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PubMed2006

The role of LRP and H-NS in transcription regulation: involvement of synergism, allostery and macromolecular crowding.

LRP has recently been shown to interact with the regulatory regions of bacterial ribosomal RNA promoters. Here we study details of the LRP-rDNA interaction by gel retardation and high-resolution footprinting techniques. We show that a second regulator for rRNA transcription, H-NS, facilitates the formation of a higher-order LRP-nucleoprotein complex, probably acting transiently as a DNA chaperone. The macromolecular crowding substance ectoine stabilizes the formation of this dynamic complex, while the amino acid leucine, as a metabolic effector, has the opposite effect. DNase I and hydroxyl radical footprint experiments with LRP-DNA complexes reveal a periodic change of the target DNA structure, which implies extensive DNA wrapping reaching into the promoter core region. We show furthermore that LRP binding is able to constrain supercoils, providing a link between DNA topology and regulation. The results support the conclusion that the bacterial DNA-binding protein LRP, assisted by H-NS, forms a repressive nucleoprotein structure involved in regulation of rRNA transcription. The formation of this regulatory structure appears to be directly affected by environmental changes.

Allosteric Regulation↗
PubMedDate not supplied

Reaction kinetics in intracellular environments with macromolecular crowding: simulations and rate laws.

We review recent evidence illustrating the fundamental difference between cytoplasmic and test tube biochemical kinetics and thermodynamics, and showing the breakdown of the law of mass action and power-law approximation in in vivo conditions. Simulations of biochemical reactions in non-homogeneous media show that as a result of anomalous diffusion and mixing of the biochemical species, reactions follow a fractal-like kinetics. Consequently, the conventional equations for biochemical pathways fail to describe the reactions in in vivo conditions. We present a modification to fractal-like kinetics following the Zipf-Mandelbrot distribution which will enable the modelling and analysis of biochemical reactions occurring in crowded intracellular environments.

Biopolymers↗
PubMed2005

Electrochemical structure of the crowded cytoplasm.

The current view of the cytoplasm as a 'bustling and well-organized metropolitan city' raises the issue of how physicochemical forces control the macromolecular interactions and transport of metabolites and energy in the cell. Motivated by studies on bacterial osmosensors, we argue that charged cytoplasmic macromolecules are stabilized electrostatically by their ionic atmospheres. The high cytoplasmic crowding (25-50% of cell volume) shapes the remaining cell volume (50-75%) into transient networks of electrolyte pathways and pools. The predicted 'semi-conductivity' of the electrolyte pathways guides the flow of biochemical ions throughout the cytoplasm. This metabolic and signaling current is powered by variable electrochemical gradients between the pools. The electrochemical gradients are brought about by cellular biochemical reactions and by extracellular stimuli. The cellular metabolism is thus vectorial not only across the membrane but also throughout the cytoplasm.

Animals↗
PubMed2002

Heterogeneous nucleation and crowding in sickle hemoglobin: an analytic approach.

Sickle hemoglobin nucleation occurs in solution as a homogeneous process or on existing polymers in a heterogeneous process. We have developed an analytic formulation to describe the solution crowding and large nonideality that affects the heterogeneous nucleation of sickle hemoglobin by using convex particle theory. The formulation successfully fits the concentration and temperature dependence of the heterogeneous nucleation process over 14 orders of magnitude. Unlike previous approaches, however, the new formulation can also accurately describe the effects of adding nonpolymerizing agents to the solution. Without additional adjustable parameters, the model now describes the data of M. Ivanova, R. Jasuja, S. Kwong, R. W. Briehl, and F. A. Ferrone, (Biophys. J. 2000, 79:1016-1022), in which up to 50% of the sickle hemoglobin is substituted by cross-linked hemoglobin A, which does not polymerize, and which substitution causes the rates to decrease by 10(5). The success of this approach provides insight into the polymerization process: from the size-dependence of the contact energy deduced here, it also appears that various contacts of unknown origin are energetically significant in the heterogeneous nucleation process.

Calorimetry↗
PubMed1986

A milling crowd model for local and long-range obstructed lateral diffusion. Mobility of excimeric probes in the membrane of intact erythrocytes.

A new model for lateral diffusion, the milling crowd model (MC), is proposed and is used to derive the dependence of the monomeric and excimeric fluorescence yields of excimeric membrane probes on their concentration. According to the MC model, probes migrate by performing spatial exchanges with a randomly chosen nearest neighbor (lipid or probe). Only nearest neighbor probes, one of which is in the excited state, may form an excimer. The exchange frequency, and hence the local lateral diffusion coefficient, may then be determined from experiment with the aid of computer simulation of the excimer formation kinetics. The same model is also used to study the long-range lateral diffusion coefficient of probes in the presence of obstacles (e.g., membrane proteins). The dependence of the monomeric and excimeric fluorescence yields of 1-pyrene-dodecanoic acid probes on their concentration in the membranes of intact erythrocytes was measured and compared with the prediction of the MC model. The analysis yields an excimer formation rate for nearest neighbor molecules of approximately 1 X 10(7) s-1 and an exchange frequency of approximately greater than 2 X 10(7) s-1, corresponding to a local diffusion coefficient of greater than 3 X 10(-8) cm2 s-1. This value is several times larger than the long-range diffusion coefficient for a similar system measured in fluorescence photobleaching recovery experiments. The difference is explained by the fact that long-range diffusion is obstructed by dispersed membrane proteins and is therefore greatly reduced when compared to free diffusion. The dependence of the diffusion coefficient on the fractional area covered by obstacles and on their size is derived from MC simulations and is compared to those of other theories lateral diffusibility.

Diffusion↗
PubMed1993

Crowding-induced organization of cytoskeletal elements: I. Spontaneous demixing of cytosolic proteins and model filaments to form filament bundles.

The theory for the effects of crowding on the behavior of reversibly self-assembling solutes is extended to mixtures containing nonassembling solutes. The theory predicts that excluded volume will cause dramatic demixing into domains of long, tightly packed, highly aligned fibers coexisting with an isotropic solution of unaggregated species. It suggests that the bundling of fibers in cells is entropically driven and that accessory binding proteins in the cytoplasm serve to modulate the process rather than create it.

Biophysical Phenomena↗
PubMed1993

Macromolecular diffusion in crowded solutions.

The effects of crowding on the self or tracer diffusion of macromolecules in concentrated solutions is an important but difficult problem, for which, so far, there has been no rigorous treatment. Muramatsu and Minton suggested a simple model to calculate the diffusion coefficient of a hard sphere among other hard spheres. In this treatment, scaled particle theory is used to evaluate the probability that the target volume for a step in a random walk is free of any macromolecules. We have improved this approach by using a more appropriate target volume which also allows the calculation to be extended to the diffusion of a hard sphere among hard spherocylinders. We conclude that, to the extent that proteins can be approximated as hard particles, the hindrance of globular proteins by other proteins is reduced when the background proteins aggregate (the more so the greater the decrease in particle surface area), the hindrance due to rod-shaped background particles is reduced slightly if the rod-like particles are aligned, and the anisotropy of the diffusion of soluble proteins among cytoskeletal proteins will normally be small.

Biophysical Phenomena↗
PubMed1997

Liquid crystalline ordering of nucleosome core particles under macromolecular crowding conditions: evidence for a discotic columnar hexagonal phase.

Macromolecular crowding conditions occurring inside the cell nucleus were reproduced experimentally with solutions of mononucleosome core particles to study their supramolecular organization. We report here that under these conditions, and over a large range of monovalent salt concentrations, mononucleosome core particles self-assemble to form a discotic liquid crystalline phase characterized in polarizing and freeze-fracture electron microscopy. Mononucleosomes are stacked on each other to form columns, which are themselves closely packed into an hexagonal array. The nucleosome concentration, estimated from the network parameters, falls in the range of values measured in cell nuclei. We suggest that these concentrated solutions, although their organization cannot be immediately compared to the organization of chromatin in vivo, may be used to investigate the nucleosome-nucleosome interactions. Furthermore, this approach may be complexified to take into account the complexity of the eucaryotic chromatin.

Animals↗
PubMed1997

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR.

Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon. It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes. This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.

Base Sequence↗
PubMed1999

Crowding effects on EcoRV kinetics and binding.

The cytosol of the cell contains high concentrations of small and large macromolecules, but experimental data are often obtained in dilute solutions that do not reflect in vivo conditions. We have studied the crowding effect that large macromolecules have on EcoRV cleavage by adding high-molecular-weight Ficoll 70 to reaction solutions. Results indicate that Ficoll has surprisingly little effect on overall EcoRV reaction velocity because of offsetting increases in V(max) and K(m), and stronger nonspecific binding. The changes in measured parameters can largely be attributed to the excluded volume effects on reactant activities and the slowing of protein diffusion. Covolume reduction upon binding appears to reinforce nonspecific binding strength, and k(cat) appears to be slowed by stronger nonspecific binding, which slows product release. The data also suggest that effective Ficoll particle volume decreases as its concentration increases above a few weight percent, which may be due to Ficoll interpenetration or compression.

Biophysical Phenomena↗
PubMed2001

Larval crowding in Drosophila melanogaster induces Hsp70 expression, and leads to increased adult longevity and adult thermal stress resistance.

In this study we show for the first time that moderate high larval density induces Hsp70 expression in Drosophila melanogaster larvae. Larval crowding led to both increased mean and maximal longevity in adults of both sexes. Two different measures of heat-stress resistance increased in adult flies developed at high density compared to flies developed at low density. The hardening-like effect of high larval density carried over to the adult life stage. The hardening memory (the period of increased resistance after hardening) was long compared to hardening of adult flies, and possibly lasts throughout life. The increase in resistance in adults following development at high larval density seemed not to be connected to Hsp70 itself, since Hsp70 expression level in adult flies after hardening was independent of whether larvae developed at low or high densities. More likely, Hsp70 may be one of many components of the stress response resulting in hardening.

Journal Article↗
PubMed2006

Use of nephrectomy at select medical centers--a case of follow the crowd?

PURPOSE: Regionalization of high risk surgical procedures to larger, teaching hospitals has been suggested as a means to improve the quality of care. We determined the extent to which the regionalization of nephrectomy has occurred and describe the potential causes and implications of any observed regionalization. MATERIALS AND METHODS: The Nationwide Inpatient Sample comprises a 20% sampling of hospital discharges in the United States yearly. Patients undergoing nephrectomy for kidney cancer between 1988 and 2002 were identified using International Classification of Disease, Ninth Revision, Clinical Modification codes. Regionalization was assessed using 6 structural hospital measures, including teaching status, urban location, discharge volume, nephrectomy volume, bed capacity and for-profit status. Adjusted models were developed to identify the significance of temporal trends in each regionalization measure. RESULTS: We identified 66,621 patients undergoing nephrectomy during the study period. Compared to procedures performed between 1988 and 1990 the likelihood of undergoing nephrectomy at teaching, high nephrectomy volume and high throughput (all diagnoses) hospitals increased by 2.0 (CI 1.9 to 2.2), 7.4 (CI 7.1 to 7.7) and 2.2 times (CI 2.1 to 2.2), respectively, in 2000 to 2002. Conversely nephrectomy was less likely to be performed at for-profit hospitals (OR 0.5, CI 0.5 to 0.6). Patients were more likely to undergo partial nephrectomy at teaching, high volume, high throughput, urban hospitals. CONCLUSIONS: Regionalization of nephrectomy to teaching and high volume (nephrectomy and all diagnoses) hospitals is currently under way. Although the implications are not entirely clear, this study provides further evidence for the crowding of complex surgical procedures into these institutions.

Aged↗
PubMed2000

Apparent string shortening concomitant with letter crowding.

In our previous studies of the crowding effect, we have observed that human observers tend to underestimate the length of a letter string (the number of letters in the string) when the letters are close to visual acuity, and the interletter spacings are small. In this study, we asked our observers to identify letters in randomly presented four-letter and five-letter strings. We found that, when a priori knowledge of the lengths of letter strings was not available, the probability of underestimating string length increased with decreasing interletter spacing. The causes of underestimation errors appeared to be the omission of an interior letter and the merging of two neighboring letters. Since our experiments were conducted in the foveal region, neither spatial uncertainty nor split attention can explain the underestimation errors. The effect of the point spread function of the eye on closely packed letter strings is discussed.

Adult↗
PubMed2003

Bilateral choroidal folds and optic neuropathy: a variant of the crowded disk syndrome?

PURPOSE: To describe the clinical features of the syndrome that includes bilateral choroidal folds and optic neuropathy and to propose a novel etiology to explain this presentation. DESIGN: Observational small case series. PARTICIPANTS: Three patients. METHODS: Three patients received an extensive clinical work-up including complete ocular examination, A and B scan ultrasonography, Humphrey visual field analysis, and imaging of the brain and orbit. Neurological consultation was obtained in each patient two of whom underwent lumbar puncture. The findings of this detailed investigation are reported. RESULTS: Each patient demonstrated bilateral choroidal folds with optic disk congestion and leakage in one eye and optic atrophy in the fellow eye. Extensive ocular and extraocular investigation failed to uncover a specific etiology. Pseudotumor cerebri, a well-documented cause of papilledema and choroidal folds, was ruled out in each case. Each patient did demonstrate variable degrees of hyperopia and shortened axial lengths. CONCLUSIONS: The syndrome of bilateral choroidal folds and optic neuropathy may have various etiologies. Pseudotumor cerebri needs to be definitively ruled out. Normal neuro-ophthalmological investigation including lumbar puncture may indicate an alternative cause. Idiopathic acquired hyperopia in middle-aged patients who are hyperopic with shortened axial lengths may be associated with choroidal folds and a constricted scleral canal causing optic disk congestion and complicated by nonarteritic anterior ischemic optic neuropathy due to a crowded disk.

Aged↗
PubMed1988

Dysthyroid optic neuropathy. The crowded orbital apex syndrome.

The authors have reviewed the clinical presentation, visual fields, color vision testing, visual-evoked potentials, and computed tomographic (CT) findings of 58 patients (95 eyes) with dysthyroid optic neuropathy. The authors compared these findings to a control group of 60 patients (119 eyes) with thyroid eye disease who underwent CT scanning and did not exhibit evidence of optic neuropathy. Clinically, dysthyroid optic neuropathy is an insidious disease; when compared with the usual Graves' orbitopathy patient, the optic neuropathy group presented at a later age and with a later onset of thyroid eye disease. The patients in this group were more likely to be male and/or diabetic, and often presented with desaturation of color vision. Asymmetrical extraocular muscle restriction and vertical tropias were more frequent in the optic neuropathy group. The most sensitive indicators of optic nerve dysfunction appeared to be visual-evoked potentials and color vision. Computed tomographic studies confirmed that apical orbital crowding was a characteristic feature of optic neuropathy. These findings should alert the clinician to a more aggressive approach to these patients.

Adult↗
PubMed2000

Medicaid and crowding out of private insurance: a re-examination using firm level data.

While previous research has identified a relationship between expanded Medicaid eligibility and falling private health insurance coverage, the exact mechanism by which this "crowding out" occurs is largely unexplained. We combine individual and firm-level data to investigate possible responses to the Medicaid expansions by firms and workers. We find no evidence that the expansions affected employer offers of insurance to workers. However, we find some evidence of an effect on the probability that a firm offers family coverage, and on the percentage of full-time workers accepting employer-sponsored coverage offered to them.

Child↗
PubMed1997

Macromolecular crowding: effects on actin polymerisation.

Dextran has been found to enhance the polymerisation of actin. This enhancement increases exponentially with increasing mass concentrations of dextran, in a manner that is consistent with excluded volume theory. Mathematical prediction of experimental results is difficult due to the fact that all participating species, namely F-actin, G-actin and dextran are best represented by differently shaped hard particles. Modelling dextran as a sphere of radius defined by an effective thermodynamic radius (Reff), we have predicted our experimental results to an acceptable degree, given the relative crudity of the model. The results imply that the highly crowded cellular environment may help to stabilise the filamentous actin network in vivo.

Actins↗
PubMed1999

Folding and unfolding of a giant duplex-DNA in a mixed solution with polycations, polyanions and crowding neutral polymers.

To understand the conformational behavior of a giant duplex-DNA chain in a mixed solution with various biopolymers with different state of ionization, the higher-order structure of the DNA chain was analyzed with a fluorescence microscope in the presence of polycations (poly-arginine), polyanions (poly-glutamic acid), and neutral polymers (poly-ethylene glycol) as a model for cellular environment. Concentrated medium with neutral polymer induced the discrete folding transition of the DNA. At the threshold condition for the transition, addition of small amounts of either the polycation or the polyanion caused marked structural changes in the folded DNAs. Based on thermodynamic considerations on the experimental results, profile of free energy of a single giant DNA chain was depicted with respect to the size, or the expansion factor alpha, in the three-dimensional structure of the DNA. The effect of the neural crowding polymer on the degree of folding of a single giant DNA chain is discussed in a semi-quantitative manner.

Journal Article↗