Agglutination Process Research Articles

FROM PROTOPLASM TO SWARMER: REGENERATION OF PROTOPLASTS FROM DISINTEGRATED CELLS OF THE MULTICELLULAR MARINE GREEN ALGA MICRODICTYON UMBILICATUM (CHLOROPHYTA)1

Protoplast regeneration from extruded cytoplasm of the multicellular marine green alga Microdictyon umbilicatum (Velley) Zanardini (Cladophorales, Anadyomenaceae) was investigated. The early process of protoplast formation is comprised of two steps: agglutination of cell organelles into protoplasmic masses followed by generation of a temporary enclosing envelope around them. Agglutination of cell organelles was mediated by a lectin–carbohydrate complementary system. Three sugars, D‐galactosamine, D‐glucosamine, and α‐D‐mannose, inhibited the agglutination process, and three complementary lectins for the above sugars, peanut agglutinin, Ricinus communis agglutinin, and concanavalin A, bound to the surfaces of chloroplasts. Agglutination assay using human erythrocytes showed the presence of lectins specific for the above sugars in the algal vacuolar sap. A fluorescent probe 1‐(4‐trimethylammoniumphenyl)‐6‐phenyl‐a, 3,5‐hexatriene revealed that the envelope initially surrounding protoplasts was not a lipid‐based cell membrane. However, this developed several hours later. Simultaneous fluorescein diacetate and propidium iodide staining showed that the primary envelope had some characteristics of cell membranes, such as semipermeability and selective transport of materials. Also, fluorescein diacetate staining showed esterase activity in the protoplast and relocation of cell organelles and compartmentalization of cytoplasm during the process of regeneration. Both pH 7–9 and salinity 400–500 mM were found to be essentially important for the development of the protoplast envelope. When the basic regeneration process was accomplished, two alternative pathways of development were seen; about 70% of one‐celled protoplasts transformed into reproductive cells within 2 weeks after wounding, whereas others began cell division and grew into typical Microdictyon thalli. Quadriflagellate swarmers were liberated from the reproductive cells, and they germinated into mature individuals. It is therefore suggested that this species may use the wound response as a method of propagation and dispersal.

Lipid extracts isolated from heat processed food show a strong agglutinating activity against human red blood cells

In this study we investigated the possibility that the lipid components of foodstuffs and mass market oils undergo oxidative and thermal changes during storage, processing and cooking and so become agglutinins. The hemagglutinating activity of several mass market oils and several lipid mixtures isolated from different food items was evaluated against human red blood cells (RBCs) and against hamster RBCs. The unheated oils and the lipid extract of the unheated foodstuff had a very low agglutination titer but lysed red cells at high concentrations. When the same foodstuff items and oils were heated (100 °C for 24 h) in air, the isolated mixture of lipids as well as the oils show a strong hemagglutinating activity. Thin layer chromatography (TLC) of the lipid mixture, isolated from the processed foodstuff, as well as the heated samples of the oils showed appearance of high molecular weight molecules, possibly dimers and polymers. Light microscopy was used to characterize and visualize the agglutination process. Agglutination without lysis or fusion was observed. Agglutination may be the result of membrane properties alteration due to the oxidation product insertion or by hydrophobic side chain insertion into adjacent RBC membranes. We conclude that oils and foodstuff items when heated in air produce hemagglutinins against human RBCs with unknown and possibly toxic effects on human health.

Biological properties of a natural agglutinin in the hemolymph of Indian white prawn, Penaeus indicus H. Milne Edwards

Biological properties of a natural agglutinin in the hemolymph of Penaeus indicus were established. Agglutination was observed against different strains of bacteria and erythrocytes and strong agglutination against a Vibrio sp. isolated from a diseased prawn gut (titer 256). Minimum concentration of agglutinin required for agglutination was 0.8 μg/ml. The agglutination reaction was affected by various sugars and ions. Agglutination and cross-linking process were inhibited by simple sugars like xylose, raffinose and trehalose, but stimulated by maltose, lactose, glucose, mannose and cellobiose. Ca 2+ and Mg 2+ ion supplementations were not essential for agglutination. Natural agglutinin was found to be a specific recognition molecule, which functions as defense protein with simultaneous agglutinating, hemolytic and antibacterial properties. Monitoring hemolymph levels of agglutinin may be helpful in disease detection.

Ultrasonic wave action upon the red blood cell agglutination in vitro

The ultrasonic wave action upon immune red blood cell (RBC) aggregation in vitro was investigated by means of the elastic light-scattering method. The RBC agglutination process in the ultrasonic wave presence was analysed as a function of irradiation time, the antiserum series, the whole blood and antiserum dilution degree and blood group types. It was experimentally shown that the ultrasonic irradiation accelerated the agglutination reaction in the blood and antiserum mixture. The sedimentation process showed a gain in the resolution of the optical method’s ability to detect the induced RBC complex formation. It revealed that the ultrasonic application led to significant diagnostic test time shortening. The results may be used to develop new diagnostic techniques and devices.

GEOCHEMISTRY OF THE SERRA DO MAR GRANITOID MAGMATISM AND TECTONIC IMPLICATIONS, SOUTHERN BRAZIL

The Neoproterozoic (580 ± 20 Ma) alkaline granitoid magmatism produced two rock associations in the Serra do Mar region, Southern Brazil. The first one (70-76% SiO 2 ) is metaluminous to weakly peraluminous, HFSE depleted relative to LILE, and includes the larger massifs mainly. The second one (62-76% SiO 2 ) comprises metaluminous as well as peralkaline rock-types and is enriched in HFSE relative to LILE. The granitoid massifs were emplaced within transtensional fault zones generated during the final stages of the agglutination process that led to the formation of the Gondwana supercontinent. As a whole, the Serra do Mar granitoid magmatism represents a transition from late orogenic calc-alkaline monzonitic to anorogenic alkaline (A-type) magmatism.

GEOCHEMISTRY OF THE SERRA DO MAR GRANITOID MAGMATISM AND TECTONIC IMPLICATIONS, SOUTHERN BRAZIL

The Neoproterozoic (580 ± 20 Ma) alkaline granitoid magmatism produced two rock associations in the Serra do Mar region, Southern Brazil. The first one (70-76% SiO 2 ) is metaluminous to weakly peraluminous, HFSE depleted relative to LILE, and includes the larger massifs mainly. The second one (62-76% SiO 2 ) comprises metaluminous as well as peralkaline rock-types and is enriched in HFSE relative to LILE. The granitoid massifs were emplaced within transtensional fault zones generated during the final stages of the agglutination process that led to the formation of the Gondwana supercontinent. As a whole, the Serra do Mar granitoid magmatism represents a transition from late erogenic calc-alkaline monzonitic to anorogenic alkaline (A-type) magmatism.

AMERICA DO SUL: QUATRO FUSÕES, QUATRO FISSÕES E O PROCESSO ACRESCIONÁRIO ANDINO

The aim of this article is try to interpret -as a first approximation- the evolution of the South American continent from the point of view of the Supercontinents Theory of the new Global Tectonics. It is preliminarly reiterated that the individualization of this lithospheric segment as a continent (geotectonic sense) is a fact younger than 100 Ma old, although we know about a crustal evolutionary history -as participant of other continental configurations as older as 3.5 Ga. Most of those previous continental configurations were larger in size than this now presented. Four major processes of (super)continental agglutination are being identified and they will be described, three of them Precambrian in age in the upper part of the Proterozoic eras (Paleoproterozoic, Mesoproterozoic and Neoproterozoic). The last of these agglutination processes took place in Early Mesozoic times, after a long and complex Paleozoic history of accretions and interior collisions. Following each of these agglutinantion events there were wide processes of continental break up (taphrogenesis) and dispersion ( drift) of the descendant lithospheric segments, as result of the dissipation of heat from astenospheric realms. The last process of dispersion, after the Triassic continental collage (Pangea supercontinent) is still under way, in terms of the present grouwth of the Atlantic Ocean. So, it has partially been coeval of the concurrent accretionary processes in the Andean Chain, which are somehow leading to a future continental agglutination (when of the disappearance of The Pacific and Caribbean oceanic segments). Such development of these two different and concurrent processes (at the western and eastern margin) is the responsible for the present shape and concept of the continent, which has to be understood just as a particular instance (and configuration) of an ongoing complex evolutionary trajectory of this continental lithospheric segment.

AMERICA DO SUL: QUATRO FUSÕES, QUATRO FISSÕES E O PROCESSO ACRESCIONÁRIO ANDINO

The aim of this article is try to interpret -as a first approximation- the evolution of the South American continent from the point of view of the Supercontinents Theory of the new Global Tectonics. It is preliminarly reiterated that the individualization of this lithospheric segment as a continent (geotectonic sense) is a fact younger than 100 Ma old, although we know about a crustal evolutionary history -as participant of other continental configurations as older as 3.5 Ga. Most of those previous continental configurations were larger in size than this now presented. Four major processes of (super)continental agglutination are being identified and they will be described, three of them Precambrian in age in the upper part of the Proterozoic eras (Paleoproterozoic, Mesoproterozoic and Neoproterozoic). The last of these agglutination processes took place in Early Mesozoic times, after a long and complex Paleozoic history of accretions and interior collisions. Following each of these agglutinantion events there were wide processes of continental break up (taphrogenesis) and dispersion ( drift) of the descendant lithospheric segments, as result of the dissipation of heat from astenospheric realms. The last process of dispersion, after the Triassic continental collage (Pangea supercontinent) is still under way, in terms of the present grouwth of the Atlantic Ocean. Só, it hás partially been coeval of the concurrent accretionary processes in the Andean Chain, which are somehow leading to a future continental agglutination (when of the disappearance of The Pacific and Caribbean oceanic segments). Such development of these two different and concurrent processes (at the western and eastern margin) is the responsible for the present shape and concept of the continent, which hás to be understood just as a particular instance (and configuration) of an ongoing complex evolutionary trajectory of this continental lithospheric segment.

Application of the quartz crystal microbalance to the monitoring of Staphylococcus epidermidis antigen–antibody agglutination

The change in solution properties due to the agglutination of an antigen with its specific antibody has previously been used as a marker of infection. This method has been modified to allow the binding activity between species to be followed using the frequency response of a quartz crystal microbalance (QCM). The Bayston agglutination plate assay for Staphylococcus epidermidis has been modified to allow the electrode of a QCM to act as a direct sensor for the change in solution properties as agglutination occurs. Antibody and antigen were introduced to the crystal surface and the agglutination process was followed as a change in crystal resonant frequency. Serum, known to be infected with the organism, gave a titre of 3.9×10 −2% v/v (−118 Hz, ±12 SD, N=9) matching that given by triplicate plate assay. Uninfected serum gave no frequency changes at this concentration, yielding a titre of 2.5×10 −2% v/v again matching the plate titre ( N=3). Infected serum gave responses 40 times faster then those of the uninfected serum. The piezoelectric quartz crystal method gave a positive or negative diagnosis in <15 min compared with the 24 h required for the plate assay.

Agglutination kinetics of F(ab′) 2 coated polymer colloids

In a previous publication we have described a simple quantitative model describing the agglutination process due to antigen–antibody interactions in IgG–latex immuno-assays. To achieve colloidal stability, however, some additives had to be used. In this paper we study the immunoreactivity of F(ab′)2 antibody physical and chemically adsorbed onto sulphonate polystyrene and chloromethylstyrene particles, respectively. This kind of latex immuno-assay does not show stability problems under physiological condi-tions. The influence on the agglutination kinetics of the protein coating level has been now studied for the whole coverage range. Also the influence of coupling procedure, storage time and ionic strength of the reaction medium have been investigated.

Cell surface properties of Aspergillus fumigatus conidia: correlation between adherence, agglutination, and rearrangements of the cell wall

Culture conditions that lead to swelling and germination dramatically influence cell surface characteristics and properties of Aspergillus fumigatus conidia. Conidial adherence to polystyrene and agglutination markedly increased during swelling, in a time-dependent manner. Agglutination appeared to be sensitive to cycloheximide and calcium. Removal of cell wall polysaccharides by lyticase or sodium metaperiodate suppressed agglutination of conidia. Proteinase K weakly decreased it whereas dithiothreitol strongly dispersed the cells. These observations suggest that both cell surface carbohydrates and proteins are involved in the agglutination process. Electron microscopic observations demonstrated that the cell wall of conidia was subject to some rearrangements during swelling, involving degradation and loss of the external convoluted layer, and subsequent exposure of underlying ligands. This was confirmed using lectins labelled with gold or fluorescein isothiocyanate, which showed that some carbohydrates, particularly those acting as ligands for peanut agglutinin, are largely exposed during the process. Finally, SDS-PAGE revealed major protein changes between resting and swollen conidia. We conclude that the ability of A. fumigatus conidia to aggregate correlates with an increase in adherence and biochemical reorganization of the cell wall.

Neutral polymers elicit and antibodies to spectrin band 4.1 protein and cytoplasmic domain of band 3 protein inhibit the concanavalin A-mediated agglutination of human erythrocytes

Concanavalin A (Con A) is known to agglutinate human erythrocytes if the cells are pre-treated with a proteinase or neuraminidase. We report that untreated cells can also be made to agglutinate with the lectin if the lectin-bound cells are treated with anti-Con A antibodies, or if a neutral polymer such as serum albumin, polyvinylpyrrolidone or Ficoll is added. Thus, Con A falls in the category of ‘incomplete’ lectins. The polymer induces Con A-agglutinability without altering the receptor number, or deformability of the cells. If the polymer is sequestered within erythrocyte ghosts, Con A is unable to agglutinate them; but the presence of the polymer only on the outer surface (as in intact cells) or on both the surfaces permits agglutinability. Thus, the site of the polymer effect resides on the outer surface of the membrane. The polymer, however, is unable to induce agglutinability in erythrocyte vesicles, whose membrane lacks skeletal proteins. The result suggests a positive role for the membrane skeleton in the process of agglutination brought about by the polymer, as is true also for the agglutination of proteinase-treated cells. In order to obtain detailed information on the proteins participating in agglutination, monospecific antibodies to spectrins, band 4.1 protein, ankyrin and the cytoplasmic domain of band 3 protein were internalized in erythrocytes. It is found that anti-spectrin and anti-band 3 cytoplasmic domain, but not their Fab's, inhibit the Con A-mediated agglutinability partially, and anti-4.1 antibodies, as well as the Fab's, inhibit the agglutinability substantially. Anti-ankyrin, however, was without any effect. The results confirm a positive role for the membrane skeleton in the Con A-mediated agglutination of normal erythrocytes in the presence of a neutral polymer, or in proteinase treated cells. We also provide evidence for requirement of Mg-ATP in the agglutination process.

Properties of a blood-meal-induced midgut lectin from the tsetse fly Glossina morsitans.

The properties of a blood-meal-induced lectin (agglutinin) from the midgut of Glossina morsitans capable of agglutinating Trypanosoma brucei were studied in vitro. The midgut homogenate from flies that had been fed twice had the highest agglutination activity, followed by that from the once-fed flies and that from the unfed insects. As compared with the bloodstream-form trypanosomes, a much lower concentration of the midgut homogenate was required for agglutination of the procyclic parasites. Furthermore, the agglutination process was specifically inhibited by D-glucosamine. Soybean trypsin inhibitor abrogated agglutination of the bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination process was temperature-sensitive, with little activity being evident between 4 degrees and 15 degrees C. Similarly, heating the midguts to 60 degrees-100 degrees C led to loss of activity. When the midgut homogenate was separated by anion-exchange chromatography, the agglutination activity co-eluted with trypsin activity at approximately 50% NaCl. These results suggest a very close relationship between midgut trypsin-like enzyme and the agglutinin. Since successful agglutination of bloodstream-form trypanosomes requires protease activity, it may be that the enzyme cleaves off some surface molecules on the parasite surface, thus exposing the lectin-binding sites.

O complexo Atuba: um cinturão paleoproterozóico intensamente retrabalhado no Neoproterozóico

Studies of terranes between the northern Ribeira and southern Dom Feliciano Belts allow the characterization of three geotectonic domains with different evolutions: the Luís Alves, Curitiba and Paranaguá terranes. The Atuba Complex occurs in the Curitiba Domain, which has a northwestern limit with metassediments of the Açungui and Setuva Groups and a southwestern limit with the granulitic gneisses of the Luis Alves domains. The contacts are expressive shear zones. The predominant rocks of the Curitiba Domain are banded, migmatitic gneisses in amphibolite grade with biotite-amphibole gneissic mesosomes and tonalitic/granodioritic to granitic leucosomes, here called the Atuba Complex. The migmatites are Paleoproterozoic (2.000 ± 200 Ma) and remigmatized in Neoproterozoic (600 ± 20 Ma). During the latter period temperatures reached more than 500º C. The structural pattem indicated shear-controlled tectonics with an important lateral component, and low-angle, south-southeastwards transport direction. The terranes of the Atuba Complex appear to represent deep-level rocks which were migmatized, granitized and then added to the border of the Luis Alves Microplate during the Neoproterozoic. This late Neoproterozoic tectonic scheme which continued to the Cambro-Ordoviciano seems to be the result of larger-scale processes of continental agglutination which ended with the formation of western Gondwanaland.

Heating unsaturated fatty acids in air produces hemagglutinins

When mono-unsaturated fatty acids are heated in air, they form hemagglutinins. When the double bond is Δ-6,7 or Δ-9,10, the titer is higher than for Δ-11,12. Stearic acid does not become a hemagglutinin on heating. Hydroxy-monounsaturated fatty acids, ricinoleic ( cis-12-OH-Δ-9) and ricinelaidic ( trans-12-OH-Δ-9) are not hemagglutinins unless they are heated. Oleic acid (Δ-9-octadecenoic acid, OA) has a very low agglutination titer but lyses red cells at higher concentrations. Rabbit and rat erythrocytes (RBC) give the highest titers but RBCs of other species are also agglutinated. OA was chosen for further study. Its specific titer against rat RBCs increases with time of heating in air. Thin-layer chromatography (TLC) and mass spectroscopy (MS) show that higher molecular weight compounds are formed and that activity is associated with these materials. Synthetic (oxidation of oleic acid with tert-butyl peroxide) and commercial preparations of oleic acid dimers (Emery and Unichema) and a commercial preparation of oleic trimer mixed with polymer (Emery) have high hemagglutination titers against rat erythrocytes. A cyclic, long-chain dicarboxylic acid, 5(6)-carboxy-4-hexyl-2-cyclohexene-1-octanoic acid (Westvaco) gives a very low titer unless heated and no lysis. Sialidase treatment of the red cells increases the titer. Removal of cations does not alter the titer but addition of Ca 2+ or Mg 2+ lowers the titer. Light microscopy was used to characterize and visualize the agglutination process with rat RBCs. Agglutination without lysis or fusion is observed for low concentrations of heated oleic acid and C-18 dimers and trimer-polymer preparations, and no large vesicles are seen. We conclude that the oligomeric fatty acids with two or more hydrophobic chains of seven or more carbons are agglutinins at physiological pH. Agglutination by dimer may be the result of the its two hydrophobic side chains inserting into adjacent RBC membranes or the result of dimer inserting completely into RBC membranes and altering their properties. The carboxyl groups may also play a role in the process by interacting with polar headgroups in the RBC membrane.

Investigation of Latex Agglutination with Different Immunoglobulin Classes of Antibodies by a Scanning Electron Microscopy

The conformation of passive latex agglutinations was investigated by a scanning electron microscopy. The complexes of 2,4-dinitrophenyl group (DNP) and bovine serum albumin (BSA) coated microspheres were incubated with monoclonal antibodies to DNP belonging to different classes of immunoglobulins. Examination with a scanning electron microscope demonstrated that the connectors between microspheres, agglutinated with IgE antibody, tended to take long filamentary forms; whereas the connectors of microspheres, agglutinated with IgG or IgM antibodies, formed shorter filaments. These phenomena were confirmed by the agglutination reactions of tick suspension-coated latex and allergic patients' sera. This morphologic difference is probably attributable to the unusually high elasticity of IgE antibody and is an evidence of the high biologic activity of the IgE antibodies. We assumed the processes of latex agglutination whereby the filamentary substances would develop, expecting that the processes could be models of in vivo and in vitro agglutination of cells.

Interactions of complement fraction C1q, fibronectin, and immunoglobulin G with polyacrylic microparticles used as solid-phase in immunoassay.

A microparticle-enhanced nephelometric immunoassay was recently described, where polyacrylic, hydrophilic, and polyfunctional microparticles are used as the solid phase. It is a one-step immunoassay based on the nephelometric quantification of microparticle agglutination. In such assays, the measurement of analytes at low concentration may be impaired by the need of using undiluted biological samples. This leads to work with high concentrations of several proteins liable to interfere with the agglutination process. In this paper, we report on a study performed with human serum and purified proteins, which were assayed by classical analytical methods. This work identified three major components of human serum specifically involved in yielding polyacrylic microparticle instability: complement fraction C1q, fibronectin, and immunoglobulins G. In this order of importance, they all showed a marked ability to be adsorbed on the microparticle's surface. Pretreatment of human serum with microparticles decreased the concentrations in C1q (82%), fibronectin (16%), and immunoglobulin G (4%) very unequally. However, it allowed the elimination of microparticle instability, consequently providing the possible use of such polyacrylic microparticles in a one-step nephelometric immunoassay of analytes at low concentration in biological samples, without washes or phase separation.

Temperature Sensitive Cytomixis in Helicanthes elastica (Desr.) Dans. (Loranthaceae).

Spontaneous occurrence of cytomixis in PMCs of H. elastica was noticed in high atmospheric temperature. The chromatin undergoes a temporary process of agglutination before migration. Cytomixis is found to have a direct correlation with other meiotic irregularities and sterility. The origin and evolutionary significance of the process is discussed.

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.

A kinetic model of the agglutination process

We propose a kinetic model of the aggregation process in a system consisting of two different types of particles. Aggregating particles (cells) are polyvalent and bear on the surface a huge number of binding sites for the other type of particles, ligands. The ligand is bivalent and has two identical active sites for binding to cells. The cross-linking of the cells by the ligands causes the aggregation phenomenon called agglutination. We obtained the analytical solution of this model task describing the time dependece of the aggregate mean size versus the composition of the system. The comparison of the analytical solution with the experimental data for the agglutination of bacterial cells by bivalent antibodies shows that the main factors affecting agglutination were correctly taken into account.