Fine-granular cationic iron colloid. Its preparation, physicochemical characteristics and histochemical use for the detection of ionized anionic groups

In order to obtain distinct and reliable information concerning the localization of ionized anionic groups in tissues, fine-granular cationic ferric hydroxide colloid solution (Fe-Cac-f) was newly devised. This can be obtained by boiling a mixture of ferric chloride and ammonium cacodylate solutions. The colloid particles of Fe-Cac-f are about 1.0 nm in size, i.e., one-fifth of the size of ferric cacodylate colloid (Fe-Cac; Seno et al. 1983a). As with Fe-Cac, Fe-Cac-f particles in the pH range of 1.6-7.6 carry a positive electric charge, but the latter show a better permeation of tissues. Using the Prussian blue reaction, Fe-Cac-f gives a distinct deep-blue color and can be used for the detection of anionic groups of acid mucopolysaccharides and proteins by light microscopy. It is also useful for detecting the exact sites of ionized anionic groups in deep tissue areas using electron microscopy.     

A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy

A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immunohistochemical researches.     

A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy

Summary A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immuno-histochemical researches.     

Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl₃) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 g/ml, whereas a 10-fold excess of iron concentration was required with FeCl₃, Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.     

Histochemical studies on the mechanism of macromolecule leakage across the glomerular capillary wall. Barrier effect of the anionic groups of the capillary wall against the serum protein leakage

For the purpose of revealing the barrier effect of the anionic groups of glomerular capillary wall against the serum protein leakage, morphologic and histochemical observations were made on the rat kidney perfused in situ with three kinds of cationic macromolecules different in chemical characteristics followed by blood flow restoration. The polyethyleneimine perfusion resulted in the complete disapperance of ionized anionic groups of glomerular capillary and the massive protein leakage through glomeruli by blood flow restoration. Cationic ferric colloid perfusion induced moderate protein leakage, and avidin perfusion was less in neutralization effect of anionic groups and the protein leakage was of least. The protein leakage from glomeruli, however, was stopped or markedly suppressed soon after the blood flow restoration by the newly formed functioning anionic barrier probably by some particular serum protein deposition. The findings indicate that the deionization of the glomerular capillary wall will not be responsible for the persistent albuminuria.     

Histochemical studies on the mechanism of macromolecule leakage across the glomerular capillary wall

Summary For the purpose of revealing the barrier effect of the anionic groups of glomerular capillary wall against the serum protein leakage, morphologic and histochemical observations were made on the rat kidney perfused in situ with three kinds of cationic macromolecules different in chemical characteristics followed by blood flow restoration. The polyethyleneimine perfusion resulted in the complete disappearance of ionized anionic groups of glomerular capillary and the massive protein leakage through glomeruli by blood flow restoration. Cationic ferric colloid perfusion induced moderate protein leakage, and avidin perfusion was less in neutralization effect of anionic groups and the protein leakage was of least. The protein leakage from glomeruli, however, was stopped or markedly suppressed soon after the blood flow restoration by the newly formed functioning anionc barrier probably by some particular serum protein deposition. The findings indicate that the deionization of the glomerular capillary wall will not be responsible for the persistent albuminuria.     

The anionic barrier system in the mesonephric renal glomerulus of the arctic lamprey,Entosphenus japonicus (Martens) (Cyclostomata)

Summary To examine the selective permeability of the nephrons of lower vertebrates, the permeability of the glomerulus in the kidney of an arctic lamprey, Entosphenus japonicus (Martens), to native anionic ferritin or cationized ferritin was studied by observing the distribution of ionized anionic groups in renal tissues. The cationized ferritin molecules injected into the dorsal aorta penetrated rapidly into the glomerular basement membrane layer through fenestrae present in the capillary endothelium and were subsequently excreted into the urinary spaces via the interstices between foot processes of the visceral epithelial cells. Native anionic ferritin, on the other hand, passed only minimally through the capillary wall. Cytochemical staining of fixed tissue or perfusion of the kidney in situ with cationic cacodylate-iron colloid revealed that the ionized anionic groups of acid mucopolysaccharides were distributed on both the luminal and abluminal surfaces of endothelial cells, and in the thick fibrous lamina rara interna of the glomerular basement membrane; they were especially dense on the surfaces of visceral epithelial cells and their foot processes. These results suggest that the mesonephric glomerulus of the arctic lamprey possesses a functionally well developed anionic barrier system comparable to that of the mammalian metanephric glomerulus.     

Asymmetry of spermiation and sperm surface charge patterns over the giant acrosome in the musk shrew Suncus murinus

Spermatozoa of the shrew Suncus murinus, a mammal with abdominal testes, exhibit four unusual features: a giant acrosome; a dorsoventral asymmetry of their spermiation; a dorsoventral asymmetry of their head surface character; and also apparent surface maturity as they enter the epididymis. A Sertoli cell-periacrosomal cisternal complex envelops the giant acrosome during spermatid maturation. Spermiation is heraled by asymmetrical disorganization of the subplasmalemmal components of this complex and is completed by retraction of the Sertoli cell from the ventral and then the dorsal face of the spermatid head. This sequence or release is correlated with an asynchronous acquisition of negative surface charges on the spermatid head-demonstrable on glutaraldehyde-stabilized cells by the binding at pH 1.8 of positively charged colloidal particles of ferric oxide. Mature epididymal spermatozoa exhibit an asymmetry in the patterns of distribution of bound colloid over the dorsal vs. ventral surfaces of the sperm head, as well as regional differences between the tail midpiece and principal piece. Surface distributions of anionic residues and lectin (Con A)-binding sites characteristic of mature Suncus spermatozoa are demonstrable within the testis, unlike the situation in most nannals where distinct modifications of the sperm surface occur during epididymal passage.     

液相沉积法制备聚(苯乙烯-丙烯酸)／铁的氧化物纳微核壳粒子

描述了一种基于液相沉积法制备纳微核壳粒子的新方法,即以聚(苯乙烯-丙烯酸)乳胶粒子为模板,利用静电相互作用和液相沉积的方法,将氢氧化铁包覆在模板粒子表面,形成聚(苯乙烯-丙烯酸)/铁的氧化物核壳纳米复合粒子。通过TEM和SEM观察,Fe2O3晶粒在模板粒子表面生长并呈草莓状,壳层厚度均匀。该特殊结构有利于通过煅烧除核的方法获得硬质空心磁球。     

Mitochondrial contacts of cardiomyocytes

The myocardium of the left and right ventricles in mature rabbits has been studied electron microscopically. The material is fixed by means of vital perfusion and/or by immersion in 2.5% glutaraldehyde with cacodylate buffer 0.05-0.1 M, pH 7.4 and treated in 1% OsO4 with the same buffer. For revealing intercellular contacts and inter-mitochondrial gaps, colloid lanthanum is applied. In order the colloid particles penetrate into cytoplasm, the model of ischemic myocardium is used. The myocardial infarction is produced by ligation of the coronary artery. The inter-mitochondrial interactions in cardiomyocytes are various and can be performed not only via hyaloplasm, but immediately by means of direct specific inter-mitochondrial contacts (IC). The IC are limited areas of maximal bringing together of the external membranes of the adjoining mitochondria. These areas are characterized by an increase electron density both of the contacting mitochondrial membranes and of the contents in the intermembranous spaces. A close topographic connection is revealed between mitochondria and cytolemma in the zone of the gap intercellular junction of the intercalated disc, where the mitochondrial nexus complex is formed. The IC can evidently ensure the metabolic and adhesive connection between separate mitochondria.     

Effect of size fractionation on the distribution of an albumin colloid in the reticuloendothelial system of the mouse

To study if by varying the particle size of a ^99mTc albumin colloid preparation its relative bone marrow accumulation could be increased, it was separated by gel filtration and different fractions were injected into mice. Particles around and smaller than the peak size of the colloid, 31 nm, exhibited a higher bone marrow/liver-spleen uptake ratio than larger particles but the uptake ratio was similar to that of the unseparated colloid. An antimony sulphide colloid showed a similar particle size distribution, but the corresponding uptake ratio was half of the albumin colloid. This indicates that characteristics other than size determine the distribution of a colloid in the reticuloendothelial system.     

Colloidal alpha-stannic acid and negative iron colloid as differential electron stains for surface proteins.

Colloidal alpha-stannic acid and a negative iron colloid obtained from ferric hydroxide and potassium ferrocyanide, both negative sols being stable within a wide pH range, were refined as surface protein electron markers. Because of the relatively small size of its particles, colloidal alpha-stannic acid was used for staining all surface proteins. According to the pH at which the negative iron colloid was applied, it revealed either all surface proteins, or because of its large colloidal particles, stained basic proteins. This differential staining capability of the iron colloidal has been demonstrated previously on various control preparations (Puvion E, Blanquet PR: J Microsc 12:171, 1971). Controls on the affinity of the two colloids to surface amino groups were carried out on rat liver, mouse fibroblasts, HeLa and KB cells, Ehrlich and Zajdela ascites cells subjected to prior enzymatic and chemical treatments (incubation with neuraminidase or phospholipase C, esterification, acetylation or lipid extraction). At any pH below 9, the two sols stained proteins in the outer hydrophilic leaflet of esterified cells with relative selectivity, but the alpha-stannic acid showed them more accurately. The iron sol did reveal at high pH protein components of high isoionic point on the surfaces of rat hepatocytes and ascites cells which had only been treated with neuraminidase.     

Attachment behaviour of Thiobacillus ferrooxidans cells to refractory gold concentrate particles

In the biooxidation of minerals, cells of Thiobacillus are distributed between the surface of the particles and the liquid. This work quantifies the kinetics of attachment, the equilibrium between attached and suspended cells, and the influence of ferric ion and particle size on this phenomenon. The attachment kinetics were fast, the equilibrium was reached in 10 to 30 minutes, depending on the initial population. The equilibrium curves showed three distinct phases, and the first two could be modelled by Langmuir equations. The maximum concentration of attached cells increases with the addition of ferric ions and decreases with particle size.     

Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

改进的免疫胶体铁细胞化学染色

本文以改进的方法制备了胶体铁。该胶体制备简单,稳定性高,易于抗体标记,在PH7.4时,胶体与抗体IgG稳定结合形成胶体抗体复合物的经以CM-Sephadex C-50代替AmberlightCG50进行标记物的纯化,方法可靠,所制备的胶体铁标记抗体用于免疫细胞化学染色,普鲁士蓝反应清晰地显示出标记抗体与相应抗原的特异性结合部位,与传统的免疫酶及免疫金银方法比较,具有敏感性高,背景干净,呈色鲜明等优点,结合免疫酶及免疫金银染色可用于抗原的双标及多标记。     

Transport of colloidal particles in lymphatics and vasculature after subcutaneous injection.

This study was designed to determine the transport of subcutaneously injected viral-size colloid particles into the lymph and the vascular system in the hind leg of the dog. Transport of two colloid particles, with average size approximately 1 and 0.41 microm, respectively, and with and without leg rotation, was tested. Leg rotation serves to enhance the lymph flow rates. The right femoral vein, lymph vessel, and left femoral artery were cannulated while the animal was under anesthesia, and samples were collected at regular intervals after subcutaneous injection of the particles at the right knee level. The number of particles in the samples were counted under fluorescence microscopy by using a hemocytometer. With and without leg rotation, both particle sets were rapidly taken up into the venous blood and into the lymph fluid. The number of particles carried away from the injection site within the first 5 min was <5% of the injected pool. Particles were also seen in arterial blood samples; this suggests reflow and a prolonged residence time in the blood. These results show that particles the size of viruses are rapidly taken up into the lymphatics and blood vessels after subcutaneous deposition.     

Packaging of a polymer by a viral capsid: the interplay between polymer length and capsid size

We report a study of the in vitro self-assembly of virus-like particles formed by the capsid protein of cowpea chlorotic mottle virus and the anionic polymer poly(styrene sulfonate) (PSS) for five molecular masses ranging from 400 kDa to 3.4 MDa. The goal is to explore the effect on capsid size of the competition between the preferred curvature of the protein and the molecular mass of the packaged cargo. The capsid size distribution for each polymer was unimodal, but two distinct sizes were observed: 22 nm for the lower molecular masses, jumping to 27 nm at a molecular mass of 2 MDa. A model is provided for the formation of the virus-like particles that accounts for both the PSS and capsid protein self-interactions and the interactions between the protein and PSS. Our study suggests that the size of the encapsidated polymer cargo is the deciding factor for the selection of one distinct capsid size from several possible sizes with the same inherent symmetry.     

THE ANATOMY OF SECRETION IN THE FOLLICULAR CELLS OF THE THYROID GLAND : II. The Effect of Acute Thyrotrophic Hormone Stimulation on the Secretory Apparatus

This paper reports a study by phase contrast and electron microscopy of changes observed in the thyroid gland of the rat at 1, 2, 12, and 24 hours following an injection of thyrotrophic hormone. Examination by phase contrast microscopy reveals that follicular cells contain numerous colloid droplets 1 and 2 hours after injection. By 12 and 24 hours, the colloid droplets are no longer present, and individual follicles appear to be subdividing into smaller units. The droplets are assumed to contain newly synthesized colloid, and their development was studied by electron microscopy. During the period of active secretion, increase in number of the Golgi vesicles leads to enlargement of this organelle. At its periphery small colloid droplets appear to form from large Golgi vesicles. As they form, their content becomes more adielectronic, and fine dense particles less than 75 A in diameter appear in their matrix. Small- and medium-sized droplets lying in the apical region of the cell contain numerous dense particles scattered in their moderately adielectronic content. Large, mature droplets in the same region have a relatively dielectronic content resembling follicular colloid and no longer contain dense particles. The follicular cells appear to utilize apical pseudopodia to release the content of mature droplets into the follicular lumen. Other droplet-like inclusions occur in follicular cells, but they do not seem to be directly concerned with secretion.     

Reaction of colloidal silica with membranes of intact mammalian cells

Colloidal silical particles were produced at a size that permitted reaction with human erythrocytes and rat macrophages without affecting cell integrity. Binding of colloid was shown by increased electrophoretic mobility of red cells and also resulted in changes in the surface topography of red cells as seen with scanning electron microscopy. The degree to which colloid binds to red cells was determined by microprobe analysis of single intact cells. Furthermore, the capacity of red cells to bind silica was increased if sialic acid residues were removed enzymatically from the cell surface.