Complete Cell Separation Research Articles

Pectin-associated mannans and xylans play distinct roles in cell-cell adhesion in pine and poplar wood

Wood particles of pine required longer treatments of acidic chlorite and dilute alkali to achieve similar proportions of cell separation as of poplar. Chemical extraction and enzymatic digestion studies indicated that rhamnogalacturonan-I (RG-I), mannan and xylan contribute to cell-cell adhesion in pine wood. Mannan associated with lignin in an acidic chlorite fraction and with xylan in a dilute alkali fraction. RG-I and xylan were extracted in both chlorite and dilute alkali in poplar. Scanning electron microscopy and a probe diffusion study using Fluorescence Recovery After Photobleaching showed that lignin, RG-I and mannan initiated cell separation at cell corners and dilute alkali extraction of xylan was required for complete cell separation in pine. Cell-cell separation increased enzymatic saccharification within 24 h by 2-fold in poplar and 3.4-fold in pine. Our results inform a strategy to reduce particle size in woody biomass and provide insights into different molecular bases of cell-cell adhesion.

Mutanase Enzyme from Paracoccus mutanolyticus RSP02: Characterization and Application as a Biocontrol Agent.

Mutanases are enzymes that have the ability to cleave α-1,3 linkages in glucan polymer. In the present investigation, mutanase enzyme purified from the culture filtrate of Paracoccus mutanolyticus was evaluated for Streptococcal biofilm degradation and antimicrobial activity against pathogenic fungi along with enzyme kinetics, activation energies, pH and thermal stability. Biochemical and molecular characterization depicted that the enzyme showed optimum activity at pH 5.5 and at 50°C. It displayed Michaelis-Menten behaviour with a Km of 1.263 ± 0.03 (mg/ml), Vmax of 2.712 ± 0.15 U/mg protein. Thermal stability studies denoted that it required 55.46 and 135.43kJmol-1 of energy for activation and deactivation in the temperature range of 30-50°C and 50-70°C respectively. Mutanase activity was enhanced ~ 50 and 75% by Fe2+ and EDTA, respectively, while presence of Hg2+ and Mn2+ inhibit > 90% of its activity. This enzyme has a molecular mass of 138kDa and showed monomeric nature by Zymography. Scanning electron microscopy analysis of mutanase treated Streptococcal cells revealed cleavage of linkages among the cells and complete separation of cells, indicating its potential in dentistry as an anticaries agent in the prophylaxis and therapy of dental caries. In addition, antifungal activity of mutanase against Colletotrichum capsici MTCC 10147 and Cladosporium cladosporioide MTCC 7371 revealed that the enzyme has potential towards biological control of phytopathogens which could be used as an alternative bio-control agent against chemical pesticides in the future.

Flow behavior analysis of Chlorella Vulgaris microalgal biomass

The processing volume of bioengineering operations requires flow properties of algal mass for effective processing techniques. Chlorella Vulgaris microalgae cultured at 25°C in Tap media under continuous illumination was considered. It showed an exponential phase of growth up to 8 days and then a stationary phase of growth from 8 days to 15 days. The rheological properties of microalgae biomass during the growth represented power law model. Microscopic analysis showed the influence of shearing on variation of algal structure from clusters to complete cell separation. The flow properties supported the microscopy analysis showing the shear thickening property at high shear rates and shear thinning nature at low shear regime. Optimal power required for the agitation of biomass based on the variations of non-Newtonian viscosity were predicted by considering the vessel geometry.

Mechanistic insight into softening of Canadian wonder common beans (Phaseolus vulgaris) during cooking

The relative contributions of cotyledons and seed coats towards hardening of common beans (Phaseolus vulgaris) were investigated and the rate-limiting process which controls bean softening during cooking was determined. Fresh or aged whole beans and cotyledons were soaked and cooked in demineralised water or 0.1 M NaHCO3 solution, and texture evolution, microstructure changes and thermal properties were studied. Fresh and aged whole beans cooked in demineralised water had significantly different softening rate constants and so did fresh and aged cotyledons. The comparable softening rate constants of aged whole beans and cotyledons indicated an insignificant role of the seed coat in hardening during storage. All samples cooked faster in 0.1 M NaHCO3 solution. Disintegration of cooked tissues followed by microscopic examination revealed a transition from cell breakage through a phase of cell breakage and separation to complete cell separation with increased cooking time wherefore texture decayed. Therefore, progressive solubilization of pectin in the middle lamella greatly promoted texture decay. While residual birefringence even after substantial cooking time suggested some molecular order of the starch, calorimetric analyses revealed complete starch gelatinisation before complete cell separation occurred. This implies an insignificant role of starch in texture decay during cooking but its hindered uncoiling into a viscous gel after gelatinisation due to the restricting cell wall could promote its retrogradation. Therefore, we suggest that the rate-determining process in bean softening relates to cell wall/middle lamella changes influencing pectin solubilization.

Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis.In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping.Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture.In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision.This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem.For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision.In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles.

Developmental biology of Streptomyces from the perspective of 100 actinobacterial genome sequences.

To illuminate the evolution and mechanisms of actinobacterial complexity, we evaluate the distribution and origins of known Streptomyces developmental genes and the developmental significance of actinobacteria-specific genes. As an aid, we developed the Actinoblast database of reciprocal blastp best hits between the Streptomyces coelicolor genome and more than 100 other actinobacterial genomes (http://streptomyces.org.uk/actinoblast/). We suggest that the emergence of morphological complexity was underpinned by special features of early actinobacteria, such as polar growth and the coupled participation of regulatory Wbl proteins and the redox-protecting thiol mycothiol in transducing a transient nitric oxide signal generated during physiologically stressful growth transitions. It seems that some cell growth and division proteins of early actinobacteria have acquired greater importance for sporulation of complex actinobacteria than for mycelial growth, in which septa are infrequent and not associated with complete cell separation. The acquisition of extracellular proteins with structural roles, a highly regulated extracellular protease cascade, and additional regulatory genes allowed early actinobacterial stationary phase processes to be redeployed in the emergence of aerial hyphae from mycelial mats and in the formation of spore chains. These extracellular proteins may have contributed to speciation. Simpler members of morphologically diverse clades have lost some developmental genes.

MICROSTRUCTURE OF BOILED YAM (DIOSCOREA SPP.) AND ITS IMPLICATION FOR ASSESSMENT OF TEXTURAL QUALITY

ABSTRACT The effects of boiling on the microstructure of yams and their association with the textural quality of “boiled yam,” arguably the most common food product from yams, were studied using six varieties with variable cooking qualities from each of the two species, Dioscorea alata L. and D. rotundata Poir. Histological studies on raw yams showed parenchyma cells of both species to be three‐dimensional and polyhedral in shape, with starch granules loosely arranged in D. rotundata but densely packed in D. alata. Generally, there was cell separation and rounding off of cells in cooked yams of D. rotundata varieties, whereas cell separation was only partial with no rounding off in D. alata. Mealy boiled yams showed complete cell separation and the “rounding off” phenomenon, whereas the waxy ones showed partial retention of textural cell integrity. These relative changes in microstructure of yam when boiled could serve as indicators of textural quality in boiled yam.

Biomagnetic separation of Escherichia coli by use of anion-exchange beads: measurement and modeling of the kinetics of cell-bead interactions.

Analyses of food-borne pathogens are of great importance in order to minimize the risk of infection for customers. These analyses should be as fast as possible. Any detection method requires enrichment and quantitative analysis of the enriched microbes. Conventional enrichment methods, which take several days, need to be replaced by faster techniques such as biomagnetic separation (BMS). This technique involves the use of paramagnetic microspheres coated with ligands that have special affinity to the microbes that have to be detected. In the studies reported here, enrichment experiments by BMS were carried out using the non-pathogenic E. coli DSM 498 as a model strain and beads coated with a polyethylenimine (PEI) anion-exchange material. The results show that the number of cells separated, as a proportion of the total, was positively correlated with the bead concentration and the length of the period they were mixed together. In addition, a mathematical model, based on the rate of impact between two different sorts of particles, was developed to describe the proportion of separated cells as a function of incubation time and the concentration, size and density of the beads and cells. This is the first mathematical description of cell-bead interactions to be based on well-understood physicochemical principles. The model was confirmed by separation experiments in which the concentration of beads and the incubation period were varied. The developed model enables optimization of the amount of beads added and the reaction period necessary for complete cell separation and thus minimization of costs in BMS.

Role of the midbody matrix in cytokinesis: RNAi and genetic rescue analysis of the mammalian motor protein CHO1.

CHO1 is a kinesin-like motor protein essential for cytokinesis in mammalian cells. To analyze how CHO1 functions, we established RNAi and genetic rescue assays. CHO1-depleted cells reached a late stage of cytokinesis but fused back to form binucleate cells because of the absence of the midbody matrix in the middle of the intercellular bridge. Expression of exogenous CHO1 restored the formation of the midbody matrix and rescued cytokinesis in siRNA-treated cells. By analyzing phenotypes rescued with different constructs, it was shown that both motor and stalk domains function in midbody formation, whereas the tail is essential for completion of cytokinesis after the midbody matrix has formed. During the terminal stage of cytokinesis, different subregions of the tail play distinctive roles in stabilizing the midbody matrix and maintaining an association between the midbody and cell cortex. These results demonstrate that CHO1 consists of functionally differentiated subregions that act in concert to ensure complete cell separation.

Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria

In the present work the effect of quenching on quantification of intracellular metabolites in Corynebacterium glutamicum was investigated. C. glutamicum showed a high sensitivity to cold shock. Quenching of the cells by −50 °C buffered methanol prior to cell separation and extraction led to drastically reduced concentrations for free intracellular amino acids compared to those for nonquenched filtration. As demonstrated for glutamate and glutamine, this was clearly due to a more than 90% loss of these compounds from the cell interior into the medium during quenching. With lower methanol concentration in the quenching solution the metabolic losses were significantly lower but still amounted to about 30%. Due to the fact that quenching with ice-cold NaCl (0.9%) also resulted in significantly lower pool sizes for intracellular amino acids, a basic cold shock phenomenon is most likely the reason for the observed effects. The results clearly demonstrate that quenching combined with cell separation for concentration of the cells and removal of the medium is not applicable for intracellular metabolite analysis in C. glutamicum. Sampling by quick filtration without quenching allows complete cell separation and authentic quantification of intracellular metabolite pools exhibiting time constants significantly larger than sampling time.

Swm1p subunit of the APC/cyclosome is required for activation of the daughter-specific gene expression program mediated by Ace2p during growth at high temperature in Saccharomyces cerevisiae.

SWM1 was originally identified for its role in the late steps of the sporulation process, being required for spore wall assembly. This protein, recently identified as one of the core subunits of the anaphase-promoting complex (APC) is also required to complete cell separation in vegetative cells during growth at high temperature. Mutants lacking SWM1 show a thermosensitive growth defect that is suppressed by osmotic support in the culture medium. At the restrictive temperature, swm1 mutants are unable to complete separation, forming chains of cells that remain associated and, with prolonged incubation times, the stability of the cell wall is compromised, resulting in cell lysis. This separation defect is due to a reduction in expression of CTS1 (the gene encoding chitinase) and a group of genes involved in cell separation (such as ENG1,SCW11, DSE1 and DSE2). Interestingly, these genes are specifically regulated by the transcription factor Ace2p, suggesting that Swm1p is required for normal expression of Ace2p-dependent genes during growth at high temperatures. Although no defect in Ace2p localization can be observed at 28 degrees C, this transcription factor is unable to enter the nucleus of the daughter cell during growth at 38 degrees C. Under these growth conditions, swm1 cells undergo a delay in exit from mitosis, as determined by analysis of Clb2p degradation and Cdc28p-Clb2p kinase assays, and this could be the reason for the cytoplasmic localization of Ace2p.

Regulation of cell separation in the dimorphic fungus Ustilago maydis.

During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree-like clusters in liquid culture and grow as ring-like (donut-shaped) colonies on solid medium. In wild-type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal-centre-kinase (GC) subfamily of Ste20-like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two-hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.

Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation.

The endothelium separates blood from tissue and actively regulates the exchange between these compartments. Throughout the body, this one-cell-thick lining of blood vessels extends over several thousand square meters. It harbors many receptors and functions that actively regulate the extravasation of nutrients, solutes, hormones, macromolecules, and leukocytes. If the barrier function of the endothelium does not perform appropriately, leakage of macromolecules occurs, causing edema formation and exposure of the interstitium to high concentrations of plasma constituents. In severe cases of vascular leakage, platelets will adhere to the exposed subendothelium and red blood cells may even accumulate in the interstitium. Vascular leakage may be desirable for recruiting plasma proteins, such as complement factors during infection, but it can become life-threatening, particularly when it occurs in the lungs or pericardium or when it causes hypovolemic shock. Furthermore, frequent or prolonged leakage may impair proper functioning of the affected tissue. In recent years, considerable progress has been made in understanding the molecular processes that contribute to the regulation of endothelial permeability. With the exception of the liver, adrenal, and bone marrow sinusoids, in which the endothelium has rather large pores, the endothelium constitutes a selective barrier between blood and tissue. In principle, this barrier consists of the EC monolayer and its matrix. Macromolecules can cross the endothelial barrier in three ways: (1) between the cells, through cell junctions (paracellular); (2) through the EC, via pores (diaphragms or fused vesicles); and (3) transcellularly, via shuttling vesicles and specific receptors. It is generally believed that the charge and compactness of the endothelial matrix (both glycocalyx and basal membrane) contribute additionally to the selectivity of the endothelial barrier toward molecules of different size and charge. Electron microscopic evidence strongly suggests that in tissue capillaries, hormones and macromolecules are shuttled across the endothelial barrier via vesicles, in …

Schizolysis of dolipore–parenthesome septa in an arthroconidial fungus associated with Dendroctonus ponderosae and in similar anamorphic fungi

An arthroconidial anamorphic fungus occurred in pupal chambers of the mountain pine beetle (Dendroctonus ponderosae) in lodgepole pine. No teleomorph was found and no suitable form genus was available for its disposition. However, in cultural characteristics it closely resembled the group 1 arthroconidial fungi, defined by other researchers as probable basidiomycete anamorphs. Septa of the pupal-chamber fungus and several of the group 1 isolates were of the dolipore–parenthesome type. Conidial separation was by septum schizolysis. Cell separation was initiated by disintegration of the dolipore wall, followed by progressive shrinkage of the fused dolipore wall. The parenthesomes retracted towards the dolipore wall and the triangular areas of the cross wall dissolved. The cross wall then split centripetally along the median electron-light layer to complete cell separation. The pupal-chamber fungus was also compared with Phlebia radiata (group 2) and with groups 3 and 4 strains of other researchers. Mauginiella scaettae was confirmed to have simple septa; thus this genus fails to accommodate arthroconidial basidiomycete anamorphs. Key words: basidiomycete anamorphs, dolipore–parenthesome septa, septum schizolysis, arthroconidiogenesis, Phlebia radiata, Mauginiella scaettae.

Testicular descent: Androgen receptors in cultured porcine gubernaculum cells

We have adapted an oil microcentrifuge assay for evaluating the binding of tritiated methyltrienolone ([ 3H]R1881) in metabolically active primary cultured porcine gubernaculum fibroblasts. Almost complete separation of cells with the bound hormone and medium with the unbound hormone can be achieved within 30s of centrifugation at 10,000 g. Specific [ 3H]R1881 binding was found in four different gubernmaculum fibroblast cultures (9,000–42,000 sites/cell) and MCF-7 human breast cancer cells (34,000 sites/cell) which were used as controls. Binding was inhibited by radio-inert R1881 and 5α-dihydrotestosterone but not by estradiol and cortisol. The results indicate that androgen receptors are present in fibroblasts from the gubernaculum during both the first and second phase of testicular descent and that they may play a role in the regulation of that process.

Vertical ascending electrophoresis of cells with a minimal stabilizing medium.

Vertical fractionation of a mixture of fixed horse and human red blood cells layered over a stabilizing support medium was done to give a valid comparison with proposed space experiments. In particular, the effects of sample thickness and concentration on zone migration rate were investigated. Electrophoretic mobilities of horse and human cells calculated from zone migration rates were compatible with those obtained by microelectrophoresis. Complete cell separation was observed when low power and effective cooling were employed.

Fatty acids and the initial events of endothelial damage seen by scanning and transmission electron microscopy.

A method was developed for observing changes in the endothelial cells in rabbit ear veins in vivo by scanning electron microscopy. Injection of fatty acids into the ear vein caused damage to the endothelium. The first signs of damage seen were marked bulges in the nuclei and loss of the rhomboidal shape of the endothelial cells. More severe damage included loss of nuclei, leaving holes in the cytoplasm. Some parts of the damaged endothelium showed complete separation of cells from each other and exposure of sub-endothelial tissue to which platelets with pseudopodia were adhering. Damage to the endothelium was produced by arachidonic, linoleic, gamma-linolenic, 8,11,14-eicosatrienoic, 5,8,11,14,-eicosatetraenoic or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acids. The effect of arachidonic acid was not prevented by pre-treating the animals with aspirin. It appears that damage produced by the fatty acids is non-specific.

Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells.

A procedure for dissociation of the guinea pig pancreas into individual cells is described which employs enzymatic digestion with pure collagenase, chymotrypsin, and hyaluronidase, utilizes an interposed chelation of divalent cations by EDTA, and is terminated by gentle shearing. Yields of cells are 50-60%, based on DNA recovered. The population comprises approximately 95% exocrine cells, the remainder consisting of endocrine, duct, and vascular endothelial cells. The exocrine cells, though spherical, retain the structural attributes of their in situ counterparts, including differentiation of the plasmalemma into zones corresponding to the former apical and basal plasmalemma, polarized distribution of organelles indicated by fields of zymogen granules in the cytoplasm underlying the former apex, central location of the Golgi complex, and placement of the rough endoplasmic reticulum and nucleus in the former basal pole of the cell. Electron microscope study of the effects of individual treatments used during dissociation indicates that digestion of basement membrane and collagen is solely due to collagenase activity and that separation of desmosomes (and possibly of zonulae adherentes) results only from exposure to low [Ca(++)] and EDTA and is not effected by the enzymes used. Gap junctions are resistant to enzymes and EDTA; tight junctions resist enzyme treatment but undergo rearrangement upon exposure to EDTA. Both junctions require mechanical shear for complete cell separation. Neither chymotrypsin nor hyaluronidase produces visible alterations in stromal or junctional elements. Dissociation requires the concerted action of enzymes, chelation of divalent cations, and mechanical shear, since the individual treatments are alone ineffective.

Single-step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque

Centrifugation of heparinized human blood on discontinuous gradients of Ficoll-Hypaque resulted in the simultaneous separation of mononuclear leukocytes, granulocytes, and erythrocytes. Nearly complete separation of cells was achieved with high recovery of each cell type. Leukocytes isolated by this method retained the ability to exclude Trypan Blue dye and granulocytes retained their chemotactic responsiveness.