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Biomedical subjects

M L Higgins

Publications and source records attributed to M L Higgins.

At least 19 recordsLinked to original sources
PubMed1998

The spoIIE locus is involved in the Spo0A-dependent switch in the location of FtsZ rings in Bacillus subtilis.

A switch in the location of FtsZ ring structures from medial to polar is one of the earliest morphological indicators of sporulation in Bacillus subtilis. This switch can be artificially caused during vegetative growth by induction of an active form, Sad67, of the transcription regulator, Spo0A (P. A. Levin and R. Losick, Genes Dev. 10:478-488, 1996). We have used immunofluorescence microscopy to show that the switch in FtsZ ring location during vegetative growth caused by Sad67 induction is blocked by a spoIIE deletion mutation. The spoIIE mutation also impaired polar FtsZ ring formation during sporulation. These results suggest that SpoIIE mediates the Spo0A-directed formation of polar FtsZ rings.

Bacillus subtilis↗
PubMed1997

The division during bacterial sporulation is symmetrically located in Sporosarcina ureae.

Immunofluorescence microscopy was used to visualize the FtsZ band that marks the site of septation in Sporosarcina ureae. Image analysis indicated that the vegetative division was symmetrically located with respect to the ends of the cells. Fusions of lacZ to the sporulation loci, spollA and cotE, of Bacillus subtilis were introduced into S. ureae by mobilization of plasmids containing the fusions from Escherichia coli. The fusions showed similar patterns of sporulation-associated expression in S. ureae to those observed in B. subtilis. Formation of beta-galactosidase encoded by the spollA-lacZ fusion made it possible to identify early sporulating cells by immunofluorescence microscopy. Analysis of the position of FtsZ bands in cells expressing spollA-lacZ indicated that the location of sporulation division was symmetrical with respect to the ends of the cells, in sharp contrast to the asymmetrical location of septation in sporulating Bacilli. It is inferred that asymmetry of location of the sporulation division is not essential for the compartmentalization of gene expression that follows the division.

Bacillus subtilis↗
PubMed1996

Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis.

A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric division. The development programs in these two cells involve the compartmentalized activities of sigma E in the larger mother cell and of sigma F in the smaller prespore. Activation of sigma E requires expression of the sigma F-directed gene spoIIR. By immunofluorescence microscopy of a strain containing a spoIIR-lacZ fusion, we have shown that spoIIR is transcribed exclusively in the prespore. By placing spoIIR under the control of PspoIIE, it was possible to express spoIIR before the spore septum was formed. Strains containing the PspoIIE-spoIIR construct activated sigma E only in the mother cell in organisms that underwent the asymmetric sporulation division. Thus, compartmentalization of sigma E activity did not require the compartmentalization of spoIIR expression. Nor did the compartmentalization of sigma E require SpoIIAA, SpoIIAB, sigma F, or sigma F-dependent transcription, all of which are required for prespore-specific gene expression. It is inferred that although sigma F and sigma E direct compartmentalized gene expression, neither of these sigma factors, nor the genes under their control, directs the process of compartmentalization.

Bacillus subtilis↗
PubMed1994

Synergism between the antifungal agents amphotericin B and alkyl glycerol ethers.

The alkyl glycerol ether rac-1-O-dodecylglycerol inhibited the growth of members of two genera of yeasts, Candida and Cryptococcus, and was strongly synergistic with amphotericin B. At one-half its MIC, dodecylglycerol decreased the MIC of amphotericin B by as much as 80-fold. This high degree of synergism between dodecylglycerol and amphotericin B was demonstrated against a number of species of yeasts including Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii. All fractional inhibitory concentrations (for all strains and species) were calculated to be less than 1, and most were less than 0.6, again demonstrating strong synergism. Other alkyl glycerol ethers with alkyl chain lengths ranging from 8 to 18 carbon atoms were also found to be synergistic with amphotericin B against C. neoformans and C. albicans. Electron microscopy experiments showed that C. neoformans grown in the presence of dodecylglycerol had severely abnormal, deformed capsules. Although the mechanism of action of dodecylglycerol is not known, dodecylglycerol was not simply acting as a detergent. The natural detergent sodium deoxycholate could not substitute for dodecylglycerol. At comparable and higher concentrations, sodium deoxycholate had no fungicidal effect on its own, nor did it potentiate the activity of amphotericin B. Dodecylglycerol did not interact synergistically with the water-soluble antifungal agent fluconazole. The lipid-soluble hydrophobic properties of amphotericin B appear to be important for this synergistic effect, in that alkyl glycerol ethers could promote synergism with amphotericin B by potentially increasing the interaction between membrane-bound ergosterol and amphotericin B.

Amphotericin B↗
PubMed1994

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.

Adhesins, Bacterial↗
PubMed1993

Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase.

When chloramphenicol was added to a culture of Bacillus subtilis in early exponential growth, microscopic observation of cells stained by 4',6-diamidino-2-phenylindole showed nucleoids that had changed in appearance from irregular spheres and dumbbells to large, brightly stained spheres and ovals. In contrast, the addition of chloramphenicol to cultures in mid- and late exponential growth showed cells with elongated nucleoids whose frequency and length increased as the culture approached stationary phase. The kinetics of nucleoid elongation after the addition of chloramphenicol to exponential-phase cultures was complex. Immediately after treatment, the rate of nucleoid elongation was very rapid. The nucleoid then elongated steadily for about 4 min, after which the rate of elongation decreased considerably. Nucleoids of cells treated with 6-(p-hydroxyphenylazo)-uracil (an inhibitor of DNA synthesis) exhibited the immediate rapid elongation upon chloramphenicol treatment but not the subsequent changes. These observations suggest that axial filament formation during stationary phase (stage I of sporulation) in the absence of chloramphenicol results from changes in nucleoid structure that are initiated earlier, during exponential growth.

Bacillus subtilis↗
PubMed1992

Septal membrane fusion--a pivotal event in bacterial spore formation?

Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro-sigma E to sigma E in the cytoplasm by exposing it to a membrane-bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why sigma E is associated with transcription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, sigma F, to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.

Bacillus subtilis↗
PubMed1991

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.

Amdinocillin↗
PubMed1990

Neuronal survival and dynamics of ultrastructural damage after dendrotomy in low calcium.

To determine the contributions of calcium to development of ultrastructural damage and neuronal death after mechanical injury, we amputated primary dendrites from over 300 cultured mammalian spinal neurons under normal (1.8 mM) or low (less than or equal to 30 microM) calcium conditions. Two general categories of early ultrastructural change were seen in both normal and low calcium: (1) a lesion-dependent gradient of damage that moved centripetally through the proximal segment and penetrated the soma within 15 min and (2) dilation of the somal Golgi/smooth endoplasmic reticulum (SER), which preceded the wave of deterioration from the lesion. Although the somal Golgi/SER changes were similar in both normal and low calcium, the damage gradient in low calcium differed from the damage gradient in normal calcium. (1) Microtubules and neurofilaments were preserved, (2) mitochondria became more electron dense but did not develop electronlucent foci or high amplitude swelling, and (3) an extensive vesicular gradient formed consisting of rows of swollen SER vesicles. Sodium ionophores have been reported to cause similar changes. Survival studies showed that calcium reduction significantly delayed neuronal death. Survival was 63 +/- 16% vs 35 +/- 8% (p less than 0.003) at 2 h and 30 +/- 7% vs 23 +/- 8% at 6 h in low and normal calcium, respectively. Dead neurons that had been lesioned in low calcium also showed greater ultrastructural preservation than neurons that died after dendrotomy in normal calcium. We hypothesize that under low calcium conditions, the large sodium injury current plays an important role in neuronal deterioration and death after mechanical trauma.

Animals↗
PubMed1990

Relationship between changes in buoyant density and formation of new sites of cell wall growth in cultures of streptococci (Enterococcus hirae ATCC 9790) undergoing a nutritional shift-up.

When the glutamate concentration of cultures of Enterococcus hirae was raised from 20 to 300 micrograms/ml, the mass doubling time decreased from ca. 85 to 45 min in 9 min, but balanced growth was not reestablished for 30 to 40 min. During the unbalanced period of growth, RNA and protein synthesis proceeded more rapidly than did peptidoglycan synthesis, buoyant density increased from ca. 1.1024 to 1.1075 g/ml, and the rate of formation of new cell wall growth sites transitorily accelerated above the new growth rate. When studied as a function of cell size, all cultures showed buoyant density to decrease around cell separation, increase as cells increased in size, and then plateau when cells reached large volumes. Greater increases in buoyant density as a function of cell size were seen after shift-up, with the greatest increases observed at 15 to 20 min after shift-up, when the rate of formation of new sites was also maximal. In a population of cells examined by electron microscopy 15 min after shift-up, buoyant density and the frequency of cells with new sites increased as old sites approached the size of two poles. These data were consistent with a model whereby buoyant density increases in the terminal stages of the cell cycle when the surface grows slower than the cytoplasm. The greater the difference in the rates of inside to outside growth, the greater the increase in buoyant density and the more frequently new sites will be initiated.

Cell Wall↗
PubMed1989

Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790.

Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.

Autoradiography↗
PubMed1989

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.

Cell Cycle↗
PubMed1987

Cytoplasmic damage gradients in dendrites after transection lesions.

Ultrastructural changes in the cytoplasm of transected dendrites have been studied with serial section electron microscopy 10 to 12 min after the lesion. Dendrites from large multipolar neurons in mouse spinal monolayer cultures were selected 4 weeks after seeding and cut with a pulsed UV laser microbeam. The data reveal cytoplasmic damage gradients which decrease in severity with distance from the lesion. The damage is expressed primarily in cytoskeletal disruption, the swelling and vacuolation of mitochondria (MC), and the swelling and vesiculation of the endoplasmic reticulum (ER). For a 3-4 microns diameter dendrite lesioned 150 microns from the soma and fixed 10 min after transection, the following results can be reported. The number of microtubules (MT) decreases almost linearly with a slope of 40 MT/10 microns until MT completely disappear 20 microns from the lesion. Neurofilaments (NF) decrease at approximately 50 NF/10 microns, with 20% of these structures still intact at or near the lesion. MC show both gradual and explosive swelling in the damaged process. The onset of these two phases of MC swelling are correlated with the initial and total loss of MT respectively. MT and NF densities in cut dendrites are drastically different from those in control processes and suggest a possible retrograde redistribution of these organelles. Damage from external shockwaves that cause process distortion but not transection results in a less severe but nevertheless similar loss of MT and NF.

Animals↗
PubMed1987

Cell cycle changes in the buoyant density of exponential-phase cells of Streptococcus faecium.

Cell buoyant densities were determined by centrifugation in Percoll gradients containing exponential-phase cells of Streptococcus faecium ATCC 9790 grown at a mass doubling time of about 33 min. This bacterium showed the highest average density values (1.13 g/ml) measured to date for any eucaryotic or procaryotic organism. Fractions having the highest densities were enriched with cells that were in the process of dividing or had just divided. These high-density fractions were also enriched with cells that had newly initiated sites of cell wall growth. It appears that S. faecium shows minimum cell densities in the midportion of its cycle.

Cell Cycle↗
PubMed1986

Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms.

Milligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37 degrees C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300-330 K molecular weight region, as well as tubulin at 51-54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross-section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three- to four-fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5-30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.

Adenosine Triphosphatases↗
PubMed1986

Rhamnolipid from Pseudomonas aeruginosa inactivates mammalian tracheal ciliary axonemes.

Isolated ciliary axonemes from pig trachea were exposed to increasing concentrations of purified Pseudomonas aeruginosa rhamnolipid. This is a defined ciliary system allowing observation of direct impairment of functional axonemes. Axonemal motility and ATPase activity were decreased in proportion to rhamnolipid concentrations. ATPase-associated proteins observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dynein arms seen in ultra-structural cross sections progressively disappeared from axonemes with exposure to rhamnolipid. These four independent measures establish that the rhamnolipid removes the ATPase-containing outer dynein arms from the ciliary axoneme, thereby rendering the axoneme immotile.

Adenosine Triphosphatases↗
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↗