Beginning Of Aggregation Research Articles

Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several μmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.

Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids.

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids.

Aggregation of freshly precipitated calcium oxalate crystals in urine of calcium stone patients and controls

Aggregation (AGN) of freshly precipitated calcium oxalate crystals was photometrically studied in urine of 30 calcium stone patients and 30 controls, in solutions containing urinary macromolecules (UMS) and in an inhibitor free control solution (CS). Crystals were produced by oxalate titration and crystallization was monitored measuring optical density (OD). Tests were repeated adding hydroxyapatite (HAP) to urine and UMS and adding citrate and pyrophosphate (PPi) to UMS of the controls. AGN was recognized as a rapid OD decrease being at least three times faster than sedimentation of single crystals (p<0.001) and used to calculate an extent of AGN (EA%). The time between the end of titration and the beginning of AGN was determined as suspension stability (SS). The main effect of urinary inhibitors was retardation of AGN without changing EA, SS being higher in urine than UMS (p<0.001) and in UMS than CS (p<0.001). In urine of 63% of controls but only in 33% of patients, no AGN was recorded (p<0.05). The high inhibitory activity of urine could not be reproduced in UMS even in combination with 3.5mM citrate or 0.05mM PPi. 0.05mg/mL HAP reduced SS in all urine samples to low values and increased the rate of rapid OD decrease, being a measure for the size of aggregates. Retarding AGN of crystals during their passage through the kidney seems to be an important mechanism to prevent stone formation during crystalluria. The promotion of AGN by HAP reveals a new role of Randall's plaques in nephrolithiasis.

Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The double W/F replacement renders apomyoglobin highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions. In this work we analyze the early stage of W7FW14F apomyoglobin aggregation following the time dependence of the process by far-UV CD, Fourier-transform infrared (FTIR) spectroscopy, and heme-binding properties. The results show that the aggregation of W7FW14F apomyoglobin starts from a native-like globin state able to bind the prosthetic group with spectroscopic properties similar to those observed for wild-type apoprotein. Nevertheless, it rapidly aggregates, forming amyloid fibrils. However, when the prosthetic group is added before the beginning of aggregation, amyloid fibrillization is inhibited, although the aggregation process is not prevented. Moreover, the apomyoglobin aggregates formed in these conditions are not cytotoxic differently from what is observed for all amyloidogenic proteins. These results open new insights into the relationship between the structure adopted by the protein into the aggregates and their ability to trigger the impairment of cell viability.

Solution behaviour and zinc complexation of di- and tripeptides with two cysteine units

The dipeptides H-CysCys-OH, H-CysCys-OEt, Ac-CysCys-OH, Ac-CysCys-OEt, Ac-lCys-dCys-OEt and the tripeptides Ac-CysCysGly-OEt, Bz-CysGlyCys-OEt and Bz-CysPheCys-OEt were prepared analytically pure in gram quantities. Potentiometric titrations in the presence of zinc nitrate have shown that most of them form ZnL and ZnL2 complexes in solution, representing ZnS2 and ZnS4 coordinations, which also occur as structural motifs in zinc enzymes. The fully protected CysCys donors Ac-CysCys-OEt and Ac-CysCysGly-OEt also form Zn2L4 and Zn3L4 species in solution, indicating the beginning of aggregation. Accordingly the isolated complexes with a 1:1 Zn:L composition are insoluble polymers, and monomers exist only in dilute solutions or in the presence of the N,N coligand neocuproin. The stability of the purely thiolate-coordinated complexes increases for L=lCys-lCys<lCys-dCys<lCys-X-lCys, indicating significant conformational effects in the chelating CysCys ligands.

Platelet activation and its role in thrombin generation in platelet-induced thrombin generation time

Platelet-induced thrombin generation time (PITT) is a newly developed global coagulation assay in which a small amount of partially anticoagulated platelet-rich plasma (PRP) is rotated in a disc-shaped cuvette within the light beam of a photometer. The time intervals from onset of rotation until aggregation and coagulation of the sample are registered. The aim of our study was to compare platelet activation with generation of thrombin during rotation of PRP in PITT system. Aliquots of PRP were taken before, 1, 3, and 8 min after the onset of rotation as well as at the beginning of aggregation and shortly before coagulation. Thrombin activity was measured with chromogenic substrate S-2238. We have also measured the level of generated prothrombin activation fragment 1+2 (F1+2), which reflects the concentration of liberated thrombin. Platelet activation was assayed by means of platelet factor 4 (PF4) and β-thromboglobulin (β-TG) concentration and registration of the aggregation. The concentrations of the F1+2, PF4, β-TG increased very slowly from the beginning of the test until aggregation occurred. From the start of aggregation, the levels of F1+2 rose rapidly. In contrast to the F1+2 measurements, thrombin activity has not been detected from onset of rotation until the end of the test. Only trace thrombin activity was detectable just after the plasma sample had been clotted in the cuvette. Our results demonstrate that there exists a close relationship between platelet activation and thrombin generation. Viable platelets, which adhered to the cuvette walls, form an active template on which thrombin can be generated from prothrombin.

Size and number density of precrystalline aggregates in lysozyme crystallization process

Using dynamic light scattering, we investigated supersaturated aqueous solutions of hen egg white lysozyme. We could observe the formation of aggregates only in solutions, from which crystals grew within a few days. The aggregates were grouped into smaller “units” and larger “clusters.” The units consisted of a few molecules, whereas the clusters grew from about 100 nm to 1 μm. At the beginning of aggregation, the number density of the units decreased, while that of the clusters increased. At this stage, unit-cluster aggregation proceeded. At the next stage, the number density of the units became constant, while that of the clusters began to decrease, which means that the units stopped aggregating and cluster-cluster aggregation started. The aggregation mechanism for the clusters fit well with the diffusion limited cluster aggregation model, but this model alone could not explain that the aggregates separated into two groups, corresponding to units and clusters, and that the units stopped aggregating during the aggregation process. We find that the observed aggregation process has several similarities to the liquid-liquid phase separation process, which occurs metastably in protein solution. Furthermore, using both models for diffusion limited aggregation and the liquid-liquid phase separation together, we could naturally explain the process of the cluster formation.

Antiplatelet effects of nitroglycerin in healthy subjects and in patients with stable angina pectoris.

The effects of nitroglycerin (NTG) on platelet aggregation are controversial. Most in vitro investigations suggest that NTG suppresses platelet aggregation only at suprapharmacologic concentrations. We investigated various aspects of the antiaggregating effects of NTG in both normal individuals and in patients with stable angina pectoris not treated with nitrates. Platelets from patients exhibited hyperresponsiveness to ADP as an inductor of aggregation. Sublingual administration to patients of NTG (300 micrograms) decreased platelet aggregability; ADP concentrations inducing 50% aggregation were 3.3 +/- 0.3 microM after NTG versus 2.1 +/- 0.1 microM before NTG (p < 0.01). Consistent with previous findings, NTG was a weak inhibitor of platelet aggregation in vitro when added before induction of aggregation. When added after the beginning of aggregation, however, NTG induced both inhibition of developing aggregation and marked disaggregation at concentrations > or = 10(-8) M NTG; concentration associated with 50% reversal of aggregation was 1.4 +/- 0.3 x 10(-6) M. Therefore, antiplatelet effects of NTG in vitro are demonstrable in low, clinically achievable concentrations; previously reported effects of NTG have been underestimated owing to suboptimum experimental conditions. Platelets from patients with angina pectoris were 100-fold less responsive to the cyclic GMP-increasing and disaggregating effects of NTG in vitro, which, together with increased aggregability, could imply reduced platelet sensitivity to endogenous sources of nitric oxide (NO) in vivo. The observed antiplatelet effects of NTG raise the question of its potential utility to reduce the risk of thrombotic complications in patients with ischemic heart disease.

Reversal of human platelet aggregation by low concentrations of nitroglycerin in vitro in normal subjects

The potential reversal of platelet aggregation in vitro by nitroglycerin in low concentrations was explored using both optical aggregometry and electron microscopy. Venous blood was collected from a cohort of normal volunteers (20 men and 10 women) aged 21 to 65 years. Aggregation in platelet-rich plasma was induced by adenosine diphosphate in concentrations just sufficient to maintain a steady state of aggregation, without a spontaneous disaggregation phase (3.5 to 5 μM). Administration of nitroglycerin after the induction of aggregation caused both inhibition of the primary wave of developing aggregation and marked disaggregation. This combined effect was maximal when nitroglycerin was added at 0.5 minute after the beginning of aggregation. The observed reversal of platelet aggregation by nitroglycerin was concentration-dependent. Significant effects occurred with nitroglycerin concentrations ≥10 −8 M. Concentration associated with 50% reversal of aggregation was 1.52 ± 0.24 (SEM) × 10 −6 M. Electron microscopy revealed that 10 −6 M nitroglycerin induced a significant reduction in both platelet clumping and morphologic changes associated with aggregation. The results of the current study suggest a beneficial antiplatelet effect of nitroglycerin in restoring homeostasis in the face of incipient platelet aggregation. The clinical use of nitroglycerin in patients with acute ischemic syndromes may rest on this action.

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells.

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.

Desmosome development in an in vitro model.

A model has been devised to study the in vitro formation of desmonsomes. The model is based on the differential labeling of two subpopulations of a desmosome-forming human cancer line (C4I). The labeled subpopulations are dispersed, preincubated separately on a shaking water bath for 24 h to allow the internalization of desmosome fragments and the repair of the cell surface, and then mixed, and allowed to aggregate. Aliquots of the mixed suspension are fixed at various intervals. The time between mixing and fixation represents the maximum age of any junction between dissimilarly labeled cells. The beginnings of desmosome formation were observed within a few minutes after the beginning of aggregation. Close apposition of cell membranes was seen immediately after mixing, followed within 15 min by the appearance of a submembrane density in one or both of the interacting cells. Intracytoplasmic filament formation takes place at between 15 and 30 min. Desmosome formation is complete by 90 min. The process is accompanied by a progressive widening of the extracellular space and the desification and organization of the extracellular material and the submembrane plaques.

Changes in morphogenesis and developmental enzyme levels in Dictyostelium discoideum after gamma irradiation.

Changes in the levels of specific activity of two enzymes believed to be involved in developmental regulation were observed after irradiating differentiating cells of Dictyostelium discoideum. Stimulation of the levels of specific activity of alkaline phosphatase occured after irradiation at the beginning of development and at the end of the aggregation period, but not after irradiation at the beginning of aggregation. A stimulation in UDP-glucose pyrophosphorylase specific activity was also observed, but to a lesser extent and only after irradiation at the end of aggregation. Dose-dependent delays in the appearance of peaks of specific activity were noted. The delay per unit dose was less when irradiation took place at the beginning of development as opposed to the beginning or end of the aggregation period. Radiation-induced delays in progression through visible developmental stages were almost identical to delays in enzyme appearance. Other radiation effects on morphogenesis included the induction of a migratory slug phase.

Independence of propagation ability and developmental processes in irradiated cellular slime-moulds.

SummaryWhen assayed for reproductive ability, the cellular slime mould Dictyostelium discoideum NC-4 is resistant to 60Co gamma-rays (D10 = 300 krads) whereas a derived mutant, γs-13, is much more sensitive (D10 = 4 krads). If ability to proceed through development to form normal-appearing spores (a process requiring neither DNA synthesis nor cell division) is assayed after irradiation of amoeboid cells, the γs-13 strain is more sensitive than NC-4, but the differential sensitivity is only about 4× at the 20 per cent level (270 krads for NC-4). When the reproductive ability of these spores is determined, those of γs-13 are mostly inviable at doses above 20 krads, while those of NC-4 are largely viable up to 200 krads. Quantitatively, the proportion of viable spores among those produced closely follows the proportion of viable cells in the irradiated population used to initiate development. The greatest diminution of spore production occurs for irradiation done at the beginning of aggregation; however, the...

Acrasin, acrasinase, and the sensitivity to acrasin in Dictyostelium discoideum

The purpose of this study was to examine the role in normal development of an acrasin (cyclic-AMP) and of an acrasinase (phophodiesterase) and the sensitivity to acrasin in the cellular slime mold Dictyostelium discoideum. Phosphodiesterase is secreted throughout the vegetative, aggregation, and migration stages. It is unclear whether or not cyclic-AMP is produced by vegetative amoebae, but there is a 100-fold increase between the beginning of aggregation and late aggregation, falling to a lower level during migration. The sensitivity to cyclic-AMP also increases 100-fold during aggregation, therefore the chemotactic system is at least 10 4 times more effective during aggregation than during the vegetative stage. Artificial patterns of amoebae may be produced by making an uneven distribution of cyclic-AMP in the agar with an even distribution of amoebae, or the reverse. The results of these experiments show: (1) the importance of phosphodiesterase in making steep gradients (and providing the basis of various acrasin assays), (2) the role of steep gradients of cyclic-AMP in producing cell adhesiveness, and possibly inhibiting centers; (3) the center-inducing capacity of a high concentration of externally applied cyclic-AMP. It is postulated that prior to normal aggregation, the distribution of chemicals affecting aggregation is homogeneous, and that this equilibrium is upset by sudden excessive sensitivity to cyclic-AMP and its production by certain cells, and the prevention of neighboring cells from having such an increase. The result, by chemotaxis, is an uneven distribution of cells which ultimately reach a new steady state in which they are massed together in groups spaced nonrandomly over the surface of the substratum.

Chemotaxis in the cellular slime molds: I. The effect of temperature

The effect of temperature on chemotaxis in the cellular slime mold Dictyostelium discoideum has been studied by incubating small populations of washed myxamoebae at different temperatures. Droplets containing a cell suspension of known density were deposited on a hydrophobic agar surface. The myxamoebae normally stayed within the boundaries of the implanted droplets, but they moved outside the margins of such droplets when they were attracted by acrasin secreted by neighboring populations. Sensitive cells in the responding populations were mainly attracted between the beginning of aggregation and its completion in the attracting populations. Secretion of acrasin before the formation of compact centers of aggregation was demonstrated at low temperatures and with time lapse photography. A decrease in temperature was correlated with: (1) an increase in the distance of attraction; (2) an increase in the time interval between the beginning of aggregation and its completion; and (3) an increase in the lapse of time between the deposition of the myxamoebae and the beginning of aggregation. The chronological age of the attracting cells did not seem to affect the chemotactic response. The increased attraction at lower temperatures was correlated with a longer period between early aggregation and its completion. Only the temperature during aggregation had an effect on the distance over which cells could be oriented; any temperature change applied during the preaggregative stages was of no consequence to the attraction at later stages.

AGGREGATION TERRITORIES IN THE CELLULAR SLIME MOLDS

The size of the area or territory which encompasses the amoebae that enter into an aggregate of a cellular slime mold is related to the process of initiation of aggregation as well as the size of the sorocarp. Each territory represents one aggregation and therefore one initiation event, and the size of any one fruiting body, or sorocarp, is primarily determined by the number of amoebae that enter an aggregation center. It is true that some of the cells may divide after the beginning of aggregation (Wilson, 1952; Bonner, 1960) but since all intake of food ceases some time before aggregation, there is no increase in bulk during the morphogenetic phases of the life-cycle, that is, while the aggregated cell mass progressively becomes transformed into a fruiting body. It will be shown that regardless of the density of the amoebae, the territory size, under controlled environmental conditions, remains constant for any one species. This means that sorocarp size is largely controlled by cell density and that the problem of initiation is identical with the problem of the establishment of these rigid aggregation territories. A simple method was used for the control of the amoeba density within a culture by controlling, in turn, the amount of bacterial food supply. Two per cent Bacto agar containing 6.2 tug./ml. of dihydrostreptomycin sulphate (Lilly) was poured into plastic Petri dishes which were marked on the bottom surface with squares of .102 cm2. A few loopfuls of Escherichia coli taken from a stock culture (grown on 1% dextrose, 1%o peptone, 2% Bacto agar) were placed in 2 to 7 ml. of sterile distilled water. The density of the suspension was then determined in a Bausch & Lomb Spectrometer 20 at 550 m/l. Appropriate dilutions were made to achieve a particular density and .09 ml. of this final suspension was evenly spread (with the help of an electric turntable and a sterile, bent glass rod) on the surface of one of the streptomycin agar Petri plates. In this way it was possible to obtain a range in food supply on the plates from approximately 500,000 bacterial cells/mm.2 (= an optical density of 6.0) to 5000 bacterial cells/mm.2 (= an optical density of 0.1). The spores of the slime mold were inoculated in three marked spots on each plate with a very fine, glass needle

Zellfunktionen und Zellfunktionswechsel in der Entwicklung vonDictyostelium discoideum. IV

The morphogenetic capacities of cells inDictyostelium discoideum, as well as of groups (agglutinates) of these cells are changed in a constant time course after food consumption has been stopped.Two questions were posed: 1. When is the capacity of aggregation at its maximum level in a culture of single suspended cells. 2. At what time do agglutinates answer an external stimulus immediately by establishing polarity.The maximum of aggregation capacity lies 8-10 hours after complete food exhaustion. This functional stage is cytologically characterized by accumulation of metachromatic granules in the cells [seeBONNER et al. (1955)]. - The polarization capacity of agglutinates depends primarily on the functional age of the cells (this means that the age of the cells is related to the time of complete food exhaustion), and secondly it depends on the age of the agglutinates. In the most favorable case the maximum response is reached 12 hours after complete food exhaustion.A difference of about 2 hours lies between the beginning of aggregation and polarization. This difference in time could explain the centerless aggregation patterns of a well synchronized cell population as well as the appearance of primary aggregation centers in non-synchronized cultures.