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Biomedical subjects

A L Koch

Publications and source records attributed to A L Koch.

At least 73 records · Page 4Linked to original sources
PubMed1986

Biophysics of pole formation of gram-positive rods.

During pole formation in Bacillus subtilis the inner and outer surfaces of the nascent pole are enlarged by almost exactly the same extent. This means that the stress is almost uniformly distributed throughout the polar wall. This differs from the situation in the cylindrical side wall, where most of the stress is exerted in the outer portions of the intact wall. Because the stress is shared more uniformly, the maximum strain in any part of the polar wall is reduced, compared with the maximum strain within the side wall. The lowered stress may account, in part, for the resistance of the polar wall to hydrolysis by autolytic enzymes under certain conditions. The shape of the newly completed pole is significantly different from the spherical shape that the hydrostatic pressure would tend to produce. It does, however, achieve the shape that maximizes the polar volume under the restrictions arising due to expansion along the circumference not being possible near the junction of cylindrical and polar wall.

Bacillus subtilis↗
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↗
PubMed1986

Estimating treatment and treatment times for special and nonspecial patients in hospital ambulatory dental clinics.

Increased time to treat the special patient is often cited as a barrier to dental care. The purpose of this study was to analyze the separate and combined effects of differences in dental services planned, services actually performed, and differences in treatment time requirements between special and nonspecial patients in a hospital ambulatory clinical setting. Data for this study were obtained from the UCLA evaluators of the RWJ-funded Hospital-Sponsored Ambulatory Dental Services Program (HSADSP). The results show that special patients require more dental treatment than nonspecial patients for advanced dental disease (i.e., periodontics, surgery, and removable prosthetics) and that they receive more of such services. The study also found that special patients should not be viewed as a homogeneous group when evaluating dental needs and required time resources for treatment. Three subgroups of special patients were identified: developmentally disabled, severely compromised, and moderately compromised. The developmentally disabled as compared to the nonspecial patients required significantly more (20 percent) provider time in completing a "representative" treatment plan.

Adult↗
PubMed1985

Inside-to-outside growth and turnover of the wall of gram-positive rods.

Gram-positive, rod-shaped bacteria, given a pulse of peptidoglycan precursors, first exhibit a lag before the second or turnover phase of peptidoglycan commences. This is because new material is inserted on the inner face of the wall and gradually displaced through the wall. Based on this experimental observation, a mathematical model was constructed and compared with experimental data obtained in several laboratories for the first and second phases of wall turnover of Bacillus subtilis. The model allows the parameters of the process to be estimated for experiments with any labeling time. According to the surface stress theory the wall which is layed down immediately outside the cytoplasmic layer is in an unextended conformation. As subsequent additions of murein occur, the wall moves outward, becomes stretched, and bears the stress due to hydrostatic pressure. Ultimately, peptide and glycosyl bonds become cleaved. At the end of the lag phase the cleavage becomes so extensive that wall fragments are liberated into the medium. This strategy permits rod-shaped growth. In some experimental situations the half-life of wall radioactivity in this second phase roughly equals the doubling time; consequently, the exponential release probably does not represent random turnover but instead is the result of expansion of the underlying wall that continues to create strain which favors autolysis action. The slower turnover of the third phase, where there is a much slower loss, is also included in the analysis.

Bacillus subtilis↗
PubMed1985

The indirect nature of interaction of glucose transport with the system transporting galactosides.

The membrane transport systems for galactosides and glucose derivatives interact in enteric microorganisms. Stop-flow experiments with a double wavelength spectrophotometer and a flow-through cuvette (designed to minimize light-scattering effects) were used to measure the speed of interaction in Escherichia coli. The in vivo hydrolysis of ortho-nitrophenol-beta-D-galactopyranoside was measured by comparing the light transmitted by cell suspensions at 420 nm with that at 500 nm. Measurements at the latter wavelength corrected for residual scattering effects. The stop-flow experiment allowed the study of the early kinetics of transport and hydrolysis. It was found with strain ML308 that there was a significant lag in the achievement of steady-state inhibition by glucose and its derivative methyl-alpha-D-glucopyranoside (alpha MG). This strain constitutively produces high levels of permease and beta-galactosidase. The absorbancy increases at 420 nm are limited by transport because the beta-galactosidase is present inside the cells in excess. From earlier results, it was not surprising that inhibition is delayed with low concentrations of the glucose compounds, but the new double wavelength technique showed no kinetic component of rapid inhibition. This result therefore excludes competition for some membrane-bound component and is consistent with the production of the dephosphorylated form of the soluble Enzyme IIIglu that binds and inhibits the permease system in the membrane.

Biological Transport↗
PubMed1985

Asymmetric distribution of charge on the cell wall of Bacillus subtilis.

The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions. The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan. In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B. subtilis 168. Detergent-extracted cell walls bound CF only on the outer wall face. Completed cell poles bound CF, but septa did not. When the walls were permitted to autolyze briefly, binding of CF occurred on both faces. In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix. When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface. Walls with carboxylates chemically neutralized also retained charge asymmetry. Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all. These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands.

Bacillus subtilis↗
PubMed1985

Cellular location of origin and terminus of replication in Bacillus subtilis.

The origin of replication of Bacillus subtilis 168 trp thy dna-1 (temperature-sensitive initiation mutant) was labeled with [3H]thymidine. Analysis of labeled cells by autoradiography revealed that most of the radioactivity was associated with cell pole areas. To label the terminus, cells that had initiated were treated with chloramphenicol to inhibit cell growth and division but to allow continued DNA synthesis. These cells were then labeled with [3H]thymidine at a time when chromosome replication was nearly complete. The distribution of radioactivity was similar to that observed in origin-labeled cells. In contrast, exponentially growing cells that were labeled for a brief time at the permissive temperature showed a random distribution of radioactivity. These data indicate that the origin and terminus of replication are located at cell poles.

Autoradiography↗
PubMed1985

A children's oral health status index based on dentists' judgment.

The children's oral health status index was developed as an integrated measure for the direct appraisal of pediatric patient populations in private practices, dental clinics, or school programs. The index is derived from a paired preference experiment with five pedodontists and five general dentists acting as judges of oral health in 200 case comparisons. Four easily measured variables are united numerically by the index: decayed teeth, occlusion, tooth position, and missing teeth (which have not been exfoliated). Clinical applications of the index in three geographic areas have all had good results.

Adolescent↗
PubMed1984

Evolution vs the number of gene copies per primitive cell.

Computer simulations are presented of the rate at which an advantageous mutant would displace the prototype in a replicating system without an accurate segregation mechanism. If the number of gene copies in the system is indefinitely large, Darwinian evolution is essentially stopped because there is no coupling of phenotype with genotype, i.e., there is no growth advantage to the advantageous gene relative to the prototype and therefore no "survival of the fittest." The inhibition of evolution due to a number of gene copies less than 100 would have been not insurmountable. Although the presence of multiple copies would have allowed replacement by an advantageous mutant, it provided a way for the primitive cell to conserve less immediately useful genes that could evolve into different or more effective genes. This possibility was lost as accurate segregation mechanisms evolved and cells with few copies of each gene, such as modern procaryotes, arose.

Biological Evolution↗
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

The variable T model for gram-negative morphology.

Gram-negative micro-organisms possess only a very thin murein sacculus to resist the stress caused by the internal hydrostatic pressure. The sacculus consists of at most one molecular layer of peptidoglycan in an extended conformation. It must grow by the insertion and cross-linking of new murein to the old before the selective cleavages of the stress-bearing murein are made which allow wall enlargement. Since insertion of new murein occurs all over the surface of Escherichia coli (even in completed poles), the internal pressure would tend to force the cells into a spherical shape and prevent both cylindrical elongation and cell division. Of course, Gram-negative bacteria do achieve a variety of shapes and do divide. Because prokaryote cells, unlike eukaryotic cells, do not have cytoskeletons and contractile proteins to transduce biochemical free energy into the mechanical work needed to achieve aspherical shapes and to divide, this paradox seems to be resolvable only by postulating that the details of the biochemical mechanism for wall growth vary in different regions of the surface, affecting the work required to enlarge the wall locally. Depending on the degree and rate of change in the biochemical energetics, it is possible to account for rod and the other more complex shapes of Gram-negative bacteria. Division occurs in Gram-negative organisms by the development of constrictions that progressively invade the cytoplasm. The work to cause these morphological processes must ultimately derive from the biochemical process of the stress-bearing wall formation. A biophysical basis for cell division in these prokaryotic organisms is proposed.

Cell Division↗
PubMed1984

Insertion and fate of the cell wall in Bacillus subtilis.

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.

Acetylglucosamine↗
PubMed1984

Shrinkage of growing Escherichia coli cells by osmotic challenge.

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.

Escherichia coli↗