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At least 145 records · Page 8Linked to original sources
PubMed1984

Pathogenesis of meningitis: experimental studies on the molecular basis of Haemophilus influenzae infection.

The successful development and implementation of rational strategies for the prevention of bacterial meningitis should be facilitated by acquiring a more detailed knowledge of its pathophysiology. We have used a biologically relevant rat model of meningitis in conjunction with classical microbial genetics and recombinant DNA technology to investigate the molecular basis of Haemophilus influenzae pathogenicity. These studies aim to define how specific bacterial genes mediate the potential of H. influenzae to colonize the nasopharynx, disseminate within the blood stream and invade the central nervous system. By identifying the state or stages in the pathogenic sequence for which the determinant is critical, this approach should also provide insight into the relevant host defense mechanisms which determine resistance or susceptibility. An understanding of the genetic basis of H. influenzae pathogenicity may develop basic knowledge relevant to the treatment and prevention of bacterial meningitis.

Animals↗
PubMed1993

Parameters influencing the productivity of recombinant E. coli cultivations.

In the past 10 to 15 years, many of the promises of microbial genetic engineering have been realized: the use of recombinant Escherichia coli has moved from the laboratory to the production facility, and the manufacture of therapeutic recombinant proteins such as human growth hormone and interleukins is a rapidly growing industry. Along with this progress, however, have come new problems to solve: bioreactor operators have discovered that large-scale cultivations of plasmid-containing bacteria do not behave in exactly the same way as those of plasmid-free cells, plasmid stability has been recognized as a major hurdle, and the protein product might not be present in a soluble form but rather as intracellular granules that resist solubilization. These and other difficulties represent a new generation of challenges for genetic engineering. However, genetic engineering can do more than solve these problems. Molecular biological techniques also have the ability to create new opportunities: to produce new compounds, to use cheaper substrates, to facilitate downstream processing, and to optimize production in new ways. The productivity of a cultivation can generally be expressed as the product of the cell density and the specific biological activity. Both of these parameters are influenced by a variety of factors. For recombinant cultivations, though, the level of biological activity, a reflection of the plasmid copy number and expression efficiency, is the more interesting and important consideration and will therefore be given more attention in our review. In this contribution, our general goal is to discuss the factors that influence the productivity of recombinant E. coli cultivations, covering parameters relating to DNA; parameters relating to protein synthesis; parameters relating to proteins; and parameters relating to downstream processing. The object is not to tell the reader how to choose the perfect plasmid, host, and cultivation conditions, but to make known the many variables involved in designing a recombinant process and to point out recent and potential advances made possible by genetic engineering. The discussion focuses on the production of a protein, but many of the same concepts apply to other cultivations of recombinant E. coli, including cases in which the desired product is not a protein or the cells have been designed for a special metabolic capability such as pollutant biodegradation.

Bacterial Proteins↗
PubMed1999

Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.

In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed.

Animals↗
PubMed1983

X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents.

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D10 values 5-10-fold of wild-type D10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D10 values less than 2-fold of wild-type D10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks.

Animals↗