinase inhibited the fungal colony size by 12-56% over the non-trangenic control. The bioassay under artificial epiphytotic conditions revealed the delay in the onset of disease as well as reduced lesion number and size in 35S-chitinase Brassica as compared to the untransformed control plants.

Alternaria↗
PubMed1995

A modified SOS-Chromotest procedure to test for genotoxicity and cytotoxicity in sediments directly without extraction.

A modified SOS-Chromotest bioassay using a chromogenic pad (pad procedure) was developed to test for genotoxicity in sediments directly without extraction. This test is based on the de novo synthesis of beta-galactosidase enzyme by a genetically-engineered E. coli strain PQ37. In the bioassay, an exponential growth phase antibiotic-containing culture of the test bacterium is introduced into a series of tubes with the first tube containing 0.1 gram of sediment. Serial dilutions are then made and the tubes of sediment plus bacterial culture are incubated at 37 degrees C for four hours, followed by placing a drop of each mixture on a chromogenic pad and additional incubation for 20 hours at 37 degrees C. The solid particulates are then washed off with tap water and positive (genotoxic) activity is noted by the presence of a distinctive blue colour on the pad. The SOS-Chromotest pad procedure may be best used as a relative measure of genotoxicity by comparing results to a reference sample. In addition it can also determine sediment cytotoxicity by comparing samples spiked with a genotoxic standard (i.e., 4-nitroquinoline-N-oxide). Preliminary results suggest that this new bioassay is highly sensitive, consistent and discriminating.

4-Nitroquinoline-1-oxide↗
PubMed1992

A simple and rapid method of preparing large fragments of dengue virus cDNA from replicative-form RNA using reverse transcriptase and PCR.

A method is described for cloning large fragments (1.5 kb to 2 kb) of dengue virus cDNA from replicative-form viral RNA. Aedes albopictus cells (C6/36 clone) infected with dengue virus contain double-stranded, replicative-form RNA molecules which were used as a template for an initial reverse transcription using a primer containing sequence homologous to regions of the genome at or near the 3' end of the gene being studied. The product was then used as a template for polymerase chain reaction (PCR) amplification using the same 3' primer and a second which hybridized to a region at the 5' end of the sequence to be cloned. Both primers were engineered to contain specific restriction enzyme cutting sites which enabled the PCR product to be cut and cloned directly into plasmids for sequencing and expression studies. We have used this method to construct clones of the envelope glycoprotein gene (E) and the non-structural genes 1 and 2a (NS1/2a) and 3 (NS3) of dengue type 2, Tonga 1974 strain, and E and NS1/2a from dengue type 3, H-87 strain, either as discrete genes or as constructs with long and short leader sequences, with or without anchor sequences. The method could be applied to the cloning of any gene from any flavivirus, directly from infected cell extracts, without the necessity for tedious virus purification steps.

Animals↗
PubMed1991

Multienzyme systems obtained by gene fusion.

The preparation of artificial bi- and polyfunctional enzymes by gene fusion appears to have great potential in enzyme technology. Their purification is facilitated compared with that of their naturally occurring counterparts and they exhibit favourable enzyme kinetics. Applications can be found in biochemical analysis, enzyme process technology and metabolic engineering.

Cloning, Molecular↗
PubMed2006

Twenty years research in cholinesterase biosensors: from basic research to practical applications.

Over the last decades, cholinesterase (ChE) biosensors have emerged as an ultra sensitive and rapid technique for toxicity analysis in environmental monitoring, food and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. Over the years, engineering of more sensitive ChE enzymes, development of more reliable immobilization protocols and progress in the area of microelectronics could allow ChE biosensors to be competitive for field analysis and extend their applications to multianalyte screening, development of small, portable instrumentations for rapid toxicity testing, and detectors in chromatographic systems. In this paper, we will review the research efforts over the last 20 years in fabricating AChE biosensors and the recent trends and challenges encounter once the sensor is used outside research laboratory for in situ real sample applications. The review will discuss the generations of cholinesterase sensors with their advantages and limitations, the existing electrode configurations and fabrication techniques and their applications for toxicity monitoring. We will focus on low-cost electrochemical sensors and the approaches used for enzyme immobilization. Recent works for achieving high sensitivity and selectivity are also discussed.

Biosensing Techniques↗
PubMed2004

Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations.

Filamentous fungi have long been used for the production of metabolites and enzymes. With developments in genetic engineering and molecular biology, filamentous fungi have also achieved increased attention as hosts for recombinant DNA. However, the production levels of non-fungal proteins are usually low. Despite the achievements obtained using molecular tools, the heterologous protein loss caused by extracellular fungal protease degradation persists. This review provides an overview of the potential bioprocessing strategies that can be applied to inhibit protease activity thereby enhancing heterologous protein production.

Bioreactors↗
PubMed2005

Macrolactonization catalyzed by the terminal thioesterase domain of the nonribosomal peptide synthetase responsible for lichenysin biosynthesis.

The excised terminal thioesterase of the lichenysin nonribosomal peptide synthetase was found to be a highly efficient and versatile enzyme. Its activity strictly requires the R configuration of the beta-hydroxy fatty acid and the side chains of aspartate-5 and isoleucine-7, but tolerates changes in five other residues of the substrate. Characterization of this enzyme facilitates future effort to engineer the lichenysin synthetase for biotechnological applications.

Catalysis↗
PubMed2006

Oxidative stress in cataracts.

Oxidative stress is the result of an imbalance of antioxidants and pro-oxidants. Since toxic free radicals are the result of normal metabolism, their destruction is imperative. Cataracts are the leading cause of blindness worldwide. Opacity of the lens is a direct result of oxidative stress. Cataracts occur primarily due to age, but also are common in diabetes where superoxide in the mitochondria is elevated as a result of hyperglycemia. This review will investigate the risk factors of cataract including diet (vitamins, fat and alcohol) as well as UV light and diabetes. The pathophysiology of lens opacification will be discussed and related to the biochemistry, especially during the aging process and in diabetes. Animal and human supplemental antioxidant studies will be reviewed and the mechanisms discussed for cataract prevention and treatment. New genetic engineering approaches to overexpress antioxidant enzymes have given intriguing results and show promise. Lastly, a new approach to target mitochondrial superoxide with antioxidant molecules will be outlined.

Journal Article↗
PubMed2001

Biomaterials integrated with electronic elements: en route to bioelectronics.

Bioelectronics is a progressing interdisciplinary research field that involves the integration of biomaterials with electronic transducers, such as electrodes, field-effect-transistors or piezoelectric crystals. Surface engineering of biomaterials, such as enzymes, antigen-antibodies or DNA on the electronic supports, controls the electrical properties of the biomaterial-transducer interface and enables the electronic transduction of biorecognition events, or biocatalyzed transformation, on the transducers. Bioelectronic sensing devices, biosensors of tailored sensitivities and specificities, are being developed.

Antigen-Antibody Reactions↗
PubMed2002

Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates.

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody-enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 microm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 microm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody-poly-HRP immunoconjugates contained an estimated number of 10(5)HRP/streptavidin microparticle and 10(6)HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 microm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5-8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.

HIV Antibodies↗
PubMed2002

Functional biosensor systems via surface-nanoengineering of electronic elements.

Bioelectronics is a progressing interdisciplinary research field that involves the integration of biomaterials with electronic transducers such as electrodes, field-effect transistors or piezoelectric crystals. Surface engineering of biomaterials such as enzymes, antigen-antibodies or DNA on the electronic supports controls the electrical properties of the biomaterial/transducer interface and enables the electronic transduction of biorecognition events, or biocatalyzed transformation, on the transducers. The development of biosensor systems of tailored sensitivities and specificities represents a major advance in bioelectronics.

Antigen-Antibody Reactions↗
PubMed2005

Discovery of enzymatic activity using stable isotope metabolite labeling and liquid chromatography-mass spectrometry.

Stable isotope labeling of an intracellular chemical precursor or metabolite allows direct detection of downstream metabolites of that precursor, arising from novel enzymatic activity of interest, using metabolite profiling liquid chromatography-mass spectrometry. This approach allows the discrimination of downstream metabolites produced from novel enzymatic activity from the unlabeled form of the metabolite arising from native metabolic processes within the cell. Even for the case in which a given product of novel enzymatic activity is a transient, the novel enzymatic activity that produced it can be demonstrated to exist indirectly by identification of product-specific downstream metabolites. Therefore, direct or indirect discovery of novel enzymatic machinery engineered within a cell can be accomplished without a requirement for direct product purification or identification. The application of this approach to confirm the presence of a novel metabolic activity, alanine 2,3-aminomutase, obtained by mutagenesis and selection are discussed. The advantages of metabolite profiling approaches to metabolic engineering in terms of accelerating enzyme discovery and development of intellectual property will also be highlighted.

Chromatography, High Pressure Liquid↗
PubMed2005

Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of beta-lactamase.

The choice of protein for use in technical and medical applications is limited by stability issues, making understanding and engineering of stability key. Here, enzyme destabilization by truncation was combined with directed evolution to create stable variants of TEM-1 beta-lactamase. This enzyme was chosen because of its implication in prodrug activation therapy, pathogen resistance to lactam antibiotics, and reporter enzyme bioassays. Removal of five N-terminal residues generated a mutant which did not confer antibiotic resistance at 37 degrees C. Accordingly, the half-life time in vitro was only 7 s at 40 degrees C. However, three cycles comprising random mutagenesis, DNA shuffling, and metabolic selection at 37 degrees C yielded mutants providing resistance levels significantly higher than that of the wild type. These mutants demonstrated increased thermoactivity and thermostability in time-resolved kinetics at various temperatures. Chemical denaturation revealed improved thermodynamic stabilities of a three-state unfolding pathway exceeding wild-type construct stability. Elongation of one optimized deletion mutant to full length increased its stability even further. Compared to that of the wild type, the temperature optimum was shifted from 35 to 50 degrees C, and the beginning of heat inactivation increased by 20 degrees C while full activity at low temperatures was maintained. We attribute these effects mainly to two independently acting boundary interface residue exchanges (M182T and A224V). Structural perturbation by terminal truncation, evolutionary compensation at physiological temperatures, and elongation is an efficient way to analyze and improve thermostability without the need for high-temperature selection, structural information, or homologous proteins.

Amino Acid Sequence↗