In vitro reaggregation of dissociated mouse cerebellar cells : I. Demonstration of different aggregation mechanisms

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca²⁺ concentrations, T and MT cells aggregated better at high Ca²⁺ concentration. Their aggregation rates were reduced by approx. 50% at low Ca²⁺ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca²⁺ concentration, being half maximal at approx. 0.1 mM Ca²⁺.The ability of M cells to aggregate at low or high Ca²⁺ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca²⁺ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca²⁺ concentrations were reduced to 35%. Variation of the Ca²⁺ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca²⁺-independent binding mechanism, whereas T or MT cells aggregate using a Ca²⁺-dependent one which may be functionally silent in M cells.     

Characterization of the chaperone-like activity of HtrA (DegP) protein from Escherichia coli under the conditions of heat shock

The protective action of chaperone-like activity of HtrA protease against protein aggregation was studied. High levels of proteolytically inactive HtrAS210A (active center serine replaced by alanine) suppressed the temperature-sensitive phenotype of the htrA mutants. The ability of HtrAS210A to alleviate the lethality of htrA bacteria at high temperatures correlated well with the observed decrease of cellular level of large protein aggregates in cells overproducing HtrAS210A. The in vitro experiments proved that HtrA was very efficient in inhibiting the unfolded substrate (lysozyme) aggregation over a wide range of temperatures (30-45 °C). HtrA was able to bind to the denatured polypeptides and as a consequence limited their ability to form large aggregates. Our results suggest that HtrA may protect the bacterial cells from deleterious effects of heat shock not only by degrading the damaged proteins but by combination of the proteolytic and chaperoning activities.     

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (R_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (R_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.     

Effect of Bacteria on Chemotaxis in the Cellular Slime Molds

The effect of chemotactic substances, secreted by Escherichia coli, on the cellular slime molds was studied by deposition of bacteria near myxamoebae populations. Droplets of a bacterial suspension and a myxamoebae suspension were placed separately, at predetermined distances from each other, on a hydrophobic agar surface of low rigidity. Myxamoebae remained confined inside the droplets, except when they were activated by the bacterial products. The sphere of attraction increased at higher bacterial concentrations. Myxamoebae could be attracted over distances as great as 5 mm. Myxamoebae in droplets close to dense bacterial populations not only were attracted toward the bacteria but also moved out in an opposite direction from the bacteria. There was a gradual decrease of attraction at increasing distances between amoebae and bacteria. The attraction by bacteria or bacterial products was reduced at lower temperatures. Light did not affect the distance over which attraction could be observed. Myxamoebae close to their aggregation phase were most sensitive to the bacterial attractants. Bacterial attractants at high concentrations could disperse aggregates, even when they were in an advanced stage. At still higher concentrations of the bacterial products, cells stopped moving altogether. The bacterial attractants activated different species of cellular slime molds. They appeared to be present not only in E. coli but also in all other bacterial species that were tested. These results are discussed in the light of earlier observations on the attraction of cells by aggregates of myxamoebae.     

Mechanism of the action of biogenic chloramines and hypochlorite on the initial aggregation of platelets

The antiaggregant effect of two reactive oxidants—N,N-dichlorotaurine (a biogenic chloramine) and sodium hypochlorite—on the initial ADP-induced aggregation of rabbit blood platelets was studied. Platelet aggregation in reconstituted platelet-rich plasma was measured nephelometrically; an increase in the intensity of small-angle light scattering served as an index of aggregation. Addition of chloramine at relatively small concentrations (no greater than 1 mM available chlorine) directly to the reconstituted platelet-rich plasma suppressed the initial aggregation (formation of small aggregates) several times more strongly than preincubation of native plasma with chloramine. This suggests that N,N-dichlorotaurine realizes its antiaggregant effect on the platelet-rich plasma by directly interacting with cells. The effects of the inhibition of platelet aggregation in two variants of addition of high concentrations of N,N-dichlorotaurine did not differ significantly. In this case, a large amount of residual unreacted chloramine remained in the plasma, which caused the suppression of platelet aggregation during subsequent reconstitution of the platelet-rich plasma. Similar data were obtained in studying the antiaggregant effect of hypochlorite. N,N-Dichlorotaurine and hypochlorite at concentrations of 0.2–0.3 and 0.15 mM, respectively, strongly inhibited the initial aggregation of isolated platelets (approximately 2·10⁸ cells/ml) preliminarily activated for 1.5 min by addition of 0.1–0.5 μM ADP. However, the antiaggregants had a more profound suppressive effect on the aggregation of unstimulated platelets. The antiaggregant effects of N,N-dichlorotaurine and hypochlorite probably stem from the oxidative modification of the sulfur-containing groups in platelet plasma membrane.     

Lipid- and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes

The lipids in cell membranes of Acholeplasma laidlawii were enriched with different fatty acids selected to produce membranes showing molecular motion discontinuities at temperatures between 10 and 35 °C. Molecular motion in these membranes was probed by ESR after labelling with 12-nitroxide stearate, and structure in these membranes was examined by electron microscopy after freeze-etching.Freeze-etching and electron microscopy showed that under certain conditions the particles in the A. laidlawii membranes aggregated, resulting in particle-rich and particle-depleted regions in the cell membrane. Depending upon the lipid content of the membrane, this aggregation could begin at temperatures well above the ESR-determined discontinuity. Aggregation increased with decreasing temperature but was completed at or near the discontinuity. However, cell membranes grown and maintained well below their ESR-determined discontinuity did not show maximum particle aggregation until after they had been exposed to temperatures at or above the discontinuity.The results show that temperatures at or near a phase transition temperature can induce aggregation of the membrane particles. This suggests that temperature-induced changes in the lipid phase of a biological membrane can induce phase separations which affect the topography of associated proteins.     

The Formation of Persister Cells in Stationary-Phase Cultures of Escherichia Coli Is Associated with the Aggregation of Endogenous Proteins

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.     

Aggregation of 3,5-dihydroxybenzyl alcohol based dendrimers and hyperbranched polymers, and encapsulation of DR1 in such dendritic aggregates

The aggregation behavior of 3,5-dihydroxybenzyl alcohol based dendritic polymers through intermolecular hydrogen bonding between periphery situated variable number of OH groups was studied using dynamic light scattering and transmission electron microscopy. Size of the aggregates was found to be dependent on the generation number of dendrimers: generations 1-3 form aggregates above critical aggregation concentration (cac), while higher generations (4-5) aggregate even at concentrations much lower than cac. Encapsulation of the dye disperse red 1 (DR1) into dendrimer aggregates was accompanied by a blue shift in the UV-Vis absorption spectrum of DR1.     

Saliva-Induced Aggregation of Oral Streptococci

Cells of several species of oral microorganisms have been shown, in earlier studies, to be aggregated by saliva. In the present study some of the basic properties of the aggregation system are examined. The observation is made that the saliva-induced aggregates of Streptococcus sanguis and S. mitis can be dissociated to stable particles which consist of about 100 cells and have a median diameter of about 4.5 μm. It is proposed that these are subunits, or core aggregates, of the large primary aggregates. Counts of the core aggregates can be taken as a precise and accurate measure of aggregation. Experiments based on this procedure show that the aggregation of S. sanguis is maximal at 10 C and at 1 meq of Ca²⁺ ions per liter and is not affected by a change in pH between 3.9 and 8.7 or by a change in the phase of growth of the microorganisms. Core aggregates diminish in number with prolonged incubation, suggesting that the aggregating factors break down with time. Formalinized cells yield stable aggregates. However, with Formalinized cell aggregation is maximal between 20 and 30 C and proceeds in the absence of calcium ions. Evidence is presented that whole saliva contains separate aggregating factors for S. sanguis and S. mitis. The factors differ in their affinity for intact cells and for hydroxyapatite and differ in their stability to dialysis. These findings suggest that many different aggregating factors exist in saliva, each of which may be capable of interacting with cells of one or several bacterial species.     

Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Rapid Aggregation of Biofuel-Producing Algae by the Bacterium Bacillus sp. Strain RP1137

Algal biofuels represent one of the most promising means of sustainably replacing liquid fuels. However, significant challenges remain before alga-based fuels become competitive with fossil fuels. One of the largest challenges is the ability to harvest the algae in an economical and low-energy manner. In this article, we describe the isolation of a bacterial strain, Bacillus sp. strain RP1137, which can rapidly aggregate several algae that are candidates for biofuel production, including a Nannochloropsis sp. This bacterium aggregates algae in a pH-dependent and reversible manner and retains its aggregation ability after paraformaldehyde fixation, opening the possibility for reuse of the cells. The optimal ratio of bacteria to algae is described, as is the robustness of aggregation at different salinities and temperatures. Aggregation is dependent on the presence of calcium or magnesium ions. The efficiency of aggregation of Nannochloropsis oceanica IMET1 is between 70 and 95% and is comparable to that obtained by other means of harvest; however, the rate of harvest is fast, with aggregates forming in 30 s.     

Characterization of full-length p53 aggregates and their kinetics of formation

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.     

Detrital aggregates in some Iowa lakes and reservoirs

Detrital aggregates in three eutrophic Iowa lakes and four eutrophic Iowa reservoirs were studied with light and scanning electron microscopy to determine if aggregate morphologies and concentrations were similar. Lake and reservoir aggregates were composed of organic and inorganic particles bound together in an organix matrix. Many of the inorganic particles were calcium carbonate. Obvious bacterial and fungal attachment to the aggregates was rare. Aggregate concentrations ranged from 4 to 274 million aggregates per liter. Aggregates smaller than 18 μm diameter dominated the hyperbolic size-frequency distribution of aggregates in all lakes. Reservoir and lake aggregate concentrations did not differ significantly, but mean aggregate concentrations were directly correlated to the mean chlorophyll a concentration of the lakes. These data strongly suggest that detrital aggregate concentrations are influenced by the trophic status of a lake. Journal Paper No. J-8705 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2051. Journal Paper No. J-8705 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2051.     

Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli

Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca²⁺-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 °C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to “non-classical” inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.     

Colonization Pattern of the Biocontrol Strain Pseudomonas chlororaphis MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein

Pseudomonas chlororaphis MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus Drechslera teres. In this study, strain MA 342 was tagged with the gfp gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The gfp-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen D. teres, which facilitates the action of the fungistatic compound(s) produced by this strain.     

Microhabitats choice in intertidal gastropods is species-, temperature- and habitat-specific

Understanding how behavioural adaptations can limit thermal stress for intertidal gastropods will be crucial for climate models. Some behavioural adaptations are already known to limit desiccation and thermal stresses as shell-lifting, shell-standing, towering, aggregation of conspecifics or habitat selection. Here we used the IRT (i.e. infrared thermography) to investigate the thermal heterogeneity of a rocky platform, with four different macrohabitats (i.e. bare rock, rock with barnacles, mussels and mussels incrusted by barnacles) over four thermally contrasted months. We investigated the body temperature of Littorina littorea and Patella vulgata found on this platform and the temperature of their microhabitat (i.e. the substratum within one body length around of each individual). We also considered the aggregation behaviour of each species and assessed the percentage of thermal microhabitat choice (i.e choice for a microhabitat with a temperature different than the surrounding substrate). We did not find any aggregation of L. littorea on the rocky platform during the four studied months. In contrast, P. vulgata were found in aggregates in all the studied periods and within each habitat, but there was no difference in body temperature between aggregated and solitary individuals. These two gastropods species were preferentially found on rock covered by barnacles in the four studied months. The presence of a thermal microhabitat choice in L. littorea and P. vulgata is habitat-dependent and also season-dependent. In June, July and November the choice was for a microhabitat with temperatures lower than the temperatures of the surrounding substrate whereas in December, individuals choose microhabitats with higher temperatures than the temperatures of their substratum. Taken together, these results suggest that gastropods species are able to explore their environment to find sustainable thermal macrohabitats and microhabitats and adapt this behaviour in function of the conditions of temperatures.     

Cell Aggregation in Cultures of <Emphasis Type="Italic">Micrococcus luteus</Emphasis>, Studied by Dynamic Light Scattering

Cell aggregation was studied using the method of dynamic light scattering in the course of growth of Micrococcus luteus cultures in a liquid medium. The method detects particles ranging in size from 0.5 to 1000 μm in samples containing no more than 10⁵ cells/ml. When grown in liquid media, M. luteus forms aggregates; during the lag phase, 80% of the cells are found in aggregates of 10–1000 μm, only minor amounts being represented by single cells. With the onset of exponential growth, the aggregates were decomposed and single cells became prevalent in the culture liquid. This observation confirms that the aggregation of the cells during the lag phase is prerequisite to the initiation of bacterial growth. The method may be used in biotechnology for monitoring the state of bacterial cultures. __________ Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 6, 2005, pp. 647–651. Original Russian Text Copyright ? 2005 by Voloshin, Kaprelyants.     

Amyloid Fibril-Like Structure Underlies the Aggregate Structure across the pH Range for β-Lactoglobulin

The protein β-lactoglobulin aggregates into two apparently distinct forms under different conditions: amyloid fibrils at pH values away from the isoelectric point, and spherical aggregates near it. To understand this apparent dichotomy in behavior, we studied the internal structure of the spherical aggregates by employing a range of biophysical approaches. Fourier transform infrared studies show the aggregates have a high β-sheet content that is distinct from the native β-lactoglobulin structure. The structures also bind the amyloidophilic dye thioflavin-T, and wide-angle x-ray diffraction showed reflections corresponding to spacings typically observed for amyloid fibrils composed of β-lactoglobulin. Combined with small-angle x-ray scattering data indicating the presence of one-dimensional linear aggregates at the molecular level, these findings indicate strongly that the aggregates contain amyloid-like substructure. Incubation of β-lactoglobulin at pH values increasingly removed from the isoelectric point resulted in the increasing appearance of fibrillar species, rather than spherical species shown by electron microscopy. Taken together, these results suggest that amyloid-like β-sheet structures underlie protein aggregation over a much broader range of conditions than previously believed. Furthermore, the results suggest that there is a continuum of β-sheet structure of varying regularity underlying the aggregate morphology, from very regular amyloid fibrils at high charge to short stretches of amyloid-like fibrils that associate together randomly to form spherical particles at low net charge.