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PubMedDate not supplied

BIOLUMBASE--the database of natural and transgenic bioluminescent organisms.

The Institute of Biophysics SB RAS hosts and maintains a specialized collection of luminous bacteria (CCIBSO 836) containing over 700 strains isolated in various regions of the world's oceans. The culture collection is a source of lux genes and biologically active substances. The wide application of bioluminescence in medicine and ecology has given importance to analysing information on the structure and functioning of bioluminescence systems in natural and transgenic microorganisms, as well as on their features that are closely interrelated with bioluminescence. The aims of our BIOLUMBASE database are: gathering information on microorganisms with lux genes, their analysis and free access, and distribution of this data throughout the global network. The database includes two sections, natural and transgenic luminous microorganisms, and is updated by our own experimental results, the published literature and internet resources. For the future, a publicly available internet site for BIOLUMBASE is planned. This will list the strains and provide comprehensive information on the properties and functions of luminous bacteria, the mechanisms of regulation of bioluminescence systems, constructs with lux genes, and applications of bioluminescence in microbiology, ecology, medicine and biotechnology. It is noteworthy that this database will also be useful for evaluation of biological hazards of transgenic strains. Users will be able to carry out bibliographic and strain searches starting from any feature of interest.

Bacteria↗
PubMed2006

Engineering TCE-degrading rhizobacteria for heavy metal accumulation and enhanced TCE degradation.

Many superfund sites are currently co-contaminated with organic pollutants such as trichloroethene (TCE) and heavy metals. A promising strategy to address these mixed-waste situations is the use of TCE-degrading rhizobacteria that will survive and thrive in soil heavily polluted with heavy metals. In this work, a gene coding for the metal-binding peptide, EC20, was introduced into rhizobacteria engineered for TCE degradation, resulting in strains with both metal accumulation and TCE degradation capabilities. EC20 was displayed onto the cell surface of Pseudomonas strain Pb2-1 and Rhizobium strain 10320D using an ice-nucleation protein (INP) anchor. Expression of EC20 was confirmed by Western blot analysis and cells with EC20 expression showed sixfold higher cadmium accumulation than non-engineered strains in the presence of 16 microM CdCl(2). As expected, the TCE degradation rate was reduced in the presence of cadmium for cells without EC20 expression. However, expression of EC20 (higher cadmium accumulation) completely restored the level of TCE degradation. These results demonstrated that EC20 expression enhanced not only cadmium accumulation but also reduced the toxic effect of cadmium on TCE degradation. We expect that similar improvements will be observed when these engineered rhizobacteria are inoculated onto plant roots.

Biodegradation, Environmental↗
PubMed2007

A comparison of high cell density fed-batch fermentations involving both induced and non-induced recombinant Escherichia coli under well-mixed small-scale and simulated poorly mixed large-scale conditions.

In this work, multi-parameter flow cytometric techniques, coupled with dual colour fluorescent staining, have been used to study the metabolic consequences of inclusion body formation in high cell density fed-batch cultures of the recombinant E. coli strain MSD3735, producing the IPTG inducible model mammalian protein, AP50. Further, we report on the development of the scale-down, two compartment (STR + PFR) experimental simulation model to study, for the first time, the effect of a changing microenvironment with respect to three of the major spatial heterogeneities that may be associated with large-scale bioprocessing (pH, glucose and dissolved oxygen concentration) on a recombinant bacterial system. Using various time points for induction and various scale-down configurations, it has been shown that inclusion body formation is followed immediately by a detrimental progressive change in individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability, resulting in a lower final biomass yield. However, the extent of this change was found to be dependent on whether the AP50 protein was induced or not, on the time of induction and on which combination of heterogeneities was being simulated. From this and previous work, it is clear that the scale-down two-compartment model can be used to study the impact of genetically modifying an organism to produce inclusion bodies and any range and combination of potential heterogeneities known to exist at the large scale.

Animals↗
PubMed2007

CD4(+) T lymphocytes mediate colitis in HLA-B27 transgenic rats monoassociated with nonpathogenic Bacteroides vulgatus.

BACKGROUND: HLA-B27/beta2 microglobulin transgenic (TG) rats develop spontaneous colitis when raised under specific pathogen-free (SPF) conditions or after mono-association with Bacteroides vulgatus (B. vulgatus), whereas germ-free TG rats fail to develop intestinal inflammation. SPF HLA-B27 TG rnu/rnu rats, which are congenitally athymic, remain disease free. These results indicate that commensal intestinal bacteria and T cells are both pivotal for the development of colitis in TG rats. However, it is not known if T cells are also required in the induction of colitis by a single bacterial strain. The aim of this study was therefore to investigate the role of T cells in the development of colitis in B. vulgatus-monoassociated HLA-B27 TG rats. METHODS: HLA-B27 TG rnu/rnu and rnu/+ rats were monoassociated with B. vulgatus for 8-12 weeks. CD4(+) T cells from mesenteric lymph nodes (MLNs) of B. vulgatus-monoassociated rnu/+ TG donor rats were transferred into B. vulgatus-monoassociated rnu/rnu TG recipients. RESULTS: B. vulgatus-monoassociated rnu/+ rats showed higher histologic inflammatory scores and elevated colonic interferon-gamma mRNA, cecal myeloperoxidase, and cecal IL-1beta levels compared to those in rnu/rnu TG rats that did not contain T cells. After transfer of CD4(+) cells from colitic B. vulgatus-monoassociated rnu/+ TG donor rats, B. vulgatus-monoassociated rnu/rnu TG recipients developed colitis that was accompanied by B. vulgatus-induced IFN-gamma production by MLN cells in vitro and inflammatory parameters similar to rnu/+ TG rats. CONCLUSIONS: These results implicate CD4(+) T cells in the development of colitis in HLA-B27 TG rats monoassociated with the nonpathogenic bacterial strain B. vulgatus.

Animals↗
PubMedDate not supplied

Patent absurdities.

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Biomedical Technology↗
PubMedDate not supplied

Water toxicity detection by a panel of stress-responsive luminescent bacteria.

A panel of Escherichia coli strains harbouring different stress-responsive promoters fused to a lux reporter system was used to assess the potential toxicity of 17 unknown model water samples. Using liquid cultures, nine out of 14 toxic samples were properly identified as toxic, whereas five were false negatives. All three non-toxic controls were identified correctly (no false positives). Two strains containing promoter-lux fusions were also tested when immobilized onto fibre-optic tips. One genotoxic sample and six toxic samples were correctly identified in this manner. The potential advantages and limitations in the use of genetically engineered bacteria as biosensors for water toxicity are discussed in view of these results.

Biosensing Techniques↗
PubMedDate not supplied

Monitoring genotoxicity during the photocatalytic degradation of p-nitrophenol.

p-Nitrophenol is a common structural unit of many pesticides and was chosen as a model compound to monitor genotoxicity during photocatalytic degradation. The genotoxicity of p-nitrophenol (PNP) and its breakdown products was measured using a bioluminescent bacterial bioassay, Vitotox. The genotoxic potential decreased with the concomitant photocatalytic degradation of the parent PNP concentration. The rate of genotoxicity reduction was slower than the rate of removal of the parent PNP, due to the formation of genotoxic by-products. After 6 h of photocatalytic treatment the total genotoxicity was removed. These results indicate that bioassays can be used as a simple and highly sensitive method for monitoring the general toxicity of chemical pollutants before, during and after photocatalytic treatment or other destructive processes.

Aliivibrio fischeri↗
PubMed2002

Generating new marine cell lines and transgenic species--conference summary.

Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment. Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others. Much still remains to be learned from these animals, and there is a growing need for new marine models. Models for expression of marine animal genes have been limited to heterologous expression systems. While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible. This has become particularly important with the development of functional genomics in marine models. These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology.

Animals↗
PubMed2002

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.

Animals↗
PubMed2005

Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867.

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Expression of a wheat cytochrome P450 monooxygenase in yeast and its inhibition by glyphosate.

Glyphosate is a non-selective herbicide which acts by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase. Wheat cytochrome P450 monooxygenase specifically catalyzes the metabolism of some sulfonylurea herbicides such as chlorsulfuron and triasulfuron. Here we report that glyphosate is an inhibitor of a wheat cytochrome (CYP71C6v1), the cDNA of which was amplified by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain had a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum, which is a typical spectral characteristic. The addition of glyphosate to the microsomal protein resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand. A spectral dissociation constant, K(s) of 70 micromol litre(-1) was observed and an IC50 of 11 micromol litre(-1) was found for glyphosate inhibition of CYP71C6v1 P450 activity.

Amino Acid Sequence↗
PubMed2006

Evaluation of oriental medicinal herbs for estrogenic and antiproliferative activities.

Herb extracts commercially used in Asia were screened for their estrogenic activity with a recombinant yeast system with both a human estrogen receptor (ER) expression plasmid and a reporter plasmid. Pueraria lobata (flower) had the highest estrogenic relative potency (RP, 17-estradiol = 1.00) (7.8e-3) (RP for + control), followed by Amomum xanthioides (1.3e-3), Glycyrrhiza uralensis, Zingiber officinale, Rheum palmatum, Curcuma aromatica, Eriobotrya japonica, Sophora flavescens, Anemarrhena asphodeloides, Polygonum multiflorum and Pueraria lobata (root) (9.5e-4-1.0e-4), and Prunus persica, Lycoppus lucidus and Adenophora stricta (9.0e-5-8.0e-5). In the antiproliferative assay, five human cancer cell lines representing different tissues (breast, lung and ovary) were used. Eriobotrya japonica showed strong cytotoxicity in ER-negative breast cancer (MDA-MB-231), cervix epitheloid (HeLa) and lung (A549) carcinoma cell lines.

Asia↗
PubMed2000

Isolation and characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase.

A 1.2-kb full-length cDNA sequence of a glyceraldehyde-3-phosphate dehydrogenase (GPD) gene was isolated from the mushroom, Pleurotus sajor-caju. The full-length cDNA of the GPD gene consists of 1248 nucleotides, predicted to encode a 36-kDa polypeptide consisting of 335 amino acid residues. Sequence analysis revealed that the GPD gene has more than 72-78% amino acid sequence homology with those of other Basidiomycetes. Expression of the GPD gene increased when P. sajor-caju was treated with various abiotic stresses, such as salt, cold, heat, and drought. There was an eightfold induction by drought treatment. Salt and cold stress induced four- and twofold induction of GPD gene expression, respectively. There was also a fivefold induction by heat stress. The GPD gene exhibits different expression patterns under different stress conditions. It reached its maximum expression level within two hours under cold or heat treatment. The mRNA levels of this gene increased proportionally to increasing treatment time under salt or dry conditions. Because the expression of GPD was significantly increased, we tested whether GPD could confer abiotic stress resistance when it was introduced into yeast cells. For this, a transgenic yeast harboring P. sajor-caju GPD was generated under the control of a constitutively expressed GAL promoter. The results from biofunctional analyses with GPD yeast transformants showed that GPD yeast transformants had significantly higher resistance to cold, salt, heat, and drought stresses.

Agaricales↗
PubMed2000

Periodic triggering of an inducible gene for control of a wild population.

A possible method of control for the management of wild populations consists of continual introgression of an inducible transgene by releasing transgenic individuals, with periodic exposure of the population to a trigger. Exposure to the trigger causes death or sterility in carriers of the transgene, but is otherwise benign. We investigate the effectiveness of various strategies for control. We show that suppression of the population density below any pre-specified level is possible using this technique. At the same time we show that too frequent or too efficient exposure to the trigger can select for non-transgenic genotypes at an intensity such that the population density will be largely unaffected by the trigger. Choices for management parameters can ensure that the latter scenario is avoided. We show that releasing individuals carrying the transgene at more than one locus facilitates density control.

Animals↗
PubMed2006

Established bioprocesses for producing antibodies as a basis for future planning.

In the early years of monoclonal antibody production for human therapy and diagnosis the methods used were arrived at by individual organisations. However, there is now an accumulating body of information on antibodies and fragments that have been produced by processes approved for human use. This information is becoming available at a time when the number of potential antibody-based medicines is growing sharply. The review addresses the reported production routes, their scale and the titres achieved. It identifies the performances of fed-batch and perfusion culture versus batch culture, and compares processes for the production of antibodies for diagnosis and for antibody fragments. The analysis defines the likely routes of future production in a sector where demanding regulations constrain new technology. It also indicates what levels of performance new approaches will need to meet to be competitive.

Animals↗
PubMed2003

The way ahead--the new technology in an old society.

Biotechnology is one of the most important scientific and technological revolutions of the last century and has greatly benefited various aspects of human life. The potentials are enormous and many breakthroughs have already been achieved in the area of healthcare, food, agricultural products and environmental production. The developments in this important area provide immediate benefits to mankind and offer environmentally friendly technologies for sustainable development. The Department of Biotechnology, Government of India, set up in 1986, has played an important catalytic role in promoting this revolutionary field. Research and development, technology validation and demonstration, technology transfer, human resource development, setting up of Centers of Excellence and promoting industry-academia interactions have been some of the major achievements during the last 15 years. A unique feature of this Department is the strong interaction with scientists and institutes across the country to promote biotechnology research and development efforts for commercialization and also to benefit the rural population for socio-economic development. A large number of research institutes/universities and organizations across the country have been supported in the areas of agriculture, healthcare, environment and industry. In addition, basic research has also been an important thrust area. In order to ensure that the benefits of biotechnology reach the masses at large, a very stringent biosafety mechanism has been adopted. India is a country rich in biodiversity with two hot spots and has a strong base of expertise available in nearly all fields--thus biotechnology could flourish leading to a Bioindustrial Revolution. We are today poised to be the leaders in this field in the 21st Century.

Agriculture↗
PubMed2004

Smart elastin-like polymers.

Elastin-like polymers are a new family of proteinaceous polymers. In these polymers converge a wide set of interesting properties that difficultly can be found together in other polymers. They are extremely biocompatible and show an acute smart and self-assembling behaviour. The increasing in complexity of the molecular design renders polymers showing combination of functionalities and complex performance. This is specially true nowadays where, taking into account their peptide nature, these polymers can be produced as recombinant proteins in genetically modified (micro)organisms. The absolute control and absence of randomness in the primary structure makes possible the realization of multifunctional polymers that can combine physical, chemical and biological functions in a desired fashion. It can be said that the molecular design is mainly limited by imagination and not by technique. This chapter is intended to show the molecular parameters that explain the smart behaviour finally observed and how the increase in complexity of the molecular designs leads to a richer behaviour of the polymer, as a way to show the enormous potential of this family in the development of advanced materials and systems for biomedicine and nanotechnology for the next decades.

Animals↗