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Biomedical subjects

M L Higgins

Publications and source records attributed to M L Higgins.

At least 37 records · Page 2Linked to original sources
PubMed1986

Effect of ciliostatic factors from Pseudomonas aeruginosa on rabbit respiratory cilia.

Heat-stable factors released by Pseudomonas aeruginosa in culture supernatants inhibit functional cilia of rabbit tracheal epithelium. Chloroform extraction removed heat-stable factors from stationary-phase culture supernatants. The extracts contained at least seven components separable by thin-layer chromatography (TLC). Cilioinhibitory components were identified as a phenazine derivative, pyo compounds (2-alkyl-4-hydroxyquinolines), and a rhamnolipid, also known as a hemolysin. Fluorescence and absorption spectra, relative migration on TLC, staining characteristics, and gas chromatography were the basis for identification. Inhibitory concentrations of each active component were established by quantitative measures of percent motility and beat frequency. Corresponding damage to ciliary ultrastructure was examined by electron microscopy. The pyo compounds produced ciliostasis at concentrations of 50 micrograms/ml, but without obvious ultrastructural lesions. The phenazine derivative also inhibited ciliary motility and caused some membrane disruption, although at substantially greater concentrations of 400 micrograms/ml. Limited exposure of tracheal explants to the rhamnolipid resulted in ciliostasis which was associated with altered ciliary membranes. More extensive exposure to rhamnolipid was associated with removal of dynein arms from axonemes. Pyocyanin at a concentration of 0.5 mg/ml did not inhibit ciliary beating under our conditions. The data suggest that the pyo compounds are the most effective per weight ciliostatic factors released by P. aeruginosa and rhamnolipid is the most destructive of cilia ultrastructure. By interfering with normal ciliary function, these ciliostatic factors may enable P. aeruginosa to more easily colonize the respiratory tract.

Animals↗
PubMed1986

Disruption of respiratory cilia by proteases including those of Pseudomonas aeruginosa.

Pseudomonad proteases disrupted the function and structure of demembranated cilia (axonemes) extracted from porcine tracheae. Proteolytic degradation by the two pseudomonad proteases elastase and alkaline protease and by trypsin and subtilisin impaired motility of ATP-activated axonemes. In addition, electron microscopic observation of negatively stained axonemes indicated that exposure to proteases caused dissociation into individual doublet or singlet microtubules. Inhibition of motility and axonemal fraying occurred when axonemes were treated with less than 5 U of proteolytic activity of any of the four proteases tested. When the effects of 2 U of each protease were compared, trypsin and subtilisin were able to produce immotility in less time than pseudomonad elastase and alkaline protease, while alkaline protease and subtilisin caused the most axonemal fraying in 10 min. Proteolytic digestion of axonemal proteins was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four proteases cleaved dynein proteins (proteins necessary for motility), though treatment with trypsin resulted in the most extensive solubilization of axonemal proteins. Trypsin and subtilisin both produced more changes in the protein profiles of treated axonemes, using fewer units of proteolytic activity, than the pseudomonad proteases. However, the limited alteration of only a few axonemal proteins by pseudomonad proteases indicates that cleavage need not be extensive to produce dysfunction. Thus, ciliary axonemes are susceptible to proteolytic attack. Degradation of axonemal proteins by pseudomonad proteases, which are released during active infection, may contribute to the impaired ciliary function associated with pseudomonad colonization of the respiratory tract.

Animals↗
PubMed1986

Inhibition of beta-lactam antibiotics at two different times in the cell cycle of Streptococcus faecium ATCC 9790.

Treatment of Streptococcus faecium ATCC 9790 with sublytic concentrations of beta-lactam antibiotics revealed two different division blocks in the cell division cycle. One block, induced by N-formimidoyl thienamycin and methicillin, occurred before the completion of chromosome replication, whereas the other, induced by cefoxitin and cephalothin, took place later in the cycle. In addition, these antibiotics gave rise to distinct morphological forms; the antibiotics acting at the earlier block point produced mainly "dumbbells," whereas those affecting the later time formed "lemons." When used in combination N-formimidoyl thienamycin and cefoxitin exerted synergistic killing on this strain. These data suggest that beta-lactam antibiotics have at least two sites of action in S. faecium.

Anti-Bacterial Agents↗
PubMed1986

Relationship of shape to initiation of new sites of envelope growth in Streptococcus faecium cells treated with beta-lactam antibiotics.

Exponential-phase cells of Streptococcus faecium were treated with concentrations of ampicillin and cephalothin which, over 60 min, had little effect on increase in culture mass but resulted in about a 65% inhibition of increase in cell numbers. The resulting drug-treated cells underwent about a doubling in cell mass and volume above that of the untreated cells. The newly divided cells produced in the presence of drugs were shown to be due to the division of central or primary sites of envelope growth present at the time of treatment. Sites that were newly initiated (secondary sites) at the time of treatment or sites initiated after treatment did not divide but enlarged in length and girth to give abnormally large cells. Although the increase in average total volume was the same after each interval of treatment with ampicillin and cephalothin, the primary growth sites of the cephalothin-treated cells grew somewhat more slowly, and their secondary sites grew somewhat more quickly, than did those of the ampicillin-treated cells. Cephalothin-treated cells initiated secondary sites at a rate similar to that of the untreated cells, whereas the ampicillin-treated cells exhibited reduced rates of secondary site initiation. Two models are presented that account for these results.

Ampicillin↗
PubMed1986

Autoradiographic studies of the synthesis of RNA and protein as a function of cell volume in Streptococcus faecium.

Mid-exponential-phase cultures were either labeled continuously with tritiated leucine and uracil or pulse-labeled with tritiated leucine. The amount of leucine and uracil incorporated into protein or RNA per cell was determined by grain counts of autoradiographs of cells seen in electron micrographs; the volume of each cell was determined by three-dimensional reconstruction. The average number of autoradiographic grains around cells continuously labeled with uracil and leucine increased linearly with cell volume. In contrast, while the average grain count around cells pulse-labeled with leucine increased in a near-linear fashion over most of the volume classes, less than the expected number of grains were seen around cells in large- and small-size classes. The distribution of grains around cells from both the continuously and pulse-labeled populations could be fit at the 5% confidence level with a Poisson distribution modified to take into consideration the volume distribution of each population of cells analyzed. These findings suggested that large changes in the density of RNA and protein do not occur in most cells as they increase in size; however, there may be decreases in the rate of protein synthesis in some large and small cells. The decrease in the rate of protein synthesis appears consistent with the hypothesis that new sites of envelope growth must be introduced into cells that are close to the division event to restore rapid growth.

Autoradiography↗
PubMed1986

Autoradiographic studies of chromosome replication during the cell cycle of Streptococcus faecium.

Analysis of the distribution of autoradiographic grains around cells of Streptococcus faecium which had been either continuously or pulse-labeled with tritiated thymidine (mass doubling time, 90 min) showed a non-Poisson distribution even when the distribution of cell sizes in the populations studied was taken into account. These non-Poisson distributions of grains were assumed to reflect the discontinuous nature of chromosome replication. To study this discontinuous process further, we fitted an equation to the grain distribution observed for the pulse-labeled cells that assumed that in any population of cells there were subpopulations in which there were zero, one, or two replicating chromosomes. This analysis predicted an average time for chromosome replication and for the period between completion of rounds of chromosome replication and division of 55 and 43 min, respectively, which were in excellent agreement with estimates made by other techniques. The present investigation extended past studies in indicating that the initiation and completion of rounds of chromosome replication are poorly phased with increases in cell volume and that the amount of chromosome replication may be different in different cell halves.

Autoradiography↗
PubMed1985

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants.

Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.

Adolescent↗
PubMed1985

In vitro simulation of neural trauma by laser.

A serious lack of knowledge about central nervous system trauma is encountered on the cellular level where the inability to create precise experimental lesions of known magnitude has limited our understanding of the reactions of single cells to injury. We used a laser cell surgery technique developed in this laboratory to manipulate neurons in a controlled environment, in order to observe pathologic reactions during and immediately after the injury. This technique is especially suited for axonal and dendritic amputations close to the perikaryon. The laser provided three different physical modes of injury to neurites: direct vaporization of cytoplasm, pressure wave damage from external vaporization of substrate material, and photobiologically-induced localized cytoskeletal destruction leading to the slow pinching of processes followed by transection. Our data indicated a great similarity between laser impact damage and the cellular damage produced by physical trauma to the central nervous system.

Animals↗
PubMed1984

Control of wall band splitting in Streptococcus faecalis.

Computer reconstructions of 659 and 1325 whole mounted, shadowed cells, randomly chosen from cultures of Streptococcus faecalis undergoing balanced growth and doubling in mass every 83 min and 30 min, respectively, were used to analyse the cell cycle. The size limits and duration of phases of the cell cycle were estimated by applying a method previously described by the authors, details of which are given here to allow others to use the method. Deeply constricted cells whose primary septal radius, Rs, was less than or equal to 0.18 micron were considered as belonging to an E-phase ending the cell cycle. The statistical parameters of these E-phase cells were used to calculate the mean and coefficient of variation of dividing cells. These latter values, in turn, predicted the moments of the total population well enough so that the method's assumptions were judged adequately satisfied. Therefore, the method was considered applicable to other phases and sub-phases of the cell cycle of these two cultures. The E-phase cells were further classified as having either 0, 1 or 2 secondary growth zones, allowing us to calculate the percentage of newborn cells without growth zones. In the slow-growing cells, 69% of the cells arose with no growth zone. On the other hand, in more rapidly growing cells 16% of the cells or less arose with no growth zone. Our calculations showed that they could exist without a growth zone for only 2 and 0.1 min, respectively. We also classified cells as possessing a 'birth site' if the volume between the two daughter bands was greater than 0, but less than 0.06 micron3. From the statistical properties of such cells with new growth zones, the mean pole time, W, was estimated. We also estimated W from the size of cells in E-phase. The major conclusion is that the pole time is only slightly greater than the mass doubling time at both growth rates. Since DNA synthesis in S. faecalis takes longer (C = 50 to 52 min) than the mass doubling time in rich medium (30 min), a new round of chromosome replication must be initiated before the old round of synthesis is completed (dichotomous replication). Consequently, wall band splitting and initiation of chromosome replication do not occur simultaneously. It was also concluded that the cell initiates wall band splitting, resulting in pole formation and cell division, when the growth zones cannot function rapidly enough to allow the increase of surface area required to accommodate continuing production of cytoplasm.

Cell Cycle↗
PubMed1984

Inability of purified Pseudomonas aeruginosa exopolysaccharide to bind selected antibiotics.

It has been proposed that the exopolysaccharide (alginate) of mucoid Pseudomonas aeruginosa strains which infect cystic fibrosis patients might bind and hence protect this pathogen from antibiotics. To test this hypothesis, we employed equilibrium dialysis to measure the binding between several antibiotics and purified Pseudomonas alginate. Binding was calculated from the residual concentrations of antibiotics in free solution by a biological assay. The detectable binding of antibiotics to alginate was consistent with expectations; the positively charged antibiotics steptomycin and tobramycin, bound to the polyanion (0.047 and 0.024 mumol/mg of alginate, respectively), whereas the neutral species, clindamycin and penicillin, bound negligibly or not at all (0.0011 and 0 mumol/mg of alginate, respectively). When these experiments were performed in the presence of physiological concentrations of saline, none of the antibiotics bound to the polysaccharide. Since the binding observed was abrogated by salt concentrations typical of the tracheobronchial secretions of cystic fibrosis patients, the data suggest that tight binding of antibiotics to the exopolysaccharide of a mucoid P. aeruginosa strain does not provide increased antibiotic resistance.

Anti-Bacterial Agents↗
PubMed1984

Analysis of initiation of sites of cell wall growth in Streptococcus faecium during a nutritional shift.

Three-dimensional reconstruction methods were applied to electron micrographs of Streptococcus faecium to study the initiation of cell wall growth sites during a nutritional shift experiment. Upon lowering the mass doubling time from 76 to 33 min by the addition of excess glutamate, the formation of new cell wall growth sites accelerated above the old steady-state rate at about the same time (10 to 15 min) as did mass, RNA, protein, cell numbers, and autolytic capacity but considerably before DNA (30 min) and peptidoglycan (20 min) synthesis did. During the shift, the average range of cell volumes over which new wall growth sites were introduced did not change significantly. However, upon the shift there was an increase in the frequency of cells having new sites, which was due to the faster-growing cells initiating more new sites in peripheral locations before division. After a transition period, the number of new sites per milliliter of culture increased at a rate that paralleled that of the culture mass. These findings support a model in which new sites are introduced when cells grow to a relatively constant, growth rate-independent size, while the rate at which sites form and grow increases with the growth rate. In this model, chromosome synthesis does not regulate the formation of new sites of cell wall growth, but existing sites cannot be completed until rounds of chromosome synthesis are completed.

Bacterial Proteins↗
PubMed1983

Protease production by Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The temporal appearance of extracellular proteases produced by Pseudomonas aeruginosa was analyzed by pH 9 and pH 4 polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Ammonium sulfate precipitates of culture supernatants from various stages of growth revealed a time-dependent increase in number and amount of proteolytically active proteins. One mucoid P. aeruginosa clinical isolate and its derived nonmucoid variant, as well as two other nonmucoid variant P. aeruginosa strains (all from cystic fibrosis patients), showed similar production of five differently migrating proteases (P1 to P5, numbered according to increasing net negative charge) in pH 9 PAGE and one protease in pH 4 PAGE. P2, P3, and P5 increased to maximum concentrations at 24 to 48 h, decreasing thereafter, whereas P4 continued increasing even at 83 h, and P1 fluctuated. P3 was identified as an elastase. P2 was possibly composed of polypeptide chains bridged by disulfide bonds, since without reduction it migrated in sodium dodecyl sulfate-PAGE as a single protein, and with reduction it migrated as three protein bands. Two-dimensional PAGE revealed multiple molecular weight species within protease-positive bands in pH 9 gel strips. Isoelectric focusing gave a pattern of protein separation that correlated with two-dimensional PAGE analysis. Thus, greater heterogeneity of active proteases than previously reported has been demonstrated in all P. aeruginosa clinical isolates studied by sensitive two-dimensional PAGE analysis.

Cystic Fibrosis↗
PubMed1983

Initiation of wall assembly sites in Streptococcus faecium.

In electron micrographs of replicas of Streptococcus faecium, sites of wall growth are located between pairs of raised equatorial bands. Analysis of cells taken from cultures with mass doubling times between 30 and 125 min indicates that rounds of wall synthesis are initiated at a time close to division, which is temporally unrelated to the initiation or termination of chromosome replication. Growth sites are initiated at a relatively constant volume independent of growth rate when the volume contained within the two segments of wall adjoining an equatorial band marker approaches ca. 0.26 micrometer 3.

Cell Division↗
PubMed1983

Cell wall assembly during inhibition of DNA synthesis in Streptococcus faecium.

Growth sites which are bounded by raised wall bands can be observed in electron micrographs of replicas of Streptococcus faecium. When mitomycin C was added to an exponential-phase culture doubling in mass every 64 min, DNA synthesis was inhibited, and eventually cell division stopped. The growth sites formed before and after inhibition of DNA synthesis enlarged until they contained about 0.25 micron3 of cell volume, at which point they ceased to increase in size. When these sites approached this 0.25-micron3 limit, new sites were initiated; this result had also been observed in untreated cells undergoing a large range of exponential-phase mass doubling times. Thus, regardless of whether chromosome replication is inhibited or uninhibited, sites have the same finite capacity to enlarge to about 0.25 micron3, and when this capacity is reached, new sites are initiated. Although initiation of new growth sites seems to be independent of normal chromosome replication, these results confirm previous studies showing that chromosome replication is necessary for the terminal events of growth site development which result in the division of a site into two separate poles. Two classes of models for the regulation of growth site initiation are discussed.

Cell Wall↗
PubMed1983

Laser microbeam surgery: ultrastructural changes associated with neurite transection in culture.

The exposure of neuronal and glial cell processes to a large number (up to 300) of 12-nsec laser pulses at a wavelength of 337 nm and energy densities below the threshold for nonlinear absorption results in a gradual, gentle process transection in the laser focus. Within 10 to 20 sec after cessation of firing, the process pinches in the target area. During this time, mitochondria become swollen and bleached, the plasma membrane develops an obvious tautness, microtubules disappear, and organelles accumulate to either side of the process constriction. Depending on the irradiation parameters, a local pinching may proceed to a transection in about 30 sec or it may reverse to yield a normal-appearing process in approximately 5 min. Severe process pinching is accompanied by a sudden depolarization that may last for 2 to 5 min and is usually followed by a repolarization to the original resting potential even if the process has transected. Spiral retraction of cut processes and cytoplasmic spillage observed after mechanical transections are not seen with this laser method. Process stretching is minimized or eliminated. Extensive vacuolization often associated with mechanical transections does not develop unless substrate involvement in the form of shock waves is apparent. For the performance of cell surgery in culture, this method appears to offer a reliable approach to morphological alteration of single cells and to the tailoring of two-dimensional neuronal networks. It should also allow more quantitative and better-controlled studies of axonotmesis, degeneration, and regeneration on the single cell level, and it may be used as a probe for the investigation of cytoskeletal dynamics. A mechanism describing the cytoskeletal changes associated with laser-induced cell process transection is proposed.

Animals↗
PubMed1982

Cell cycle dynamics inferred from the static properties of cells in balanced growth.

The duration of a morphological phase of the cell cycle is reflected in the steady state distribution of the sizes of cells in that phase. Relationships presented here provide a method for estimating the timing and variability of any cell cycle phase. It is shown that the mean size of cells initiating and finishing any phase can be estimated from (1) the frequency of cells exhibiting the distinguishing morphological or autoradiographic features of the phase; (2) the mean size of cells in the phase; and (3) their coefficient of variation. The calculations are based on a submodel of the Koch-Schaechter Growth Controlled Model which assumes that (i) the distribution of division sizes is Gaussian; (ii) there is no correlation in division sizes between successive generations; and (iii) every cell division gives rise to two daughter cells of equal size. The calculations should be useful for a wider range of models, however, because the extrapolation factors are not sensitive to the chosen model. Criteria are proposed to allow the user to check the method's applicability for any experimental case. The method also provides a more efficient test of the dependence of growth on cell size than does the Collins-Richmond method. This is because the method uses the mean and coefficient of variation of the size of the total population, in conjunction with those of the cells in a final phase of the cell cycle, to test potential growth laws. For Escherichia coli populations studied by electron microscopy, an exponential growth model provided much better agreement than did a linear growth model. The computer simulations were used to generate rules for three types of cell phases: those that end at cell division, those that start at cell division, and those totally contained within a single cell cycle. For the last type, additional criteria are proposed to establish if the phase is well enough contained for the formulae and graphs to be used. The most useful rule emerging from these computer studies is that the fraction of the cell cycle time occupied by a phase is the product of the frequency of the phase and the ratio of the mean size of cells in that phase to the mean size of all cells in the population. A further advantage of the techniques presented here is that they use the 'extant' distributions that were actually measured, and not hypothesized distributions nor the special distributions needed for Collins-Richmond method that can only be calculated from the observed distributions of dividing or newborn cells on the basis of an assumed growth law.

Bacteria↗
PubMed1982

The role of surface stress in the morphology of microbes.

The shapes of many prokaryotes can be understood by the assumption that the cell wall expands in response to tension created by the osmotically derived hydrostatic pressure. Different organisms have different shapes because wall growth takes place in different regions. A previous paper (Koch et al., 1981 a) considered the simplest case of prokaryotic growth, i.e. that of Streptococcus faecium. In the present paper, an elaboration of this theory is applied to two further cases - the more perfectly spherical cocci and the rod-shaped bacteria. These cases are more complex mathematically, because growth over a considerable fraction of the surface must be considered. Such diffuse growth cannot be treated analytically, but can be simulated on a computer or handled by geometric arguments. The spherical form of the cocci may result from either diffuse growth over their entire external surface, or from zonal growth in which the addition of new material only occurs in the immediate vicinity of the splitting septum. In the zonal model, it must be assumed that the least amount of previously laid down septal peptidoglycan consistent with wall growth is reworked in the formation of the new external wall. For Gram-positive rods, where the body of the rod is truly cylindrical, three kinds of growth zones are required: (1) the inward edge of the ingrowing septum, (2) the junction of septum and nascent pole, and (3) the cylindrical walls. Two modes for cylindrical elongation ara possible: (a) new wall is added in one or a few narrow annular zones, or (b) new wall material is added continuously all over the innermost surface and the outer layer is degraded. It is shown that the latter case applies to Bacillus subtilis. Also summarized in this paper are results, developed in more detail elsewhere, concerning the morphology of fusiform bacteria, Gram-negative rods and the hyphal tips of fungi.

Bacillus subtilis↗