A computational model of chemotaxis-based cell aggregation

We present a computational model that successfully captures the cell behaviors that play important roles in 2-D cell aggregation. A virtual cell in our model is designed as an independent, discrete unit with a set of parameters and actions. Each cell is defined by its location, size, rates of chemoattractant emission and response, age, life cycle stage, proliferation rate and number of attached cells. All cells are capable of emitting and sensing a chemoattractant chemical, moving, attaching to other cells, dividing, aging and dying. We validated and fine-tuned our in silico model by comparing simulated 24-h aggregation experiments with data derived from in vitro experiments using PC12 pheochromocytoma cells. Quantitative comparisons of the aggregate size distributions from the two experiments are produced using the Earth Mover's Distance (EMD) metric. We compared the two size distributions produced after 24 h of in vitro cell aggregation and the corresponding computer simulated process. Iteratively modifying the model's parameter values and measuring the difference between the in silico and in vitro results allow us to determine the optimal values that produce simulated aggregation outcomes closely matched to the PC12 experiments. Simulation results demonstrate the ability of the model to recreate large-scale aggregation behaviors seen in live cell experiments.     

Stereoselective synthesis of β-arabino glycosyl sulfones as potential inhibitors of mycobacterial cell wall biosynthesis

A series of β-arabino glycosyl sulfones with varying alkyl chain lengths were synthesised in a stereoselective fashion as putative mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Biological testing against Mycobacterium bovis BCG revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which was maximal for a C-12 chain.     

Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.     

Identification of cell culture conditions to control protein aggregation of IgG fusion proteins expressed in Chinese hamster ovary cells

The presence of aggregated forms of proteins can be problematic for therapeutics due to the potential for immunogenic and pharmacokinetic issues. Although downstream processing can remove the aggregated forms, inhibiting aggregate formation upstream during the cell culture stage could reduce the burden on downstream processing and potentially improve process yields. This study first examined the effects of environmental factors (temperature, pH, and dissolved oxygen) and medium components (bivalent copper ion, cysteine, and cystine) on the aggregation of two different recombinant fusion proteins expressed by Chinese hamster ovary (CHO) cells. Any strategy to reduce protein aggregation upstream during cell culture must also consider potential effects on critical upstream parameters such as cell growth, harvest titer, and protein sialylation levels. Manipulating the culture temperature shift and cystine concentration in the medium were both identified as effective and practical strategies for reducing protein aggregation in both CHO-cell expression systems. Furthermore, a combination of both strategies was more effective in reducing protein aggregation levels compared to either approach individually; and without any negative effects on harvest titer and protein sialylation. This study demonstrates a practical methodology for decreasing protein aggregation during upstream processing and emphasizes the importance of process understanding to ensure production of recombinant glycoprotein therapeutics with consistent product quality.     

Chemical probes for tagging mycobacterial lipids

Mycobacteria, which cause tuberculosis and related diseases, possess a diverse set of complex envelope lipids that provide remarkable tolerance to antibiotics and are major virulence factors that drive pathogenesis. Recently, metabolic labeling and bio-orthogonal chemistry have been harnessed to develop chemical probes for tagging specific lipids in live mycobacteria, enabling a range of new basic and translational research avenues. A toolbox of probes has been developed for labeling mycolic acids and their derivatives, including trehalose-, arabinogalactan-, and protein-linked mycolates, as well as newer probes for labeling phthiocerol dimycocerosates (PDIMs) and potentially other envelope lipids. These lipid-centric tools have yielded fresh insights into mycobacterial growth and host interactions, provided new avenues for drug target discovery and characterization, and inspired innovative diagnostic and therapeutic strategies.     

Identification of mycobacterial HSP70 reactive human T cell clones discriminating between M. tuberculosis and M. leprae

M. tuberculosis reactive CD4⁺ T cell clones were established from a BCG vaccinated donor and tested for proliferative responses against complex mycobacterial antigens like M. tuberculosis , M. leprae , and PPD, as well as the recombinant M. tuberculosis HSP70 and HSP65 antigens from both M. tuberculosis and M. leprae . This screening permitted the identification of T cell clones specifically recognizing the mycobacterial HSP70 or HSP65 antigen. All HSP65 reactive T cell clones were cross-reactive for M. tuberculosis and M. leprae , whereas three HSP70 reactive T cell clones only recognized M. tuberculosis . In addition, HLA typing and blocking experiments with anti-HLA antibodies revealed that antigen presentation to all M. tuberculosis reactive T cell clones was restricted by HLA-DR3 molecules. We have thereby demonstrated the presence of human T cell specificities directed against the mycobacterial HSP70 antigen that are able to discriminate between M. tuberculosis and M. leprae .     

Identification of a novel galactosyl transferase involved in biosynthesis of the mycobacterial cell wall

The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the "possible arabinogalactan biosynthetic gene cluster" of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl-N-acetylglucosaminyl-rhamnose (C(50)-P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[(14)C]galactopyranose as the immediate precursor of UDP-[(14)C]galactofuranose, the ultimate source of all of the galactofuranose (Galf) units of galactan. Obvious increased and selective synthesis of C(50)-P-P-GlcNAc-Rha-Galf-Galf, the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C(50)-P-P-GlcNAc-Rha-Galf, followed by immediate conversion to C(50)-P-P-GlcNAc-Rha-Galf-Galf. Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.     

Electron microscopical characterization of sponge aggregation factors

Aggregation factors, purified from 14 sponges which belong to the classes Tetraxonida and Cornacuspongia, were visualized electron microscopically. Two types of basic structural forms were detected; first, circular structures from the species Ancorina cerebrum, Mycale massa, Hemimycale columella, Crella rosea, Clathria coralloides, Axinella cannabina, Pellina semitubulosa, Ircinia muscarum, Hippospongia communis, Verongia aerophoba and second, rod-like structures from Tethya lyncurium, Tedania anhelans, Hymeniacidon sanguinea, Dysidea tupha. In most of the cases the structures carry side chains.     

Teratocarcinoma cell adhesion: Identification of a cell-surface protein involved in calcium-dependent cell aggregation

Teratocarcinoma cells have a Ca²⁺-dependent cell-cell adhesion site (t-CDS) that is unique in being inactivated with trypsin in the absence of Ca²⁺ but not in the presence of Ca²⁺. Fab fragments of antibodies raised against teratocarcinoma F9 cells dissociated by treatment with trypsin and calcium (anti-TC-F9) inhibit the aggregation of teratocarcinoma cells mediated by t-CDS. This inhibitory effect of Fab is removed when anti-TC-F9 is absorbed with F9 cells treated with trypsin and calcium (TC-F9), but not when it is absorbed with F9 cells treated with trypsin and EGTA (TE-F9). Comparisons of cell-surface antigens reactive to anti-TC-F9 in TC-F9 cells with those in TE-F9 cells reveal that only one component, with an approximate molecular weight of 140,000 (p140), is detected specifically on the surface of TC-F9 cells. When TC-F9 cells are retrypsinized in the absence of Ca²⁺, a substance with an approximate molecular weight of 34,000 (p34) is released that can neutralize the aggregation-inhibitory effect of the Fab. This p34 interferes with the immunoprecipitation of p140 with anti-TC-F9, suggesting that p34 is a tryptic fragment of p140. Anti-TC-F9 Fab causes the dissociation of the monolayers of teratocarcinoma cells. This effect is removed by absorption of the Fab with p34 as well as with TC-F9 cells, but not with TE-F9 cells. These results suggest that p140 is essential for the function of t-CDS, and that this is an actual cell-adhesion molecule active in the establishment of monolayers of teratocarcinoma cells.     

Identification of a cell surface glycoprotein involved in cell aggregation in D. discoideum.

Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoper-oxidase-catalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gp150 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gp150 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gp150 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fa'b derived from anti-gp150, when added to aggregation-competent cells, blocks the cells' ability to reaggregate. Fab's derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gp150 serves an intimate role in cell adhesion.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.     

Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes

Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.     

Correlation between cell aggregation and antibody production in IgE-producing plasma cells

Allergic conditions result in the increase of immunoglobulin (Ig)E-producing plasma cells (IgE-PCs); however, it is unclear how IgE production is qualitatively controlled. In this study, we found that IgE-PCs in spleen of immunized mice formed homotypic cell aggregates. By employing IgE-producing hybridomas (IgE-hybridomas) as a model of IgE-PCs, we showed that these cells formed aggregates in the presence of specific antigens (Ags). The formation of the Ag-induced cell aggregation involved secreted IgE and Fcγ receptor (FcγR)II/FcγRIII, but not FcεRs. Ag-induced cell aggregation plus lipopolysaccharide signaling resulted in an enhancement of IgE production in aggregated IgE-hybridomas. Furthermore, the administration of anti-FcγRII/FcγRIII antagonistic monoclonal antibody to immunized mice tended to reduce the splenic IgE-PC aggregation as well as the serum IgE levels. Taken together, our results suggested that Ag-IgE complexes induced IgE-PCs aggregation via FcγRII/FcγRIII, leading to the enhancement of IgE production. These findings suggest the presence of a novel mechanism for regulation of IgE production.     

A novel method for the isolation of mycobacterial DNA

Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to −70 °C, then incubated at 65 °C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 × 10⁹) or more cells) including Mycobacterium neoaurum M. fortuitum M. phlei and M. smegmatis . Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis .     

Polyhistidine tract expansions in HOXA1 result in intranuclear aggregation and increased cell death

HOXA1 gene is part of a cluster of homeotic selector genes that regulates the anteroposterior patterning of mammals during embryonic development. HOXA1 encodes two alternatively spliced mRNAs with two isoforms, A and B, the former contains the homeodomain and expressed in early embryonic development. HOXA1 contains a string of 10 histidine repeats. However, individuals heterozygous for 7, 9, 11, and 12 histidine repeat variants were present among the Japanese population, notably in some autism cases. To determine the biological implications of the different polyhistidine repeat lengths, we expressed these variants in COS-7 and a human neuroblastoma cell line (SK-N-SH). Expression of expanded variants of HOXA1 isoform A, containing 11 and 12 polyhistidine, resulted in early and great degree of protein aggregation in the nucleus. This aggregation resulted in accelerated cell death in cells expressing 11 and 12 expanded variants compared to those transfected with 7 and 10 polyhistidine variants. Furthermore, we showed that these aggregates were ubiquitinated and were inhibited by a histidine-modifying compound, DEPC. These data suggest that HOXA1 protein with polyhistidine tract expansions misfold, aggregate, and have a toxic effect on cell.     

The influence of light and different ATP concentrations on cell aggregation in cyclic AMP sensitive and insensitive species of the cellular slime molds

The influence of light and different concentrations of ATP on cell aggregation in cyclic AMP sensitive (Dictyostelium mucoroides, D. purpureum) and cyclic AMP insensitive species (Polysphondylium violaceum, P. pallidum, D. lacteum) of the cellular slime molds was observed in small and in large amoebal populations.Both light and ATP (optimal concentration:10^-5M) accelerated cell aggregation and increased the number of aggregating centers in large populations. For cyclic AMP sensitive species the effect of ATP in large populations was more pronounced than for the species that do not react to cyclic AMP.A possible explanation for the similar effect of light and ATP has been discussed.     

The mechanism of the dextran-induced red blood cell aggregation

In order to clarify the mechanism of dextran-induced aggregation, the effect of the ionic strength (I) on the minimal shear stress (tau(c)) required to rupture RBC doublets was studied for suspensions with the external media containing 76 and 298 kDa dextrans. At low and high ionic strengths, tau(c) increases with increasing I, whereas at intermediate I values, tau(c) versus I dependencies reveal a plateau step. The non-monotonous shape of these curves disagrees with the depletion model of RBC aggregation and is consistent with the predictions of the bridging mechanism. Literature reports point out that elastic behavior of dextran molecules in low and high I regions is fairly typical of Hookean springs and hence predict an increase in tau(c) with increasing I. A plateau step is accounted for by the enthalpic component of the dextran elasticity due to the shear-induced chair-boat transition of the dextran's glucopyranose rings. A longer plateau step for suspensions with a higher molecular weight dextran is explained by a larger contribution of the enthalpic component to the dextran elasticity. Thus, the results reported in this study provide evidence that RBC aggregation is caused by the formation of dextran bridges between the cells.     

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation

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. Proteins 2010. © 2010 Wiley‐Liss, Inc.