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PubMed2001

Crowding and hydration effects on protein conformation: a study with sol-gel encapsulated proteins.

We are developing an experimental system for testing the effects of macromolecular crowding and molecular confinement on protein structure. In the present study, solvent effects on the secondary structure of two proteins were examined by circular dichroism following encapsulation in the hydrated pores of a silica glass matrix by the sol-gel method. Changes in the unfolded conformations of encapsulated apomyoglobin and reduced serum albumin were analyzed after equilibration with aqueous solutions of natural osmolytes, short-chain alcohols, polyethylene glycol, and a complete series of Hofmeister cations. In many instances, the alpha-helical content of the encapsulated protein was increased by addition of solutes at concentrations that have no effect on the protein in the absence of the glass. The results are discussed from the perspective of water structure. We argue that perturbed water at the silica interface causes an increase in the average free energy of the bulk water phase which, consequently, diminishes the strength of the hydrophobic effect inside the glass matrix and destabilizes the conformation of encapsulated proteins. We propose that solutes can increase the strength of the hydrophobic effect and influence folding equilibria without directly interacting with the protein. A hypothesis is provided for the apparent paradox that kosmotropic (strongly water binding) anions favor native protein structure, whereas chaotropic (weakly water binding) cations enhance native protein structure. The encapsulation results suggest that macromolecular crowding and molecular confinement are accompanied by hydration effects that may oppose or potentiate the stabilizing effects of excluded volume on protein structure, depending on the surface chemistry of the crowding agent and its influence on bulk water structure. In the crowded environment of a living cell, excluded volume effects, surface-induced water structure, and compatible solutes are expected to complement the dominant forces in protein folding.

Apoproteins↗
PubMed2002

Anterior crowding--just an esthetic problem?

BACKGROUND: One frequent late sequel of frontal crowding is the development of gingival recessions manifesting themselves especially at rotated anterior teeth. MATERIAL: This correlation is discussed with regard to the literature and to the authors' own clinical findings and experimental studies. RESULTS: According to present-day knowledge the following statements can be made: In cases with frontal crowding the tangential stiffening of the anterior teeth is inadequate. Thus translatory and rotational force components resulting from the elastic deformation of the mandible under functional loading effect the movement of rotated front teeth during maximum jaw opening and mandibular protrusion. Also tongue thrusting and frontolateral bruxism lead to force vectors in vestibular direction affecting the delicate periodontal tissues. These statements are confirmed by the authors' own in-vivo experimental studies measuring the amount of tooth displacement during test persons' functional movements. Furthermore, in the long-term anterior crowding will mostly trigger incorrect anterior guidance, i.e. guidance predominantly by the rotated or ectopically positioned front tooth with subsequent overloading of the antagonist. CONCLUSION: Orthodontic therapy of frontal crowding is not just an esthetic problem but can be of special importance in the prevention of stress-related reactions in the masticatory system.

Esthetics, Dental↗
PubMed1999

Crowding Norms in Marine Settings: A Case Study of Snorkeling on the Great Barrier Reef.

/ Research on crowding in natural environments has traditionally been concerned with encounters in terrestrial settings. Increased visitation to tropical marine environments, however, has meant that evaluations of aesthetic quality are increasingly becoming issues for managers of marine parks. In this study, we used image-capture techniques to develop a series of above- and below-water images depicting different numbers of people snorkeling in acoral reef setting. The presence of safety facilities in the above-water settings was manipulated to examine the influence of human-made structures on perception of crowding. Four respondent groups-a scuba-diving club, local residents, tourists, and US university students-representing different levels of experience in marine recreation on the Great Barrier Reef, were asked to rate the acceptability of each image. Ratings were significantly influenced by the number of people in the images, the prior experience and gender of the respondents, and the presence of safety infrastructure. Experienced scuba divers preferred scenes without people or infrastructure, while novices regarded the presence of both as more acceptable. The results suggest that evaluations of social density and crowding may vary between below-water scenes and the more familiar above-water setting. A lack of concordance between how respondents rated the images and their nominated preferences for the number of other people in the settings highlights a need for more research on how perceptions of resource conditions should be measured in marine environments.KEY WORDS: Recreation; Great Barrier Reef Marine Park; Image capture technology; Crowding norms; Snorkelinghttp://link.springer-ny.com/link/service/journals/00267/bibs/24n3p369.html

Journal Article↗
PubMed1982

The adaptive value of dental crowding: a consideration of the biologic basis of malocclusion.

Malocclusion, an ambiguous concept that refers to structural disharmony of the teeth and jaws, can be defined only in reference to normal occlusion. If normal occlusion is synonymous only with ideal occlusion, then normal occlusion becomes a rare occurrence. A biologically valid concept of normal occlusion includes a range of variation in the relevant occlusal variables that is compatible with health and unimpaired function. The difficulty in defining malocclusion is the determination of the point at which normal variation becomes abnormal. A clearer concept of malocclusion is obtained if the occlusal variables that it comprises are considered. Dental crowding is endemic among technologically advanced populations and uncommon in primitive groups. The significant elements in the development of most dental crowding are mesial migration and the lack of interproximal attrition. Mesial migration of the posterior teeth provides the functional replacement for the tooth surface lost to attrition because of the rigors of a primitive diet. In modern man there is little attrition of the teeth because of a soft, processed diet; this can result in dental crowding and impaction of the third molars. It is postulated that the tooth-jaw size discrepancy apparent in modern man as dental crowding is, in primitive man, a crucial biologic adaptation imposed by the selection pressures of a demanding diet that maintains sufficient chewing surface area for long-term survival. Selection pressures for teeth large enough to withstand a rigorous diet have been relaxed only recently in advanced populations, and the slow pace of evolutionary change has not yet brought the teeth and jaws into harmonious relationship.

Adaptation, Biological↗
PubMed1980

Crowding depression of UV-mutagenesis in E. coli.

Strains of E. coli B/r were exposed to UV radiation and assayed for reversion mutation, using a standard selection medium. If more irradiated bacteria were assayed per petri dish, a proportional increase in the number of indicated reversion mutants was found only up to a limiting plating density. Beyond a density of about 10(8) viable bacteria per petri dish, the number of indicated revertants per viable bacteria assayed (the mutation frequency) decreased as the plating density was increased. The crowding depression of mutagenesis was more severe for de novo and converted suppressor mutations, the mutation frequency being reduced 100-fold at a plating density of about 6 x 10(9) viable bacteria per plate. The effect on backmutation was 10 times less. Crowding depression of mutagenesis occurred in excision-proficient and -deficient strains, with identical effects in the 2 strains on de novo and converted suppressor mutation, but different effects on backmutations. There were no accompanying effects on viability. Irreversible loss of potential mutants during crowded growth was indicated in wash-off experiments. The kinetics suggested a half-life of approximately 1 h. Kinetics for accumulation by the bacteria of the limiting metabolite (tyrosine) on the assay plate indicated a short period of time for protein synthesis, but direct examination of the proteins synthesized during early growth on a crowded plate demonstrated successful induction of recA protein. The results suggested a possible disruption in the rec/lex respondency system somewhere between induction of recA protein and the various end points, including mutational repair, in cells plated close to one another.

Bacterial Proteins↗
PubMed1981

cis-Platinum(II)diamminodichloride-induced mutagenesis in E. coli K12: crowding depression of mutagenesis.

Cis-Platinum(II)diamminodichloride (cis-PDD)-induced mutations to prototrophy were studied in Escherichia coli. Mutagenesis was not detected in a recA nor in a lexA mutant, but was greater in a uvrA strain than in a repair-proficient strain, at a given treatment of cis-PDD. Increasing the plating density above 10(5) cells per plate did not give an equivalent increase in revertants per plate [crowding depression of mutagenesis (Bockrath et al., 1980)]. Growth rates were similar at different plating densities and crowding depression of mutagenesis was observed in both excision-proficient and excision-deficient strains. A filtrate of a plate wash from crowded plates, of either treated or untreated cultures, further reduced the mutation frequencies over that due to crowding depression of mutagenesis.

Cisplatin↗
PubMed1995

Effects of housing on male and female rats: crowding stresses male but calm females.

Housing conditions affect behavioral and biological responses of animals. Effects of same-sex grouped, crowded, or individually housed conditions on plasma corticosterone levels of male and female Wistar rats were examined in two experiments. Experiment 1 examined the effects of individual vs. crowded housing conditions on corticosterone, a biochemical index of stress, in seven male and seven female rats. Experiment 2 extended the findings of Experiment 1 by separately manipulating spatial and population aspects of housing with 50 male and 50 female rats. Male rats had higher corticosterone levels under crowded conditions. In contrast, female rats had higher levels when individually housed. Spatial crowding was the key variable for males, whereas the number of other animals was more important for females. These results indicate that investigators must consider housing conditions as an intervening variable that is likely to differentially affect behaviors of male and female rats.

Analysis of Variance↗
PubMed1988

Correlations between lower incisor crowding and lower incisor position and lateral craniofacial morphology.

Crowding of the lower incisors is a problem encountered frequently in orthodontic practice. Successful therapy may depend on the orthodontist's ability to evaluate factors contributing to the overall pattern. Two of these factors, vertical skeletal morphology and lower incisor position in the lateral cephalogram, were evaluated. Dental casts and cephalograms of 100 children with primary mandibular incisor crowding (that is, primary discrepancy between mesiodistal tooth width and available space of the dental alveolar process and apical base) were examined in this study. Results indicated the following: (1) all vertical skeletal and lower incisor position measurements closely duplicated published norms, (2) no correlation was found between lower incisor crowding and either skeletal morphology or lower incisor position, (3) a factor analysis did demonstrate, however, that other selected variables were interrelated, and (4) the cause of lower incisor crowding must be attributed to factors not examined in this study.

Cephalometry↗
PubMed1988

Dental crowding and its relationship to mesiodistal crown diameters and arch dimensions.

An investigation was carried out to determine the correlation among cumulative mesiodistal crown widths, arch dimensions, and the degree of primary dental arch crowding. Two different ethnic groups were studied--namely, indigenous British and Pakistani immigrant groups--to determine whether similar correlations existed as part of a larger study of ethnic comparisons. Correlation matrix computation and multiple regression analysis were among the tests used. The results showed that in both ethnic groups there were very significant correlations between certain arch dimensions and the degree of crowding. There was no significant correlation between cumulative mesiodistal crown widths and dental crowding when considered in isolation. In combination with the other parameters, however, cumulative mesiodistal crown widths contributed significantly to the overall regression equation. This suggested a complex interrelationship among cumulative mesiodistal crown widths, the various arch dimensions, and primary dental crowding.

Adolescent↗
PubMed2005

Intra-arch occlusal indicators of crowding in the permanent dentition.

INTRODUCTION: The objective of this study was to identify the intra-arch occlusal characteristics that best discriminated 3 groups with different grades of dental arch discrepancies. This cross-sectional analysis was conducted in Lima, Peru, in 2003. METHODS: Intra-arch measurements were made on 150 sets of dental casts of high school students (aged 12-16; 75 boys, 75 girls). Stepwise multiple discriminant analysis (SMDA) was used to obtain a better understanding of the morphological relationships between tooth and dental-arch variables and their relationship with crowding. RESULTS: Mesiodistal tooth sizes and crown proportions of some teeth differed among significantly crowded, mild-to-moderately crowded, and spaced dental arches. Buccolingual tooth sizes were similar in the 3 groups. Of the arch dimensions evaluated, only intermolar arch width and arch length differed between the groups. An SMDA was developed to classify dental-arch discrepancies in the permanent dentition based on several intra-arch occlusal characteristics. The variable with the highest discriminatory capability between groups was arch length. When arch length was taken out of the SMDA, the explanatory capability from the variability on the dental arch discrepancies diminished from 51% to 14%. When the remaining arch dimension variable (intermolar width) was taken out, the explanatory capability diminished more (from 14% to 8%). CONCLUSIONS: Although other tooth-size and arch dimensions are indicators of crowding, arch length is the most important factor.

Adolescent↗
PubMed2004

Transcript profiling of pacifastin-like peptide precursors in crowd- and isolated-reared desert locusts.

Locusts have fascinated researchers for several decades, because they have the remarkable ability to undergo phase transition from the harmless solitary to the swarm-forming gregarious phase. However, the physiological and endocrine mechanisms, underlying phase polymorphism, are only partially unravelled. Nevertheless, besides the 'classical' hormones, pacifastin-related peptides have been suggested to play a role in phase transition. Here, we present the first quantitative and comparative analysis of locust transcripts, in particular pacifastin-related precursor (SGPP-1-3) mRNAs, between isolated-reared (solitary) and crowd-reared (gregarious) desert locusts, revealing a phase-dependent transcriptional regulation of the corresponding genes. While the SGPP-1 and SGPP-3 transcripts were most abundant in fat body from crowd-reared males, corresponding to significantly higher levels than in isolated-reared males, the SGPP-2 transcript was detected most abundantly in brain from crowd-reared male locusts. Furthermore, SGPP-2 transcript levels in brain, testes, fat body, and accessory glands from crowd-reared males significantly exceeded the levels in solitary locusts.

Adaptation, Physiological↗
PubMed2006

Translational and rotational motions of proteins in a protein crowded environment.

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.

Apoproteins↗
PubMed2006

Expanding to fill the gap: a possible role for inert biopolymers in regulating the extent of the 'macromolecular crowding' effect.

We discuss the potential for inert biopolymers existing in cells to play a role in regulating the macromolecular crowding effect via their ability to undergo shape changing structural transitions. We have explored this possibility by the use of theory and experiment. The theoretical component utilized Monte-Carlo based simulations to examine the folding of a hypothetical protein in a concentrated environment of hard spheres which are themselves capable of reversible expansion and contraction. The experimental component of the study involved examination of the effect of different sized crowding agents on the thermally induced denaturation of cytochrome c [in phosphate buffered saline solution containing 1.0M guanidinium hydrochloride at pH 7.0]. On the basis of our findings we suggest that in a crowded solution environment the presence of a non-reactive polymer capable of reversible expansion/contraction via folding and unfolding may alter the excluded volume component of the solution. This ability would confer on the non-reactive polymer a novel role in influencing other processes in solution affected by macromolecular crowding.

Animals↗
PubMed2002

Search for peptidic molecular markers in hemolymph of crowd-(gregarious) and isolated-reared (solitary) desert locusts, Schistocerca gregaria.

An HPLC analysis of hemolymph extracts was undertaken to uncover differences between desert locusts, Schistocerca gregaria, reared under either crowded or isolated conditions. Some differences in the chromatographic pattern could be detected. One of the major peaks in the hemolymph of crowd-reared adults was found to be a minor one in isolated-reared individuals, whereas other peaks increased after solitarization. The differences became even more pronounced after several generations of isolated rearing. The dominant chromatographic peak in hemolymph extracts of the crowd-reared animals was identified as a novel peptide with a molecular mass of 6080Da. Edman degradation in combination with enzymatic fragmentation and quadrupole-time of flight (Q-Tof) mass spectrometry revealed the full sequence: DNADEDTICVAADNKFYLYANSLKLYTCYNQLPKVYVVKPKSQCRSSLSDCPTS. This 54 aa-peptide is very abundant in hemolymph of crowd-reared adults. Its concentration in hemolymph amounts to 0.1mM. To uncover the function, its effects were investigated in several bioassays, so far without positive results. One of the other peaks differentially expressed in the individuals of the two phases was identified as SGPI-2 (MW=3794Da), which is a serine protease inhibitor in locusts.

Amino Acid Sequence↗
PubMed2005

Mixing crowded biological solutions in milliseconds.

In vitro studies of biological reactions are rarely performed in conditions that reflect their native intracellular environments where macromolecular crowding can drastically change reaction rates. Kinetics experiments require reactants to be mixed on a time scale faster than that of the reaction. Unfortunately, highly concentrated solutions of crowding agents such as bovine serum albumin and hemoglobin that are viscous and sticky are extremely difficult to mix rapidly. We demonstrate a new droplet-based microfluidic mixer that induces chaotic mixing of crowded solutions in milliseconds due to protrusions of the microchannel walls that generate oscillating interfacial shear within the droplets. Mixing in the microfluidic mixer is characterized, mechanisms underlying mixing are discussed, and evidence of biocompatibility is presented. This microfluidic platform will allow for the first kinetic studies of biological reactions with millisecond time resolution under conditions of macromolecular crowding similar to those within cells.

Biocompatible Materials↗
PubMed2001

Effects of crowding by mono-, di-, and tetrasaccharides on cytochrome c-cytochrome c peroxidase binding: comparing experiment to theory.

In cells, protein-protein interactions occur in an environment that is crowded with other molecules, but in vitro studies are almost exclusively performed in dilute solution. To gain information about the effects of crowding on protein complex formation, we used isothermal titration calorimetry to measure the stoichiometry, the free energy change, and the enthalpy change for the binding of yeast iso-1-ferricytochrome c to yeast ferricytochrome c peroxidase in dilute solution and in solutions crowded with the sugars glucose, sucrose, and stachyose. The stoichiometry is 1:1 under all conditions. The sugars stabilize the complex, but by only 0.1-0.5 kcal.mol(-)(1), and the increased stability is not correlated with the change in enthalpy of complex formation. We then compared the measured stability changes to values obtained from several analyses that are currently used to predict crowding-induced changes in biomolecular equilibria. None of the analyses are completely successful by themselves, and the results suggest that a complete analysis must account for both excluded-volume and chemical interactions.

Calorimetry↗
PubMed2002

Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding.

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.

Chaperonin 10↗
PubMed2001

Effects of macromolecular crowding on the intrinsically disordered proteins c-Fos and p27(Kip1).

A number of biologically active proteins exhibit intrinsic structural disorder in vitro under thermodynamically ideal conditions. In vivo, however, proteins exist in a crowded, thermodynamically nonideal environment. We tested the hypothesis that intrinsically disordered proteins adopt stable structure under crowded conditions in which excluded volume is predicted to stabilize compact, native conformations. In the presence of macromolecular crowding agents, neither the intrinsically disordered C-terminal activation domain of c-Fos nor the kinase-inhibition domain of p27(Kip1) undergoes any significant conformational change that is detected by changes in either circular dichroism or fluorescence spectra. We conclude that molecular crowding effects are not necessarily sufficient to induce ordered structure in intrinsically disordered proteins.

Cell Cycle Proteins↗