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Alzheimer's disease is a progressive fatal neurodegenerative disease with no cure or effective treatments. The hallmarks of disease include extracellular plaques and intracellular tangles of aggregated protein. The intracellular tangles consist of the microtubule associated protein tau. Preventing the pathological aggregation of tau may be an important therapeutic approach to treat disease. In this study we show that small heat shock protein 22 kDa (Hsp22) can prevent the aggregation of tau in vitro. Additionally, tau can undergo liquid–liquid phase separation (LLPS) in the presence of crowding reagents which causes it to have an increased aggregation rate. We show that Hsp22 can modulate both the aggregation and LLPS behavior of tau in vitro.

Protein aggregation has two aspects, namely, mechanistic and kinetics. Understanding protein aggregation kinetics is critical for prediction of progression of diseases caused by amyloidosis, accumulation of aggregates in biotherapeutics during storage and engineering commercial nano-biomaterials. In this work, we have collected experimentally determined absolute protein aggregation rates and developed an SVM based regression model to predict absolute rates of protein and peptide aggregation near-physiological conditions. The regression model achieved a correlation coefficient of 0.72 with MAE of 0.91 (natural log of kapp, where kapp is in hour-1) using leave-one-out cross-validation on a dataset of 82 non-redundant proteins/peptides. The model accounts for the experimental conditions (such as temperature, pH, ionic and protein concentration) and sequence-based properties. The amino acid sequence features revealed by this model as being important for aggregation kinetics, are also associated with the aggregation mechanism. In particular, inherent aggregation propensity of the protein/peptide sequence and number of aggregation prone regions (APRs) unpunctuated by the gatekeeping residues, were found to play important roles in the prediction of the absolute aggregation rates. This analysis shows that mechanism and kinetics of protein aggregation are coupled via common sequence attributes. The aggregation kinetic prediction method developed in this work is available at https://web.iitm.ac.in/bioinfo2/absolurate-pred/index.html.

Cardoon flower extract is a vegetable coagulant used in the manufacture of some highly appreciated Mediterranean Protected Designation of Origin (PDO) sheep cheeses, due to their typical flavor and smooth texture. Flowers from 6 cardoon plants (1M–6M), selected on the basis of the total and specific A0, A and B cardosin concentrations were characterized using chromatographic and electrophoretic analysis. Extracts of these flowers were used to investigate the relationship between the biochemical profile and the proteolytic activity on caseins, coagulation properties and cheese texture. Milk-clotting activity and coagulation were measured using an Optigraph. Cheese making trials were done using the traditional protocol of Serra da Estrela PDO cheese manufacture. A multivariate exploratory PCA analysis showed that plants 4M, 5M and 6M with the highest total concentration of cardosins, the prevalence of cardosin A, proteolytic action with Ƙ-casein, and the lowest time of coagulation yielded cheeses with a firmer paste. Plants 1M, 2M and 3M, without the presence of cardosin A, were negatively influenced by the proteolytic action with β-casein, starting flocculation and gel consistency. Plant 3M showed a slow micellar aggregation rate and low gel firmness during milk coagulation, which was reflected in the smooth texture of the cheese. The specific characterization of natural biochemical diversity of cardoon flowers and their influence on cheese texture may help producers to choose the appropriated cardoon flowers to obtain a desired texture.

Single amino acid mutations in amyloid-beta (Aβ) peptides can lead to early onset and increased severity of Alzheimer's disease. An example is the Osaka mutation (Aβ1-40E22D), which is more toxic than wild-type Aβ1-40. This mutant quickly forms early stage fibrils, one of the hallmarks of the disease, and these fibrils can even seed fibrilization of wild-type monomers. Using molecular dynamic simulations, we show that because of formation of various intra- and intermolecular salt bridges the Osaka mutant fibrils are more stable than wild-type fibrils. The mutant fibril also has a wider water channel with increased water flow than the wild type. These two observations can explain the higher toxicity and aggregation rate of the Osaka mutant over the wild type.

Amyloid proteins of different aminoacidic composition share the tendency to misfold and aggregate in a similar way, following common aggregation steps. The process includes the formation of dimers, trimers, and low molecular weight prefibrillar oligomers, characterized by the typical morphology of globules less than 10 nm diameter. The globules spontaneously form linear or annular structures and, eventually, mature fibers. The rate of this process depends on characteristics intrinsic to the different proteins and to environmental conditions (i.e., pH, ionic strength, solvent composition, temperature). In the case of neurodegenerative diseases, it is now generally agreed that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. However, the molecular mechanism by which these oligomers trigger neuronal damage is still unclear. In particular, it is not clear if there is a peculiar structure at the basis of the neurotoxic effect and how this structure interacts with neurons. This review will focus on the results we obtained using salmon Calcitonin, an amyloid protein characterized by a very slow aggregation rate, which allowed us to closely monitor the aggregation process. We used it as a tool to investigate the characteristics of amyloid oligomers formation and their interactions with neuronal cells. Our results indicate that small globules of about 6 nm could be the responsible for the neurotoxic effects. Moreover, our data suggest that the rich content in lipid rafts of neuronal cell plasma membrane may render neurons particularly vulnerable to the amyloid protein toxic effect.

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation.

The interaction between urea and guanidinium chloride (GuHCl) on lysozyme refolding was investigated in this work. Live micrococcus lysodeikticus was successfully introduced into a refolding system. Lysozyme can be refolded from the GuHCl-denatured, DTT-reduced state in a good yield of 96.54% at final protein concentration as high as 0.2 mg·mL−1. A model could be employed to elucidate refolding kinetics behavior and the kinetics constants were studied. In the coexistence of GuHCl and urea, the aggregation rate decreased by increasing urea concentration to a proper value. The cooperation of GuHCl and urea not only suppressed the competition of the aggregation reaction but also increased the yield of refolding efficiently.

In experiments on Wistar male rats, on a specially constructed computer installation, O2 consumption by the animals in comparison with changes of hematological, biochemical, and rheological blood properties is studied after anemization--acute bloodletting (12-15% of the total blood mass). An increase of the O2 consumption by the organism and tissues by 18-28% has been revealed for the first 7 days after the bloodletting, in spite of a pronounced decrease of hematocrit, and of the amount of erythrocytes and hemoglobin in peripheral blood by 20-25% of the initial level. There was a 5-10-fold increase of the content of immature erythrocyte forms--reticulocytes and a progressive rise of cell acidic resistance, which is characteristic of young erythrocyte forms. An increase of O2 consumption at a decrease of the blood oxygen capacity (a low hemoglobin level) seems to be due to the more efficient transport and yield of O2 to tissues. At the 3rd 7th day after the bloodletting, activity of Na,K-ATPase has been shown to increase by 60% and 20%, respectively. Analysis of the erythrocyte rheological properties has shown that the maximal firmness of aggregates (Uq) and the aggregation rate (1/T) decrease progressively beginning from 3 days after the bloodletting; index of deformability (I(max)) turned out to be elevated by 7-11%, probably due to an increase of the cell membrane elasticity. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed to optimization of blood circulation in large vessels and the capillary network.

Beta-casein (beta-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel beta-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k(c), microM(-1)), thermal aggregation constant (k(T), degrees C(-1)) and aggregation rate constant (k(t), min(-1)) were measured. Bovine beta-CN displays greater chaperone activity than camel beta-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine beta-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel beta-CN. Greater relative hydrophobicity of bovine beta-CN than camel beta-CN may be a factor responsible for stronger interactions of bovine beta-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine beta-CN.

Biosynthesis of silver nanoparticles in an aqueous mixture of fig (Ficus carica) leaf extract and AgNO3 solution exposed to a set of irradiances at different wavelengths are studied. Nanoparticle formation for irradiances between 6.5 mW/cm(2) and 13.3 mW/cm(2) in the 330-550 nm wavelength range is investigated and the results are compared to those of the nanoparticles synthesized in the dark and under direct sunlight. Ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy, along with particle size analysis and transmission electron microscopy are employed for the characterization of samples and extracts. Irradiance is found to have profound influence on the reduction rates. However, size and spherical shape of the nanoparticles are persistent, irrespective of irradiance and wavelength. Irradiance is discussed to influence the particle formation and aggregation rates through the formation of free radicals in the fig extract.

In experiments on rats of the Wistar line and on spontaneously hypertensive animals of the SHR line, a complex study was performed of rheological (deformability and aggregation properties) and hematological parameters of erythrocytes at the single (40 min) and multiple (20–30 min for 7 and 14 days) exercise load (a forced tredmill running at a rate of 14 m/min). It has been shown that rats of both lines respond to the single, sufficiently light exercise load by a manifestation of stress-reaction. After the repeated exercise load, all stress-induced changes were leveled and replaced by adaptation to this action in Wistar rats, but not in the SHR line rats. The hypertensive rats were characterized by a greater fatigue and refusal of the tredmill running; therefore, this load to them had to be shortened to 20 min. For the 1st week of running, no statistically significant differences in the studied parameters were found between rats of the Wistar and SHR lines; however, by the 14th day of training, substantial differences between these lines were noticed: the Wistar rats were easily adapted to the tredmill running, whereas the SHR animals completely refused to run. At the single running, the stress-polycythemia, an increased hematocrit, a decreased hemoglobin concentration per erythrocyte, an increased plasma corticosterone level, and changes of blood rheological characteristics were shown, the erythrocyte deformability and the spontaneous aggregation rate under the stress conditions being in the direct positive correlational dependence that was absent in control. The Wistar rat training to the exercise load for 14 days led to normalization of the parameters, which indicates a favorable effect of trainings on the animal health state.

We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the alpha/beta protein acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16-31 and 87-98, each of which has a relatively high hydrophobicity and propensity to form beta-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and beta-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.

Familial British Dementia (FBD) is the early-onset autosomal dominant disorders characterized by cognitive impairment. The ABri is a 34-mer peptide which is the main component of amyloid deposits in FBD. As a first step toward shedding light for the discovery of amyloid inhibition drugs for FBD, we set the optimal conditions for quantification ABri peptide by the fluorescamine and alpha imager assay, which can provide a precise peptide quantity for determination of amyloid inhibition rate at micromolecular level and estimate aggregation rate by measuring monomer and dimer quantity which are filtered through 10,000 Da MW cutoff filter.

The tendency of equine IgG to form soluble aggregates at low pH was a function of elution time during purification by anion exchange chromatography on DEAE-Sepharose, with later eluting fractions showing faster aggregation rate and higher aggregate levels in the pH range 3·5–4·0. An ELISA assay using antibodies to different equine IgG subclasses was used to show that subclasses have different elution profiles, with IgGb being slowest to elute. Separation of the soluble aggregates by polyethylene glycol precipitation and analysis for subclass composition showed that aggregates were enriched in IgGb. ANS binding experiments demonstrated that later eluting fractions have a considerably greater exposure of surface hydrophobic groups at pH 3·7. The results underscore the importance of careful control of the ‘cut-off’ volume during ion exchange chromatography of polyclonal IgG in preparing a consistent purified product.

The present investigation is aimed to understand the physiological significance of bilirubin interaction with collagen. In human skin, collagen absorbs both free bilirubin and serum bound bilirubin from the human system. Interaction between bilirubin and collagen depends on time, temperature and concentration of bilirubin. There is an increase in the aggregation rate of collagen in the presence of biliruibin. At physiological condition, 125 nM of bilirubin is the maximum concentration absorbed by per mg of collagen molecule. Bilirubin accelerates the lateral growth of collagen fibrils by shifting its rate of nucleation. Moreover, collagen-bilirubin complex exhibit a tendency to undergo adsorption onto the surface of the fibroblast cells, showing detrimental effects on fibroblasts proliferations. Based on the collagen binding assays, the binding of bilirubin to collagen is found to be electrostatic in nature, which confirms binding between the amino acid fragment of α1 (I) region of collagen and carboxyl group of bilirubin. The biotinylated bilirubin derivatives show better binding to α1 (I) chain rather than α2 (I) chains which clearly designates that bilirubin shows greater affinity to α1 chains of collagen. This novel approach directs to reduce the occurrence of bilirubin in hyperbilirubinemia patients.

Therapeutic proteins must be generally formulated to reduce unwanted aggregation. Fusion proteins, which comprise domains assembled from separate proteins, may require unique formulation strategies in order to maximize their stability. A fusion protein of human serum albumin (HSA) and human granulocyte colony stimulating factor (GCSF; HSA–GCSF) was used as a model to test the hypothesis that formulations that increase the thermodynamic conformational stability of the least stable domain of a fusion protein will stabilize the entire fusion protein against aggregation. Conformational stability of HSA–GCSF was modulated by addition of octanoic acid, which was previously shown to increase the conformational stability of HSA, the least stable domain. Contrary to our hypothesis, increased conformational stability of the HSA domain did not result in increased resistance to aggregation of HSA–GCSF. These results for HSA–GCSF were also compared with similar studies conducted previously on a therapeutic protein formed by the fusion of HSA and human growth hormone (hGH; HSA–hGH). © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2009–2016, 2012

The current challenge in the amyloid field is to obtain structural information on the progression of associated aggregation pathways, relate them to the cause of disease, and specifically point to the species truly responsible for neurotoxicity. Huntington's disease (HD) is linked to the aggregation of mutant huntingtin (htt) peptide, which contains an abnormally long polyglutamine (polyGln) repeat that leads to the formation of fibrils with stable, β-sheet rich structures. Analogous to other neurodegenerative diseases like Alzheimer's, mature htt fibrils are thought to be overall protective while the earlier smaller intermediates are proposed to be toxic to neuronal cells. We performed time-resolved small-angle neutron scattering (SANS) experiments to monitor the structural kinetics for htt peptides having polyGln repeats in the normal (22 Glns) and pathological (42 Glns) range. In addition to observing the expected faster aggregation rate for longer polyGln repeats, we find distinct conformational differences between normal and pathological htt. We also present, for the first time, three-dimensional structures of intermediates formed at the earliest stages of htt aggregation as detected by SANS and obtained using ab initio shape reconstruction methods. Finally, we are able to investigate the internal structure of the mature fibrils, where the mass-per-length of pathological htt fibrils is considerably less than normal htt fibrils. These findings provide the first steps toward characterizing the oligomers formed by htt peptides and illustrate the utility of SANS for identifying different aggregate intermediates formed in the development of neurodegenerative diseases.

Aluminium has been widely recognized as a neurotoxic element, as demonstrated in relation to long-term dialysis treatment in uremic patients. More uncertain is its potential role as a factor/cofactor in the etiopathogenesis of Alzheimer's disease (AD) as well as of other neurodegenerative diseases. The distinction between neurodegeneration and neurotoxicity is crucial to gaining a clear understanding of the role of aluminium in the field of neuroscience.This chapter critically reviews the recent findings correlating aluminium to AD, highlighting in particular what remains from the old “aluminium hypothesis”. Moreover, recent studies performed by our group, on the interaction between aluminium and β-amyloid peptide (Aβ), shed some light on the potential involvement of Al in particular in the pathogenesis of Alzheimer's disease, as reported herein. The involvement of metal ions as a potential key factor in conformational modifications and aggregation of Aβ is widely recognized. We thus investigated the potential and differential role of some metal ions (e.g., aluminium, copper, iron and zinc) in affecting the fibrillogenesis of Aβ. Data from our laboratory demonstrated that the aggregational profile of the Aβ peptide was differently affected by the presence of various metal ions. In particular, aluminium was the most effective, among the tested metals, in promoting a conformational modification of Aβ which resulted in an increased aggregation rate and exposure of hydrophobic clusters. This modification stabilized the peptide in an oligomeric (apparently 8–9mers) state which was highly toxic to neurons. Moreover, the genotoxic role of Aβ and Aβ–metal complexes was also investigated in human neuroblastoma cell culture, indicating that aluminium, when complexed with Aβ, can stimulate genes directly correlated with AD.A better understanding of the interaction of Al and Aβ will certainly help in clarifying its potential involvement, if any, in AD etiology.

The conversion of proteins into structured fibrillar aggregates is a central problem in protein chemistry, biotechnology, biology and medicine. It is generally accepted that aggregation takes place from partially structured states of proteins. However, the role of the residual structure present in such conformational states is not yet understood. In particular, it is not yet clear as to whether the α-helical structure represents a productive or counteracting structural element for protein aggregation. We have addressed this issue by studying the aggregation of pH-unfolded HypF-N. It has previously been shown that the two native α-helices of HypF-N retain a partial α-helical structure in the pH-unfolded state and that these regions are also involved in the formation of the cross-β structure of the aggregates. We have introduced mutations in such stretches of the sequence, with the aim of increasing the α-helical structure in the key regions of the pH-unfolded state, while minimizing the changes of other factors known to influence protein aggregation, such as hydrophobicity, β-Sheet propensity, etc. The resulting HypF-N mutants have higher contents of α-helical structure at the site(s) of mutation in their pH-unfolded states, but such an increase does not correlate with a change of aggregation rate. The results suggest that stabilisation of α-helical structure in amyloidogenic regions of the sequence of highly dynamic states does not have remarkable effects on the rate of protein aggregation from such conformational states. Comparison with other protein systems indicate that the effect of increasing α-helical propensity can vary if the stabilised helices are in non-amyloidogenic stretches of initially unstructured peptides (accelerating effect), in amyloidogenic stretches of initially unstructured peptides (no effect) or in amyloidogenic stretches of initially stable helices (decelerating effect).

In amyloid fibrils, beta-strand conformations of polypeptide chains, or segments thereof, are perpendicular to the fibril axis, but knowledge of their three dimensional structure at atomic level of detail is scarce. Two types of computational approaches have been developed recently for investigating the aggregation propensity of peptides and proteins and identifying the segments most prone to form fibrils (hot spots). The physicochemical properties of the natural amino acids (e.g. beta-propensity, hydrophobicity, aromatic content and charge) have been used to derive phenomenological models able to predict changes in aggregation rate upon mutation, as well as absolute rates and hot spots. Applications of these models to entire proteomes have provided evidence that intrinsically disordered proteins are less amyloidogenic than globular proteins. In the second type of approach, amyloidogenic polypeptides have been decomposed into overlapping segments, and atomistic simulations of three or more copies of each segment have been performed to obtain insights into aggregation propensity and structural details of the ordered aggregates (e.g. turn regions).

The reliable identification of beta-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. Here, a model based on physicochemical properties and computational design of beta-aggregating peptide sequences is shown to be able to predict the aggregation rate over a large set of natural polypeptide sequences. Furthermore, the model identifies aggregation-prone fragments within proteins and predicts the parallel or anti-parallel beta-sheet organization in fibrils. The model recognizes different beta-aggregating segments in mammalian and nonmammalian prion proteins, providing insights into the species barrier for the transmission of the prion disease.

The conformational stability of beta-lactoglobulin increases in D2O over that in H2O. This is concluded from an increase in peak temperature by about 3 degrees C of differential scanning calorimetry (DSC) thermograms and from a decrease in overall aggregation rate. However, effects of pH and salt concentration on the heat-induced aggregation (reaction kinetics, DSC thermograms and aggregate growth) are similar in H2O and D2O. This indicates that the mechanism of heat-induced aggregation of beta-lactoglobulin is not significantly affected by replacement of H2O with D2O.

Prion proteins are known to misfold into a range of different aggregated forms, showing different phenotypic and pathological states. Understanding strain specificities is an important problem in the field of prion disease. Little is known about which PrPSc structural properties and molecular mechanisms determine prion replication, disease progression and strain phenotype. The aim of this work is to investigate, through a mathematical model, how the structural stability of different aggregated forms can influence the kinetics of prion replication. The model-based results suggest that prion strains with different conformational stability undergoing in vivo replication are characterizable in primis by means of different rates of breakage. A further role seems to be played by the aggregation rate (i.e. the rate at which a prion fibril grows). The kinetic variability introduced in the model by these two parameters allows us to reproduce the different characteristic features of the various strains (e.g., fibrils' mean length) and is coherent with all experimental observations concerning strain-specific behavior., Author Summary Prion diseases are caused by the accumulation of a cellular prion protein with an altered conformation, which acts as a catalyst for the further recruitment and the modification of the normal form of the protein. Protein polymerization appears to have a central role in the progression of the disease, an aspect shared with several other neurodegenerative diseases. The aim of this work is to investigate at the kinetic level the “prion strain phenomenon”, i.e., the ability of prion proteins to misfold into a range of different aggregated forms exhibiting different replication and propagation properties. The dynamics of prion replication is investigated with the help of a mathematical model. We relate a measurement accessible in vitro (prion structural stability) to a mathematical description of the fibrils' kinetics in vivo. The analysis of the model suggests that the replication kinetics of the different prion strains is characterizable by means of two parameters, representing the rates of breakage and aggregation. This result is coherent with various experimental findings concerning strain-specific behavior, such as, for example, the observation of the fibril mean length of the various strains.

BIOCHEMISTRY In fully folded proteins, hydrophobic amino acids are hidden in the protein core. But when a protein is unfolded or partially folded, these residues render it prone to aggregation. Chiti et al. now provide much-needed insights into the molecular basis of aggregate formation. They introduced point mutations in the protein acylphosphatase (AcP) and determined the relative aggregation rates of the mutants. Mutations in two particular regions of the protein, which possess high hydrophobicity and propensity to form β-sheet structure, led to increased rates of aggregate formation. Mutational “hot spots” in protein misfolding diseases may thus result from changes in the aggregation behavior. The regions determining the aggregation rate are distinct from those that determine protein folding. Only a small number of residues is involved in setting the scene for aggregation, raising the hope that aggregation is governed by relatively simple principles. — JU Nature Struct. Biol. 10.1038/nsb752.

The aggregation kinetics of African green monkey kidney cells CV1 and of the SV40 transformed derivative COS1 cells that had been incubated at 37 degrees C or 43.5 degrees C was studied using the shaking flask system. COS1 cells show a three fold decrease in aggregation rate compared to CV1 cells when both cell types were incubated and aggregated at 37 degrees C. When these cell types were incubated at 43.5 degrees C for 5 hours, then aggregated at 37 degrees C showed a faster aggregation kinetics than before. Their aggregation at 43.5 degrees C with prior incubation at 37 degrees C or 43.5 degrees C reached the aggregation kinetics of 43.5 degrees C incubated cells aggregated at 37 degrees C. The addition of serum in the aggregation medium did not influence extensively the aggregation rates of both cell types.

Three conditions which influence both the aggregation rate and the final size of aggregates of dissociated embryonic chick retina cells were studied to determine whether there was a strict relationship between these two variables. The length of trypsinization time, age of the embryo, and concentration of chondroitin sulfate in the medium under some conditions showed an inverse relationship between their influence on aggregation rate and the final size of the aggregates; conditions favoring more rapid aggregation reduced the size of aggregates formed by 24 hr. Embryonic liver cells also showed this inverse relationship when cultured with chondroitin sulfate. These observations and other similar observations in the literature emphasize that when interpreting the action of exogenous promoters or inhibitors of cell aggregation, attention should be given to the possibility that there might not be a direct relationship between initial rate and final size.