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PubMed2000

Implication of gene distribution in the bacterial chromosome for the bacterial cell factory.

As bacterial genome sequences accumulate, more and more pieces of data suggest that there is a significant correlation between the distribution of genes along the chromosome and the physical architecture of the cell, suggesting that the map of the cell is in the chromosome. Considering sequences and experimental data indicative of cell compartmentalisation, mRNA folding and turnover, as well as known structural features of protein and membrane complexes, we show that preliminary in silico analysis of whole genome sequences strongly substantiates this hypothesis. If there is a correlation between the genome sequence and the cell architecture, it must derive from some selection pressure in the organisms growing in the wild. As a consequence, the underlying constraints should be optimised in genetically modified organisms if one is to expect high product yields. Consequences in terms of gene expression for biotechnology are straightforward: knocking genes out and in genomes should not be randomly performed, but should follow the rules of chromosome organisation.

Bacteria↗
PubMed2003

Plant cell factories and mucosal vaccines.

Many advances continue to be made in the field of plant-derived vaccines. Plants have been shown capable of expressing a multicomponent vaccine that when orally delivered induces a T-helper cell subset 1 response and enables passive immunization. Furthermore, a plant-derived vaccine has been shown to protect against challenge in the target host. Increased antigen expression levels (up to 4.1% total soluble protein) have been obtained through transformation of the chloroplast genome. In view of these findings, plant-derived vaccines have been proved as valuable commodities to the world's health system; however, before their application, studies need to focus on optimization of immunization strategies and to investigate antigen stability.

Administration, Oral↗
PubMed1999

Mesonephric kidney--a stem cell factory?

Mesonephros is a vestige, transient renal organ that functions only during embryonic development. The anatomy, position and even cellular fate of the mesonephric kidney varies drastically among mammalian species. The origin of mesonephros from intermediate mesoderm and the dependence of its differentiation on the nephric or Wolffian duct have been well established. Commonly accepted is also the mesonephric origin of epididymal ducts of the male reproductive tract. Recently, upon the more profound understanding of the molecular mechanisms involved in the development of the permanent mammalian kidney, some light has been shed over the molecular events taking place during the mesonephric development as well. Because of the functional and structural similarities between the mesonephric and metanephric kidneys, it is not surprising that many molecules regulating metanephric development are also activated during mesonephric development. However, the multifunctional nature of mesonephros has been unexpected. First, it serves as an embryonic secretory organ, in some mammalian species more so than in others. It is thereafter removed by programmed cell death. Second, it is a source of multiple stem cells including somatic cells in the male gonad, vascular endothelial cells, and hematopoietic stem cells. Thus, mesonephros is a challenging model for studies on epithelial differentiation and organogenesis, regulation of apoptosis, sex determination and stem cell differentiation. In this review, we focus in the molecular and stem cell aspects in the differentiation of the mammalian mesonephros.

Animals↗
PubMed2000

Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering.

Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.

Journal Article↗
PubMed2002

Filamentous fungi as cell factories for heterologous protein production.

Filamentous fungi have been used as sources of metabolites and enzymes for centuries. For about two decades, molecular genetic tools have enabled us to use these organisms to express extra copies of both endogenous and exogenous genes. This review of current practice reveals that molecular tools have enabled several new developments. But it has been process development that has driven the final breakthrough to achieving commercially relevant quantities of protein. Recent research into gene expression in filamentous fungi has explored their wealth of genetic diversity with a view to exploiting them as expression hosts and as a source of new genes. Inevitably, the progress in the 'genomics' technology will further develop high-throughput technologies for these organisms.

Fermentation↗
PubMed2000

Clinical-scale production of granulocyte progenitor and post-progenitor cells using daniplestim, leridistim, Progenipoietin, Promegapoietin and autologous plasma.

BACKGROUND: Supplementation of PBPC autografts with ex vivo expanded PBMC may significantly reduce or eliminate the period of neutropenia associated with high-dose chemotherapy. METHODS: Unmanipulated growth-factor mobilized PBMC were expanded in media containing daniplestim, leridistim, Promegapoietin, and Progenipoietin (DLPP) and 2% autologous plasma at 4 x 10(5) PBMC/mL, first in 25 cm(2) T-flasks, with sampling on Days 7, 10, 13 and 15, and then in 1264 cm(2) Nunclon Cell Factories, with sampling on Days 7 and 13. RESULTS: In T25-flasks, maximal CFU-GM expansion ([38.2 +/- 9.5]-fold) occurred on Day 10, whereas maximal total cell expansion ([6.7 +/- 1.1]-fold) occurred on Day 15. Production of CD15(+)CD11b(-) and CD15(+)CD11b(+) granulocytic post-progenitors (3.0 +/- 0.4 x 10(6) and 3.7 +/- 0.9 x 10(6), respectively) was also maximal at Day 15. Compared with the previously studied combination of Flt3L, PIXY321, G-CSF, GM-CSF and Epo, the DLPP cocktail performed similarly, with the exception of yielding larger GM colonies at Day 10 and fewer granulocyte post-progenitors on Day 15. In Cell Factories, CFU-GM were expanded (31.6 +/- 14.5)-fold, while total nonadherent cells were expanded (2.6 +/- 0.5)-fold. The two stack Cell Factory cultures seeded with 1.0 x 10(8) unselected PBMC produced approximately 3.3 x 10(6) CFU-GM and 1.3 x 10(8) myeloid post-progenitors. DISCUSSION: Whereas expansion of cell numbers, CFU-GM and granulocytic post-progenitors in Cell Factories mirrored that achieved in T25-flasks, future preclinical studies with the DLPP cytokine combination may be performed in small volumes, with subsequent translation to the larger volume Cell Factories. Sufficient expansion can be achieved using the DLPP cytokine combination in the Cell Factories to provide the numbers of progenitors required for clinical trials.

Cell Culture Techniques↗
PubMedDate not supplied

Videomicrofluorometry on living cells and discriminant factorial analysis to study cell cycle distributions.

After a rapid overview of the approaches used to study cell cycle, a fluorescent digital imaging microscopy method is proposed. This method is improved by a factorial analysis relying on the evaluation of several parameters recorded on each living cell. Single lympho-blastoid living cells are labeled with three fluorescent markers: Hoechst 33342 for nuclear DNA, Rhodamine 123 for mitochondria and Nile Red for plasma membrane. For each cell, morphological and functional information parameters are obtained. A typological analysis is used to separate control cells into four groups: G0-G1, S, G2+M and polyploid cells Gn. These control cells define a learning population used to analyze untreated and adriamycine treated cells as supplementary individuals in a discriminant factorial analysis. Such an approach allows to accurately evidence the change of the values of some cellular parameters.

Benzimidazoles↗
PubMedDate not supplied

Determination of manganese in airborne particulates in a dry-cell battery factory using X-ray fluorescence technique.

Feasibility of using X-ray fluorescence techniques for the determination of airborne manganese in dry-cell battery factories was investigated. Both Energy Dispersive (EDX) and Wavelength Dispersive (WDX) X-ray fluorescence techniques of analysis were used. The minimum detectable quantity of Mn by the EDX method was found to be 50 micrograms for membrane filter (AA type) and 100 micrograms for cellulose filter (pore size 2.5 microns), respectively, with a counting time of 2000 s. The most suitable exciting source was 1.11 GBq (30 mCi) Pu-238. The minimum detectable quantity of Mn by the WDX method was 10 micrograms, with a counting time of 100 s. Results of the X-ray fluorescence and the AA methods were found to be in good agreement. Field measurements were carried out in a dry-cell battery factory. Concentrations of airborne manganese in the factory were found to be 0.02-41.1 mg/m3.

Air Pollutants, Occupational↗
PubMedDate not supplied

Enzymatic characterization of purified recombinant human renin.

Renin is a highly specific aspartyl protease of the renin-angiotensin system initially synthesized as preprorenin. Recombinant human prorenin was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line. The equivalent of 10-15 mg of recombinant human renin was secreted in the supernatant from each cell factory. Following a single affinity chromatography step using a renin inhibitor as the ligand, a 181-fold purification was achieved with 81% recovery of the renin activity. This highly pure recombinant enzyme having a specific activity of 3.44 mg angiotensin I.mg protein-1.h-1 was used for kinetic analysis. The kinetic parameters were determined with the natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp1-Asn14, at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both Km and kcat values for the peptidic substrate (13.3 microM and 8.1 s-1, respectively), when compared with values for the natural substrate (2.04 microM and 1.41 s-1), the catalytic efficiency (0.69 microM-1.s-1) of the enzyme for both substrates was the same. However, the kcat/Km value with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum pH 5.5. The recombinant human renin displayed similar optimum pH and kinetic parameters with angiotensinogen and the tetradecapeptide substrate when compared with human kidney renin.

Animals↗