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Biomedical subjects

N B Grover

Publications and source records attributed to N B Grover.

At least 37 records · Page 2Linked to original sources

Relationship between size of parent at cell division and relative size of its progeny in Escherichia coli.

This article examines the empirical basis for the assumption of independence between the relative size (length or surface area) of a newborn cell w and the absolute size of its mother at cell division. Random samples from two strains of Escherichia coli B/r cells in steady-state exponential growth, covering a range of doubling times, were fixed in osmium tetroxide and prepared for electron microscopy by agar filtration. Length and diameter of over 3000 constricted cells were measured from the electron micrographs and cell surface area computed by assuming an idealized geometry of right circular cylinders with hemispherical polar caps. In general, these strains were found to divide into two daughter cells with a precision that is independent of the size of the mother. In addition, both a normal and a symmetrical beta-distribution were shown to fit the observed size distributions of w rather well; theoretical grounds for preferring the latter are discussed.

Cell Division↗

Analysis of a model for minichromosome segregation in Escherichia coli.

The present article contains a theoretical, quantitative analysis of the implications of the Helmstetter-Leonard model (1987, J. molec. Biol. 197, 195-204.) for the segregation of chromosomal DNA in Escherichia coli, on the expected copy-number distribution of minichromosomes in a culture in steady-state exponential growth. According to the model, two determinants are involved in the mechanism of chromosome segregation: a partition system that assures the equal allotment of chromosomes between daughter cells at cell division, and a locus within the minimal oriC region that specifies the attachment site of the chromosomes to the cell envelope at initiation of replication. There are many parameters that must be taken into account in such a study, and since some of them are probabilistic in nature, a strictly analytical approach is not feasible and we had to resort to computer simulation. A wide range of parameter values was tested, in all combinations. The minichromosome copy-number distributions obtained all had a prominent mode equal to the number of oriC binding sites and their main features were determined essentially by that and very little by any of the other parameters of the model. In order to avoid the unrealistic situation in which this one feature completely dominates the results, the original model was modified so that each individual minichromosome is no longer required to replicate during every cell generation, by introducing a limit to the number of unsuccessful attempts to locate a suitable binding site. The copy-number distributions predicted by this version of the model are quantitatively and qualitatively very different and depend on all the components of the model. The simulation results are sufficiently well-behaved to allow consideration as to whether a particular empirical minichromosome copy-number distribution--when such data become available--could in fact be governed by the proposed model; it may even be possible to get a rough estimate for the different parameters involved.

Computer Simulation↗

Surface-limited growth: a model for the synchronization of a growing bacterial culture through periodic starvation.

This article analyses the Surface-Limited Growth Model put forward to explain the very tight synchrony, over more than ten division cycles, obtained experimentally by subjecting a growing bacterial culture to alternating periods of starvation and dilution, using inorganic phosphate as the limiting substrate. The Model states that when an essential nutrient is in limited supply, the rate of growth of an individual cell will be proportional to its surface area (and the current concentration of the limiting substance) rather than to its volume. This decrease in dimensionality from volume to surface is expected to favor the smaller cells and so result ultimately in a narrower size distribution. The Surface-Limited Growth Model deals with cell growth under unusual nutritional conditions, and its predictions depend on how the cell replication cycle is assumed to behave under these same circumstances. Two alternatives are considered: the volume at which cells divide is the same during the starvation phase as during steady-state exponential growth, and the cells adjust immediately to the changing growth rate. In the latter case, we have tested both C + D constant with time and C + D variable (where C + D is the time between initiation of chromosome replication and the corresponding cell division), the incremental value at any instant being computed separately for each individual cell from its current effective growth rate. The simulation results are of two sorts depending on the auxiliary assumptions used. Either the dilution-starvation cycles have no effect whatsoever on the cell volume distribution, or the width of the distribution decreases gradually with time, approaching zero slowly and asymptotically, but the mean cell volume decreases as well--directly contradicting experimental observations. We conclude that the Surface-Limited Growth Model is incapable of explaining the synchronization of cells by periodic starvation of a growing bacterial culture.

Bacteria↗

Predicted steady-state cell size distributions for various growth models.

The question of how an individual bacterial cell grows during its life cycle remains controversial. In 1962 Collins and Richmond derived a very general expression relating the size distributions of newborn, dividing and extant cells in steady-state growth and their growth rate; it represents the most powerful framework currently available for the analysis of bacterial growth kinetics. The Collins-Richmond equation is in effect a statement of the conservation of cell numbers for populations in steady-state exponential growth. It has usually been used to calculate the growth rate from a measured cell size distribution under various assumptions regarding the dividing and newborn cell distributions, but can also be applied in reverse--to compute the theoretical cell size distribution from a specified growth law. This has the advantage that it is not limited to models in which growth rate is a deterministic function of cell size, such as in simple exponential or linear growth, but permits evaluation of far more sophisticated hypotheses. Here we employed this reverse approach to obtain theoretical cell size distributions for two exponential and six linear growth models. The former differ as to whether there exists in each cell a minimal size that does not contribute to growth, the latter as to when the presumptive doubling of the growth rate takes place: in the linear age models, it is taken to occur at a particular cell age, at a fixed time prior to division, or at division itself; in the linear size models, the growth rate is considered to double with a constant probability from cell birth, with a constant probability but only after the cell has reached a minimal size, or after the minimal size has been attained but with a probability that increases linearly with cell size. Each model contains a small number of adjustable parameters but no assumptions other than that all cells obey the same growth law. In the present article, the various growth laws are described and rigorous mathematical expressions developed to predict the size distribution of extant cells in steady-state exponential growth; in the following paper, these predictions are tested against high-quality experimental data.

Bacteria↗

Elongation and surface extension of individual cells of Escherichia coli B/r: comparison of theoretical and experimental size distributions.

The way individual cells grow and divide uniquely determines the (time-invariant) cell size distribution of populations in steady-state exponential growth. In the preceding article, theoretical distributions were derived for two exponential and six linear models containing a small number of adjustable parameters but no assumptions other than that all cells obey the same growth law. The linear models differ from each other with respect to the timing of the presumptive doubling in their growth rate, the exponential models--according to whether there is or is not a part of the cell that does not contribute to the growth rate. Here we compared the size distributions predicted by each of these models with those of cell length and surface area measured by electron microscopy; the quality of the fit, as determined by the mean-square successive-differences test and the chi 2 goodness-of-fit test, was taken as a measure of the adequacy of the model. The actual data came from two slow-growing E. coli B/r cultures, an A strain (pi = 125 min) and a K strain (pi = 106 min), and a correction was introduced in each to account for the distortion caused by the finite size of the picture frame. The parameter estimates produced by the various models are quite reliable (cv less than 0.1%); we discuss them briefly and compare their values in the two strains. All the length extension models were rejected outright whereas most of the surface growth versions were not. When the same models were tested on A-strain data from a faster growing culture (tau = 21 min), those models that provided an adequate fit to the cell surface area data proved equally satisfactory in the case of cell length. These findings are evaluated and shown to be consistent with cell surface area rather than cell length being the dimension under active control. Three surface area models, all linear, are rejected--those in which doubling of the growth rate occurs with a constant probability from cell birth, at a particular cell age, and precisely at cell division. The evidence in the literature that appears to contradict this last result, rejection of the simple linear surface growth model, is shown to be faulty. The 16 original models are here reduced to five, two involving exponential surface growth and three linear, and possible reasons are presented for our inability to discriminate further at this stage.

Cell Division↗

Initiation of chromosome replication in bacteria: analysis of an inhibitor control model.

This article contains an analysis of a version of the well-known inhibitor-dilution model for the control of initiation of chromosome replication in bacteria. According to this model, an unstable inhibitor interacts with an initiation primer in a hit-and-destroy fashion to prevent successful initiation; both constituents are presumed to be RNA species that are synthesized constitutively. The model further postulates that the inhibitor interacts cooperatively with the primer, that the inhibitor gene is removed some distance from the origin of replication, and that an eclipse period exists during which the chromosome origin is not able to reinitiate. This unstable-inhibitor version is characterized by four parameters: the inhibitor half-life, the cooperativity index, the location of the inhibitor gene, and the eclipse period; computer simulations are used to study the effect of each of these on the DNA and interdivision time distributions in exponentially growing steady-state cultures. In neither case was any combination of parameter values found that could provide even moderately satisfactory agreement between the simulation results and experimental data. From the examples furnished and the associated discussion, it appears that there are none--that no combination of parameter values exists that can reasonably be expected to produce a significantly better fit than those tested. We conclude that the model in its present form cannot be a valid description of chromosome replication control in bacteria. It is pointed out that this does not necessarily apply to negative initiation control models in general, or even to all inhibitor-dilution systems, merely to the particular ColE1-like mechanism considered here. Nevertheless, recent experimental results, which can only be understood in terms of a very high degree of initiation synchrony within individual cells, offer strong evidence against stochastic models of this kind for the control of chromosome replication.

Bacteria↗

Initiation of DNA replication in bacteria: analysis of an autorepressor control model.

The precise mechanism by which the initiation of chromosome replication in bacteria is controlled has not yet been established, and several theoretical models have been proposed in an attempt to provide a conceptual framework for the accumulated experimental evidence. The present article contains a detailed quantitative analysis, using computer simulation, of the control model first put forward schematically by Sompayrac & Maaløe in 1973, in which a single operon codes for both the initiator protein and an autorepressor. By comparing the predictions of the model with what is known about the physiology and molecular biology of Escherichia coli under different growth conditions, we are able to delineate the characteristics that such a control system would need to possess in order to be capable of regulating chromosome replication: the control operon has to lie fairly near the origin of replication and contain a moderate to strong promoter and an operator that competes for its repressor with other equally specific binding sites along the chromosome in an interaction that is somewhat weaker than usual; in addition, the messenger molecules encoded for by the repressor gene must have a relatively ineffective ribosome binding site and not too long a halflife.

Binding, Competitive↗

Intravenous albumin administration in acid aspiration syndrome in rabbits.

The possible value of albumin in a rabbit model of the acid aspiration syndrome was studied. Hydrochloric acid was instilled into the respiratory tracts of three groups of rabbits: Group A received a high intravenous dose of human albumin (1.5 gm/kg body weight); Group B (control) was given Hartmann's solution, and Group C a low dose of albumin (0.25 gm/kg body weight). The total amount of intravenous fluids was identical in all groups. Serum and pulmonary edema fluid (PEF) concentrations of total protein were highest in Group A. Simultaneous concentration gradients between serum and PEF for total protein, human and rabbit albumin, and globulin fractions were not statistically different in the three groups. In Group A, PEF appeared first and PaO2, static compliance, and hematocrit decreased significantly more than those of the two other groups. Survival of animals in Group C was highest. An additional Group (D) of rabbits received the same high dose of albumin without acid aspiration. In Group D hematocrit decreased while serum total protein and pulmonary function remained unchanged. It seems that a high dose of albumin causes a further deterioration of lung function following acid aspiration because of extravasation into the interstitial space. The administration of low doses of albumin was not different than Hartmann's solution, but led to the best survival in our model.

Animals↗

Sensitivity of exponentially growing populations of Escherichia coli to photo-induced psoralen-DNA interstrand crosslinks.

Experimental survival curves for Escherichia coli K 12 (CR 34) were determined after exposure to 4,5',8-trimethylpsoralen and near ultraviolet light. The lethal action was shown to arise exclusively from interstrand crosslinks, cell vulnerability increasing markedly with the doubling time of the culture. To account for these results, two quite different models are considered. The first assumes that a cell survives as long as at least one copy of its genome remains undamaged; a variant of this permits repair by DNA strand exchange. The second model allows for a limited period of time during which DNA repair can take place. A crosslink in a stretch of DNA due to be replicated within this interval constitutes a fatal lesion. Theoretical survival curves are computed for bacterial populations with defined age distributions and chromosome configurations. While the first model completely fails to provide a satisfactory description of the experimental results, the second model does predict the presence of a shoulder in the survival curves and, in one of its forms, it seems to agree rather well with the measured data over a wide range of crosslink concentrations and doubling times.

DNA Repair↗

The influence of splenic tissue implantation upon platelet population in rabbits after splenectomy.

An investigation of the behavior of the blood platelet population following splenectomy and autologous splenic tissue implantation has been done on the rabbit. A bimodal rise in platelet size and count is observed in the postoperative period. After splenectomy, a 722 per cent increase in the platelet count and a 21.9 percent rise in the platelet size are found. These increases are abolished by the implantation of splenic tissue. The existence of a splenic humoral factor that controls platelet production in the bone marrow is possible.

Animals↗

Three-dimensional organization of collagen fibres in tendon.

Electron microscopic observations are presented on thin sections of excised chicken breast tendon following the introduction and diffusion of aqueous solutions of heavy metal salts. The dark banded regions of the collagen fibrils are seen to be in near-perfect register throughout the diameter of each fibril and, in many cases, to be continuous across the intervening ground substance. Clusters of uranyl ions form well-defined chains extending across the interfibrillar space between neighbouring fibrils, a distance of several hundred nanometres. It is suggested that the high degree of organization characteristic of collagen fibrils in tissue may perhaps be a property not only of the protein but also of the ground substance in which it is embedded, the fibres merely rendering visible a lattice pattern of their surroundings to which they have conformed.

Animals↗

Effects of adenosine diphosphate, colchicine and temperature on size of human platelets.

An improved measuring system based on the Coulter principle and developed in our laboratory is used to size human blood platelets. The mean volume of platelets in 24 healthy subjects is found to be 8.45 micron 3 with a standard deviation of 1.07 micron 3; the typical size-distribution curve is unimodal and asymmetrical, with a marked skew to the right. The effects of different reagents on platelet size (shape factor x volume) are evaluated. Platelets increase in size by 23% following suspension in isotonic, phosphate-buffered saline and incubation with 10 microM adenosine diphosphate; no change is observed when the suspending medium is autologous plasma. Cooling the platelets to 0-4 degrees C results in a size increase of 25%; rewarming to 37 degrees C restores them to their initial size within 2 hr. A similar increase occurs when the platelets are incubated with 1-10 mM colchicine. It is proposed that these reagents, which are known to produce changes in the orientation of the marginal bundle of microtubules, cause platelets to undergo disc-sphere transformations. Calculations are made which show that such transformations increase platelet size by 27% as measured electrically, and we conclude that the so-called volume changes reported in the literature reflect shape changes only and that no true volume increase actually takes place.

Adenosine Diphosphate↗

The lymphocyte production pathway in bone marrow: possible significance of the size spectrum of lymphocytes and their precursors.

It is now generally accepted that transitional ('lymphoid') cells are the precursors of small lymphocytes. Such cells have a heterogeneous size spectrum and show high proliferative capacity. To facilitate the study of the kinetics of lymphocyte production, a detailed investigation of cell sizes of the transitional cell-lymphocyte compartment was carried out using a Coulter counter modified to permit a very rapid and accurate examination of cells in suspension. Enriched populations of 'lymphoid' cells, obtained after 10 d rebound from hypoxia at half an atmosphere, were enriched further by bovine albumin and Ficoll gradients to give density fractions containing two types of cells. Differential counts of stained smears of these fractions enabled a comparison to be made between the size distribution and the specific cell types. Four distinct cell types were characterized in terms of volume and density: small and intermediate-sized lymphocytes (volume 53-59 fl, albumin fractions 19-23%), small transitional cells (154-160 fl, 21-23%), medium transitional cells (206-218 fl, 17-19%) and large transitional cells (350-400 fl, 21-27%). These findings are consistent with the view that there are at least three mitoses in the course of the lymphocyte production pathway in the bone marrow.

Animals↗