Biomedical subjects

S W Peretti

Publications and source records attributed to S W Peretti.

6 recordsLinked to original sources

PubMed1998

Coordinated clearance of periciliary liquid and mucus from airway surfaces.

Airway surface liquid is comprised of mucus and an underlying, watery periciliary liquid (PCL). In contrast to the well-described axial transport of mucus along airway surfaces via ciliary action, theoretical analyses predict that the PCL is nearly stationary. Conventional and confocal microscopy of fluorescent microspheres and photoactivated fluorescent dyes were used with well-differentiated human tracheobronchial epithelial cell cultures exhibiting spontaneous, radial mucociliary transport to study the movements of mucus and PCL. These studies showed that the entire PCL is transported at approximately the same rate as mucus, 39.2+/-4.7 and 39.8+/-4.2 micrometer/sec, respectively. Removing the mucus layer reduced PCL transport by > 80%, to 4.8+/-0.6 micrometer/sec, a value close to that predicted from theoretical analyses of the ciliary beat cycle. Hence, the rapid movement of PCL is dependent upon the transport of mucus. Mucus-dependent PCL transport was spatially uniform and exceeded the rate expected for pure frictional coupling with the overlying mucus layer; hence, ciliary mixing most likely accelerates the diffusion of momentum from mucus into the PCL. The cephalad movement of PCL along airway epithelial surfaces makes this mucus-driven transport an important component of salt and water physiology in the lung in health and disease.

Biological Transport↗

PubMed1998

Continuous culture dynamics for aniline metabolism by Pseudomonas sp. CIT1.

Inhibition by toxic substrates enables multiple steady states to arise in biodegradation systems. This phenomenon was investigated for the continuous metabolism of aniline by Pseudomonas sp. CIT1. Differences of various metabolic parameters between the two growth regimes (uninhibited and inhibited) and the transient response to a step-up in dilution rate were determined. Regulatory mechanisms consistent with the experimental evidence are proposed. Aniline is the transcriptional inducer of a metabolic pathway that converts aniline to TCA cycle intermediates. The suite of enzymes is coordinately expressed from a single promoter. We followed the level of the pathway mRNA using a fragment containing the catechol 2,3 dioxygenase gene (andioxB) and monitored the pathway enzyme activity using catechol 2,3 dioxygenase (C23D). The inhibited regime resulted in a 60% lower growth yield, near constant levels of C23D monomer, but a 50% reduction in the specific activity of C23D, increased RNA synthesis rates (total and aniline pathway mRNA), and elevated RNA decay rates. Elucidation of regulatory mechanisms indicates that C23D is noncompetitively inhibited by aniline and subject to feedback inhibition by 2-hydroxymuconic semialdehyde (HMS). During uninhibited growth regime operation, metabolism of HMS is the rate-limiting step; in contrast, conversion of aniline to catechol limits growth in the inhibited regime.

Aniline Compounds↗

PubMed1998

Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. I. Experiments.

A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization.

Bacillus↗

PubMed1998

Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. II. Modeling.

A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited.

Bacillus↗

PubMed1986

Studies of host-plasmid interactions in recombinant microorganisms.

Plasmid genes redirect some components of cellular metabolism into synthesis of plasmid gene products and additional plasmids. The stoichiometric and kinetic implications of these host-plasmid interactions have been investigated theoretically and experimentally. Using known pathway energetics, maximum theoretical yield factors based on ATP, glucose, and O2 have been estimated for recombinant Escherichia coli and compared with corresponding estimates for host cells alone, indicating major changes in carbon and energetic stoichiometry in recombinant cells in cases of high cloned gene expression. The influence of the number of plasmids in recombinant E. coli has been experimentally characterized using a series of pMB1 derivatives stably propagated at copy numbers from 12 to 408. Recombinant cell growth rate declines monotonically as plasmid content increases as does efficiency of plasmid gene expression. A detailed metabolically structured single-cell model for E. coli has successfully simulated these trends. Interrelationships among number of plasmids per cell, induction of expression of a plasmid gene, and recombinant population growth rate have been experimentally delineated for Saccharomyces cerevisiae containing plasmid pLGSD5 and derivatives in which the 2-micron origin of replication has been replaced by a cloned ARS1 sequence or its deletion fragments. The CEN4 centromere sequence has been included in some of these plasmids to provide more regular segregation. Specific growth rate of these recombinant yeasts exhibits a maximum as a function of plasmid content, an effect attributed to the interplay between beneficial effects of the plasmid in selective medium and parasitic effects on metabolism at larger plasmid content or with more plasmid gene expression activity. The yeast strains investigated exhibit substantial segregational instability that was characterized using a rapid-flow cytometry measurement based upon single-cell deletion of E. coli beta-galactosidase activity in recombinant cells.

DNA, Recombinant↗

PubMedDate not supplied

Lambda vectors for stable cloned gene expression.

The bacteriophage lambda offers a unique opportunity concurrently to minimize segregational instability in recombinant systems by chromosomal integration of the cloned gene and to achieve high cloned gene expression during an abortive lytic phase. Lysis leads approximately to a 100-fold amplification of the cloned gene. Cell lysis in the lytic state is blocked by a specific mutation (Sam), allowing the cell to maintain its integrity, and lambda DNA packaging is blocked by other mutations (Wam, Eam) that keep cloned genes open to transcription. In the presence of these mutations, extremely high levels of cloned beta-galactosidase (more than 15% of total cell protein) have been obtained during abortive lysis from vectors found to be essentially 100% stable for over 75 generations in the lysogenic phase.

Bacteriophage lambda↗