Showing papers in "Molecular Biotechnology in 2007"

An overview of structural DNA nanotechnology.

Abstract: Structural DNA Nanotechnology uses unusual DNA motifs to build target shapes and arrangements. These unusual motifs are generated by reciprocal exchange of DNA backbones, leading to branched systems with many strands and multiple helical domains. The motifs may be combined by sticky ended cohesion, involving hydrogen bonding or covalent interactions. Other forms of cohesion involve edge-sharing or paranemic interactions of double helices. A large number of individual species have been developed by this approach, including polyhedral catenanes, a variety of single-stranded knots, and Borromean rings. In addition to these static species, DNA-based nanomechanical devices have been produced that are ultimately targeted to lead to nanorobotics. Many of the key goals of structural DNA nanotechnology entail the use of periodic arrays. A variety of 2D DNA arrays have been produced with tunable features, such as patterns and cavities. DNA molecules have be used successfully in DNA-based computation as molecular representations of Wang tiles, whose self-assembly can be programmed to perform a calculation. About 4 years ago, on the fiftieth anniversary of the double helix, the area appeared to be at the cusp of a truly exciting explosion of applications; this was a correct assessment, and much progress has been made in the intervening period.

Bioenergetic and Antioxidant Properties of Coenzyme Q10: Recent Developments

Abstract: For a number of years, coenzyme Q (CoQ10 in humans) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in plasma, and extensively investigated its antioxidant role. These two functions constitute the basis on which research supporting the clinical use of CoQ10 is founded. Also at the inner mitochondrial membrane level, coenzyme Q is recognized as an obligatory co-factor for the function of uncoupling proteins and a modulator of the transition pore. Furthermore, recent data reveal that CoQ10 affects expression of genes involved in human cell signalling, metabolism, and transport and some of the effects of exogenously administered CoQ10 may be due to this property. Coenzyme Q is the only lipid soluble antioxidant synthesized endogenously. In its reduced form, CoQH2, ubiquinol, inhibits protein and DNA oxidation but it is the effect on lipid peroxidation that has been most deeply studied. Ubiquinol inhibits the peroxidation of cell membrane lipids and also that of lipoprotein lipids present in the circulation. Dietary supplementation with CoQ10 results in increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoproteins to the initiation of lipid peroxidation. Moreover, CoQ10 has a direct anti-atherogenic effect, which has been demonstrated in apolipoprotein E-deficient mice fed with a high-fat diet. In this model, supplementation with CoQ10 at pharmacological doses was capable of decreasing the absolute concentration of lipid hydroperoxides in atherosclerotic lesions and of minimizing the size of atherosclerotic lesions in the whole aorta. Whether these protective effects are only due to the antioxidant properties of coenzyme Q remains to be established; recent data point out that CoQ10 could have a direct effect on endothelial function. In patients with stable moderate CHF, oral CoQ10 supplementation was shown to ameliorate cardiac contractility and endothelial dysfunction. Recent data from our laboratory showed a strong correlation between endothelium bound extra cellular SOD (ecSOD) and flow-dependent endothelial-mediated dilation, a functional parameter commonly used as a biomarker of vascular function. The study also highlighted that supplementation with CoQ10 that significantly affects endothelium-bound ecSOD activity. Furthermore, we showed a significant correlation between increase in endothelial bound ecSOD activity and improvement in FMD after CoQ10 supplementation. The effect was more pronounced in patients with low basal values of ecSOD. Finally, we summarize the findings, also from our laboratory, on the implications of CoQ10 in seminal fluid integrity and sperm cell motility.

Gene delivery by lentivirus vectors

Abstract: The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.

Adenoviral vectors for gene therapy

Abstract: Vectors based on human adenovirus serotypes 2 (Ad2) and 5 (Ad5) of species C possess a number of features that have favored their widespread employment for gene delivery both in vitro and in vivo. However, the use of recombinant Ad2- and Ad5-based vectors for gene therapy also suffers from a number of disadvantages. These vectors possess the tropism of the parental viruses, which infect all cells that possess the appropriate surface receptors, precluding the targeting of specific cell types. Conversely, some cell types that represent important targets for gene transfer express only low levels of the cellular receptors, which lead to inefficient infection. Another major disadvantage of Ad2- and Ad5-based vectors in vivo is the elicitation of both an innate and an acquired immune response. Considerable attention has therefore been focused on strategies to overcome these limitations, thereby permitting the full potential of adenoviral vectors to be realized.

History of plant tissue culture

Abstract: Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the begining of the 20th century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those that were already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. The historical development of these in vitro technologies and their applications are the focus of this chapter.

Carotenoids and Flavonoids Contribute to Nutritional Protection against Skin Damage from Sunlight

Abstract: The concept of photoprotection by dietary means is gaining momentum. Plant constituents such as carotenoids and flavonoids are involved in protection against excess light in plants and contribute to the prevention of UV damage in humans. As micronutrients, they are ingested with the diet and are distributed into light-exposed tissues, such as skin or the eye where they provide systemic photoprotection. β-Carotene and lycopene prevent UV-induced erythema formation. Likewise, dietary flavanols exhibit photoprotection. After about 10–12 weeks of dietary intervention, a decrease in the sensitivity toward UV-induced erythema was observed in volunteers. Dietary micronutrients may contribute to life-long protection against harmful UV radiation.

Alterations in Antioxidant Enzymes During Aging in Humans

Abstract: The oxidative stress theory of aging offers the best mechanistic elucidation of the aging phenomenon and other age-related diseases. The susceptibility of an individual depends on the antioxidant status of the body. In humans, the antioxidant system includes a number of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), nonenzymatic antioxidants such as glutathione (GSH), protein -SH, ascorbic acid, and uric acid, and dietary antioxidants. Antioxidant enzymes form the first line of defense against reactive oxygen species. In an earlier report, we showed that the plasma antioxidant potential in humans decreases as a function of age and that there are compensatory mechanisms operating in the body which are induced to maintain the antioxidant capacity during aging. In the present study, we report the relationship between human aging and antioxidant enzymes SOD and CAT; we also correlate the activity of these enzymes with the antioxidant capacity of the plasma. Our results show a significantly higher plasma SOD and CAT activity in older individuals than in younger individuals. The induction in activity of SOD and CAT during human aging may be a compensatory response of the individual to an increased oxidative stress.

A comparison of protein quantitation assays for biopharmaceutical applications.

Abstract: Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA™), DC, Fluorescamine and Quant-iT™) were compared to the ‘gold standard’ assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.

Carbofuran-Induced Oxidative Stress in Mammalian Brain

Abstract: Chronic exposure to carbofuran, a carbamate pesticide, via oral administration has been reported to generate reactive oxygen species (ROS) in rat brain. However, information regarding the effect of short-term intraperitoneal (i.p.) carbofuran intoxication on oxidative stress is lacking. In the present study, the effect of carbofuran on oxidative indices in brain of Wistar rats has been determined by exposing the animals to three subacute concentrations (0.2, 0.4 and 0.8 mg/kg body weight) equivalent to 10, 20, and 40%, respectively, of its LD50 (i.p.) for 24 h. Rat liver has been used as a positive control. The results demonstrated that carbofuran treatment at the 3 concentrations tested caused significant increase in lipid peroxidation (LPO) by 12.50, 34.38, and 59.38%, respectively. The increased oxidative stress at same pesticide concentrations significantly induced activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase in rat brain; the impact on catalase being more marked only at high-pesticide doses (0.4 and 0.8 mg/kg body weight). Carbofuran also caused reduction in protein content of rat tissues tested. Rat brain was more severely affected by carbofuran than liver. The results clearly demonstrated that i.p. administration of carbofuran accelerated oxidative stress in rat brain in a dose-dependent manner.

The important role of lipid peroxidation processes in aging and age dependent diseases.

Abstract: Any change in the cell membrane structure activates lipoxygenases (LOX). LOX transform polyunsaturated fatty acids (PUFAs) to lipidhydroperoxide molecules (LOOHs). When cells are severely wounded, this physiological process switches to a non-enzymatic lipid peroxidation (LPO) process producing LOO· radicals. These oxidize nearly all-biological molecules such as lipids, sugars, and proteins. The LOO· induced degradations proceed by transfer of the radicals from cell to cell like an infection. The chemical reactions induced by LO· and LOO· radicals seem to be responsible for aging and induction of age dependent diseases.

Anti-oxidants from green tea and pomegranate for chemoprevention of prostate cancer.

Abstract: Among males, prostate cancer has become the second leading cause of cancer-related deaths in North America, with similar trends in many Western and developing countries. One way to control prostate cancer is through chemoprevention, which refers to the administration of synthetic or naturally occurring agents to block, reverse, or delay the process of carcinogenesis. For a variety of reasons, the most important of which is human acceptance, for chemopreventive intervention, naturally occurring diet-based agents are preferred. Prostate cancer is an ideal candidate disease for chemopreventive intervention, because it grows very slowly, likely for decades, before symptoms arise and a diagnosis is finally established, it has a long latency period, and it is typically diagnosed in men >50 years of age. Most chemopreventive agents are antioxidant in nature. We have been defining the usefulness of dietary anti-oxidants for chemoprevention of prostate and other cancers. It is increasingly appreciated that some of these dietary anti-oxidants are nature’s gift molecules endowed with cancer preventive and therapeutic properties. This review will focus on prostate cancer chemopreventive effects of polyphenolic anti-oxidants derived from green tea and pomegranate. It is a challenge to custom-tailor these gift molecules as cocktails in concentrations that can easily be consumed by humans for delaying prostate and other cancers.

An improved method for single step purification of metagenomic DNA

Abstract: An improved method for purification of intact metagenomic DNA from soil has been developed using Q-Sepharose, which purified the DNA from phenolic and humic acid contaminants in a single step. The entire procedure for purification took only 45 min. A total of 81% of DNA was recovered after purification and there was 84% reduction in humic acid contents. The purified DNA was readily digested with restriction enzymes and can be further used for molecular applications.

Signaling Functions of Free Radicals Superoxide & Nitric Oxide under Physiological & Pathological Conditions

Abstract: Superoxide and nitric oxide are ubiquitous physiological free radicals that are responsible for many pathological disorders. Both radicals by themselves are relatively harmless but are the precursors of many toxic species such as peroxy and hydroxyl radicals, hydrogen peroxide, and peroxynitrite. However, it has been shown now that both superoxide and nitric oxide are also able to perform important signaling functions in physiological and pathophysiological processes. Wrongly named "superoxide," the radical anion of dioxygen is not a super-oxidant but the strong super-nucleophile, an efficient catalyst of heterogenic nucleophilic reaction. Due to this, superoxide plays an important role in many enzymatic processes such as the phosphorylation and activation of numerous protein kinases. On the other hand, superoxide inhibits the activation of phosphatases, the enzymes catalyzed by dephosphorylation of protein kinases. We suggest that superoxide catalyzes these enzymatic processes as a result of its nucleophilic properties. Another important physiological function of superoxide and nitric oxide is their competition for the interaction with mitochondrial cytochrome c oxidase. Disturbance of superoxide/nitric oxide balance leads to the dysfunction of mitochondria and the enhancement of apoptosis and oxidative stress, which are primary causes of various pathological disorders and aging. In conclusion, interplay between superoxide and nitric oxide, one of major factors of aging development, is considered.

Methods and goals for the use of in vitro and in vivo chemosensitivity testing.

Abstract: Sensitive, specific, and accurate methods to assay chemosensitivity are needed to (1) screen new therapeutic agents, (2) identify patterns of chemosensitivity for different tumor types, (3) establish patterns of cross-resistance and sensitivity in treatment of naive and relapsing tumors, (4) identify genomic and proteomic profiles associated with sensivity, (5) correlate in vitro response with preclinical in vivo effects and clinical outcomes for a particular therapeutic agent, and (6) tailor chemotherapy regimens to individual patients. Various methods are available to achieve these end points, including several in vitro clonogenic and proliferation assays, cell metabolic activity assays, molecular assay to monitor expression of markers for responsiveness, drug resistance, and for induction of apoptosis, in vivo tumor growth and survival assays in metastatic and orthotopic models, and in vivo imaging assays. The advantages and disadvantages of the specific assays are discussed. A summary of research questions related to chemosensitivity testing is also included.

ASK Family Proteins in Stress Response and Disease

Abstract: Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

Abstract: A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 104 copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.

A comparison of methods for successful triggering of gene silencing in Coprinus cinereus.

Abstract: Post-transcriptional gene-silencing methods (PTGS), including RNAi, are becoming increasingly pervasive in functional genomics. To advance analysis of the recently sequenced Coprinus cinereus genome, a high throughput gene silencing method is essential. We have exploited the GFP reporter gene to evaluate and quantify efficacy of three different silencing strategies. Modular constructs that encompassed antisense, untranslatable sense, and RNAi-mediating hairpin sequences, were transformed into a GFP-expressing host strain. Transformants exhibiting strong downregulation and partial suppression of GFP were recovered with all three constructs. Analyses of protein and transcriptional nucleic acids revealed that the antisense and hairpin sequences yielded similar levels of GFP suppression, and were both more efficient than untranslatable sense sequences. Our antisense vectors will expedite functional characterisation of C. cinereus and the modular nature of the constructs should permit exploitation of directional cDNA libraries for high throughput screening.

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm

Abstract: Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Isolation and Purification of RNA from Tissues Rich in Polyphenols, Polysaccharides, and Pigments of Annatto (Bixa orellana L.)

Abstract: The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system.

Abstract: Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ- 10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-beta-D-thiogalactopyranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.

Production of 1,3-propanediol from glycerol by recombinant E. coli using incompatible plasmids system.

Abstract: 1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare, primitive hematopoietic cells from patients with chronic myeloid leukemia

Abstract: Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib.

Microarray analysis: basic strategies for successful experiments.

Abstract: Microarrays offer a powerful approach to the analysis of gene expression that can be used for a wide variety of experimental purposes. However, there are several types of microarray platforms that are available. In addition, microarray experiments are expensive and generate complicated data sets that can be difficult to interpret. Success with microarray approaches requires a sound experimental design and a coordinated and appropriate use of statistical tools. Here, the advantages and pitfalls of utilizing microarrays are discussed, as are practical strategies to help novice users succeed with this method that can empower them with the ability to assay changes in gene expression at the whole genome level.

Use of thermolysin in the diagnosis of prion diseases

Abstract: The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPc into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27–30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.

Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli

Abstract: Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l(-1) isopropyl-beta-D- thiogalactopyranoside (IPTG) at 20 degrees C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.

Revealing differentially expressed proteins in two morphological forms of Spirulina platensis by proteomic analysis.

Abstract: Spirulina is distinguished from other cyanobacteria by its spiral morphology; however, this cyanobacterium has frequently been observed with a linear morphology in laboratory and industrial conditions. In our laboratory conditions, the simultaneously presence of the linear and spiral forms has also been observed. In the present study, the two forms of S. platensis C1 were separated and grown as axenic cultures in order to study the proteins that were differentially expressed in the soluble and insoluble protein fractions of the spiral and the linear forms. Two dimensional-differential gel electrophoresis (2D-DIGE) was performed to separate differentially expressed proteins that were subsequently identified by mass spectrometry. The differentially expressed proteins suggested two points. First, the morphological change is possibly induced by various environmental stresses such as oxygen level, carbon dioxide level, nutrient availability, and light. Second, the change of cell-shape might be a result of the change in a cell shape determination mechanism. Thus, this study is the first to show evidence at the protein level that may explain this morphological transformation in Spirulina.

RNAi-mediated male sterility of tobacco by silencing TA29.

Abstract: The superior performance of F1 hybrids has a significant impact on agricultural productivity For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco TA29 is expressed exclusively in anthers at the time of microspore development About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile Transgenic plants were phenotypically indistinguishable from non-transgenic plants At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed

Comparison of Alexa Fluor® and CyDye™ for practical DNA microarray use

Abstract: Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy™3 and Cy™5, are not ideal due to the decreased stability and fluorescence intensity of the Cy™5 dye relative to the Cy™3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor® 555 and Alexa Fluor® 647 are increasingly promoted as replacements for CyDye™ in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye™ resulted in significantly higher signal intensities (P 0.05). This translated to greater levels of differential gene expression with CyDye™ than with the Alexa Fluor® counterparts. However, CyDye™ fluorophores and in particular Cy™5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Abstract: Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369-375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.

Role of Ascorbic Acid in Scavenging Free Radicals and Lead Toxicity from Biosystems

Abstract: Free radicals are reactive species that are responsible for damaging normal cells and creating diseases in humans Antioxidants from natural resources or as supplements can scavenge these radicals A MedLine search indicates that vitamin C is the most investigated antioxidant responsible for the elimination of free radicals Its chelating property for the removal of neurotoxic lead, which creates oxidative stress in the human biosystem, was investigated and results indicate its great potential as a lead-detoxifying agent