Crystal-cell interaction and apoptosis in oxalate-associated injury of renal epithelial cells.

Two renal epithelial cell lines, LLC-PK1 and Madin-Darby canine kidney (MDCK), were grown in monolayers and exposed to oxalate (Ox) and/or calcium oxalate (CaOx) crystals to investigate cellular responses to these challenges. In addition, LLC-PK1 cells were exposed to high concentrations of Ox for various time periods to investigate the role of apoptosis in Ox-associated cell injury. Both cell types showed signs of damage when exposed to Ox. However, LLC-PK1 cells appeared more sensitive than MDCK cells. There was a significant increase in release of lactate dehydrogenase into the medium and decrease in trypan blue exclusion by cells in the monolayer. Most noticeable was the detachment of cells from the substrate. Exposure of cells to CaOx crystals resulted in their attachment to cell surfaces followed by internalization. Using flow cytometry for quantification of apoptotic cells, transmission electron microscopy for morphology, and electrophoresis for DNA laddering detection, we observed significant apoptotic changes including condensation and margination of nuclear chromatin, DNA fragmentation, and migration of phosphatidylserine of the plasma membrane from inside to the cell surface. However, these cells also showed some necrotic changes such as loss of plasma membrane integrity and release of lactate dehydrogenase, indicating that the apoptotic process was interrupted.

The genotoxicity and cytotoxicity of dermally-administered cadmium: effects of dermal cadmium administration.

Cadmium, unlike zinc, selenium and copper, has no known biological importance, and therefore, it is classified as a carcinogen in humans, as well as in animals. The effect(s) of levels of dermally-administered cadmium on cadmium genotoxicity and cytotoxicity was investigated in Harlan Sprague-Dawley rats for 14, 21, 28, 35 and 42 days at concentrations of 14 and 28 mg/kg/day. Exposure of rats to cadmium via dermal application caused lesions on the skin (hyperkeratosis, acanthosis and scabbing, alopecia and erythema) and tumors in the scrotum. Anatomical changes, such as distention of the stomach, atrophy of kidney and liver and loss of body weight were also observed in these rats. The toxic effects of cadmium on cell ultrastructure were nuclear membrane damage, chromatin condensation, regression of mitochondrial cristae and ultimately cell death. Analyses of the brain, kidney and liver cells of rats exposed to cadmium, clearly showed DNA damage. Of the three organs examined, DNA from kidney cells sustained the most damage followed by DNA in liver cells. There is a positive correlation between Cd dose(s) and duration of exposure and the extent of DNA damage.

Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes.

The purpose of this study was to elucidate the interaction of cationic liposomes and plasmid cDNA by examining their ultrastructure, zeta potential, stability in aqueous media and protection from DNaseI digestion; their potential for hemolysis and platelet aggregation was evaluated as it may serve as an in vitro toxicity screen. Liposomes consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and dioleylphosphatidylethanolamine (DOPE) were complexed with plasmid constructs of ovine prostaglandin G/H synthase (pCMV4-PGH) or human alpha 1-antitrypsin (pCMV4-AAT) at lipid:plasmid (L/P) ratios of 3:1-8:1 (w/w). The electron micrographs showed bead-like attachment of liposomes to cDNA and coating of plasmid strands. The zeta potential showed isoelectric points at L/P ratios of 3.5-4 (DOTMA/DOPE) and 5.5-6.5, corresponding to a pKa of 6.45 (DC-Chol/DOPE). Liposome cDNA complexes were stable in water, saline and 5% dextrose for 48 h, but precipitated instantaneously in PBS. An increase in the L/P ratio corresponded with increased protection from DNaseI digestion. DOTMA/DOPE liposomes alone were highly hemolytic and DC-Chol/DOPE liposomes moderately hemolytic; hemolysis was abolished by cDNA complexation, with the exception of very high (> or = 7:1) L/P ratios. Both liposomes alone and cDNA complexes caused transient serum turbidity, while none caused platelet aggregation. It was concluded that current cationic lipid cDNA formulations are metastable and appear to have very little if any toxicity with respect to hemolytic potential and untoward interaction with other blood components.

Intraerythrocytic inclusions associated with iridoviral infection in a fer de lance (Bothrops moojeni) snake.

Intraerythrocytic inclusions associated with infection by an iridovirus were observed in a fer de lance (Bothrops moojeni) snake that was being evaluated for the presence of renal carcinoma. The erythrocytes contained two types of inclusions, one viral and one crystalline, usually concomitantly. The snake was markedly anemic and exhibited a marked regenerative response. Ultrastructural analysis identified the virus to be an iridovirus consistent with snake erythrocyte virus and the crystalline structures to be of a different nature than hemoglobin.

Localization of 14-3-3 proteins in the nuclei of arabidopsis and maize.

It has been demonstrated that 14-3-3 proteins are present in the nuclei of Arabidopsis thaliana and Zea mays cells using laser scanning confocal microscopy and immunocytochemistry with monoclonal antibodies against plant 14-3-3 proteins. Confirmation of nuclear localization provides insight into the range of functions normally attributed to 14-3-3 proteins, especially since the association of 14-3-3s with transcription factors is (thus far) a phenomenon unique to plants, and since 14-3-3 proteins do not possess a recognizable nuclear targeting sequence.

The influence of molecular conformation upon the self-assembly of cyclohexane diamide diacids.

BACKGROUND: Information regarding the self-association of small peptide motifs can be used in the design of peptide microstructures. Previous work in our laboratories illustrated the self-association of certain diamide diacids into microcapsules. In this report a series of cyclohexane diamide diacids are investigated. The cyclohexylene (R-C6H10-R) system (with its axial and equatorial requirements) provided an opportunity to study the influence of molecular conformation upon the self-aggregation process. RESULTS: Condensation of the respective cis- and trans-1,2-, 1,3-, and 1,4- cyclohexane dicarboxylic acid platforms with two equivalents of a L-Phe ester followed by deprotection gave the desired diamide diacids. Basic solutions of cis-1,2-, trans-1,3-, and cis-1,4-diamide diacids generated solid microspheres when acidified to pH 2.4. Molecular modeling revealed that 1,3-diaxial interactions favor a helical turn within these diamides. CONCLUSIONS: Access to 'complementary' molecular geometries is needed to self-associate into microscopic architectures.

Self-assembly of the brain MAP-2 microtubule-binding region into polymeric structures resembling Alzheimer filaments.

The neuronal microtubule-associated protein known as MAP-2 has not been considered to be a subunit of paired helical filaments (PHFs) in neurofibrillary tangles seen in Alzheimer's Disease. We now describe the assembly of paired helical filament-like structures from MAP-2's 203-residue microtubule-binding region (MTBR). SDS gel electrophoresis and equilibrium ultracentrifugation suggest that a dimeric form, cross-linked by an interchain disulfide, is involved in polymerization. MAP-2 MTBR polymers bind thioflavin-S, a dye used to histochemically localize Alzheimer neurofibrillary tangles. Our finding that PHF-like structures assemble from a MAP-2 fragment raises new questions about MAP-2's role in the etiology of Alzheimer's Disease.

SP75 is encoded by the DP87 gene and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins.

Highly purified spore coats of Dictyostelium discoideum each contained about 5 x 10(6) protein molecules as determined by amino acid composition analysis. By two-dimensional gel electrophoresis the coats were found to contain nine major-abundance and numerous minor protein species, most of which were highly enriched relative to the adjacent interspore matrix. Protein was nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that it was not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free thiol groups have been prealkylated, it was concluded that the D. discoideum spore coat proteins are disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this finding was supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned were associated with the outer layer of the coat. In coats missing one or more of four of these proteins as a result of gene disruption, there were physical changes but, with one exception, the other major coat proteins appeared to be incorporated normally. Sequence analysis showed that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH2-terminal subdomain of epidermal growth factor modules and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation.

Identification of a new spore coat protein gene in the cellular slime mold Dictyostelium discoideum.

Genomic DNA encoding the prespore cell-specific PL3 cDNA was cloned and sequenced, revealing that the gene consists of three exons separated by short 100-bp introns. The single long open reading frame predicts a primary translation product of 70 kDa after removal of a cleavable signal peptide, two-thirds of which consists of four kinds of amino acid repeat elements, including two found in other spore coat proteins. The 85-kDa PL3 protein synthesized in vivo accumulates specifically in regulated secretory vesicles of prespore cells (prespore vesicles), as determined microscopically using antibody against a PL3 gene fusion product expressed in Escherichia coli. The protein later accumulates extracellularly in the spore coat, which is formed during sporulation, as determined ultrastructurally and by Western blot analysis of SDS-PAGE gels. In addition to its high proportion of repeat elements, the PL3 protein has the following properties which distinguish it from other spore coat proteins: (1) it is located at the outer extent of the middle layer, beneath the outer layer, (2) its dissociation from the coat requires the presence of protein denaturants and reducing agents at elevated temperature, and (3) a large proportion of the protein is not dissociated from the coat even under these conditions, as determined by ultrastructural analysis of the extracted coat. The PL3 protein may contribute to the structure of the coat at the interface between the middle, cellulosic layer and the outer, electron-dense, proteinaceous layer.

Fluctuations and ultrastructural localization of oncoproteins and cell cycle regulatory proteins during growth and apoptosis of synchronized AGF cells.

AGF cells were synchronized by blocking the cell cycle at the G1/S boundary with high concentrations of thymidine (thymidine block) for 11 h. Prolongation of the thymidine block from 11 h to 20 h resulted in apoptosis. Early changes in cellular and nuclear morphology were monitored by confocal microscopy, transmission electron microscopy, and scanning electron microscopy. The fluctuations in the levels of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c-myc, and p53 proteins were monitored in synchronized cultures and in cells undergoing apoptosis by immunofluorescence staining, flow cytometry, and Western immunoblotting. When assayed by immunofluorescence staining and flow cytometry, the levels of cyclin A and PCNA increased about 2-fold during the S phase, and the level of CDC-2 was fairly constant during S and slightly decreased during late S/G2. The level of c-myc also increased about 2-fold during the S phase, whereas the level of p53 increased only slightly during S. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2, and PCNA increased 50%-150% during apoptosis compared to the levels observed in cells at G1/S. In contrast, the levels of actin and vimentin, although increased during S, were decreased during apoptosis compared to the levels observed at G1/S. Western blot analysis of the steady state levels of PCNA, cyclin A, and CDC-2 revealed an increase in the levels of all three proteins during S, with higher levels of these proteins observed in apoptotic cells compared to the levels observed in cells at G1/S. Similarly, the levels of p53 and c-myc proteins increased during S and were also high in apoptotic cells. Interestingly, high levels of these two proteins were observed also in cells arrested at G1/S. AGF cells undergoing apoptosis were immunostained for c-myc, p53, PCNA, cyclin A, and CDC-2 and were viewed by confocal microscopy. Apoptotic cells exhibited increased staining for c-myc and p53 in the blebbing nuclei. Furthermore, we observed for the first time that CDC-2, cyclin A, and PCNA proteins were associated mostly with the plasma membrane and the cytoplasm of log phase cells. However, in cells undergoing apoptosis, these proteins were found exclusively in the nuclei of apoptotic cells. These results suggest a possible active role for c-myc, p53, and the cell cycle regulatory proteins in the process of nuclear blebbing and apoptosis.

Ultrastructural localization and fluctuation in the level of the proliferating cell nuclear antigen and myc oncoproteins in synchronized neuroblastoma cells.

A method for rapid synchronization of neuroblastoma cells was developed using the thymidine block to arrest cells in the G1-S boundary. Following release from the thymidine block, cells traversed to G2-M in 7-8 h with 85% cell synchrony. Determination of the steady-state level of proliferating cell nuclear antigen (PCNA) mRNA and protein by Northern and Western blots revealed an accumulation of the PCNA messenger RNA transcripts and PCNA protein at G1-S and a rapid decrease when cells entered S phase. The level of both the messenger RNA transcripts and protein increased as the cells moved to late-S and G2-M. Similarly, the steady-state level of c-myc and N-myc messenger RNA transcripts and proteins increased during the G1-S block, decreased when the cells entered S, and increased as the cells moved through S phase to G2-M. However, immunofluorescence staining for PCNA and myc protein indicated a low level of staining for all three proteins at G1-S and a significant increase in staining intensity during S phase. Similarly, immunoelectron microscopy revealed low levels of N-myc and c-myc staining during G1-S and increased staining during mid-S and late S phase of the cell cycle. These results suggest differential cell cycle-dependent accessibility of myc protein and PCNA to staining in the intact cells compared to the whole cell extract. Furthermore, using immunofluorescence staining, confocal microscopy, and immunoelectron microscopy, we demonstrate for the first time that myc proteins are associated with the chromosomes during mitosis.

Development of a two color immunofluorescence stain and immunolocalization method for N-myc and c-myc oncoproteins with a newly generated mouse IgM anti N-myc antibody.

A new mouse monoclonal antibody specific for N-myc oncoprotein was generated and used in combination with an anti-c-myc antibody to develop two color immunofluorescence staining and ultrastructural immunolocalization of N-myc and c-myc in well established (SK-N-SH; CHP 126) and in newly established neuroblastoma (NB) cell lines. Analysis and quantitation of c-myc and N-myc in dually stained cells was done by flow cytometry. Immunolocalization was done by staining with immunogold secondary antibodies and transmission electron microscopy. The results obtained from analysis of 13 newly established NB cell lines revealed, great heterogeneity in the expression of N-myc oncoprotein with 10/13 cell lines over expressing the protein. C-myc oncoprotein was also expressed in all cell lines, however, the level of expression was 4-10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc whereas N-myc was localized to the nucleus and to the cytoplasm.

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.

The production of antibacterial tubing, sutures, and bandages by in situ precipitation of metallic salts.

Two procedures were used to modify gauze bandages, polyester sutures, silicone tubing, and polyvinyl chloride tubing. In one procedure, the materials were first modified by in situ precipitation of metallic hydroxides and then used to adsorb silver ions. In the second procedure, the materials were soaked in sodium pyrophosphate or sodium chloride, dried, and then soaked in silver nitrate. These procedures produced materials with silver deposited on the surface of the tubing and sutures and both on the surface and within the gauze fibers. The modified materials inhibited the growth of Pseudomonas aeruginosa. Escherichia coli, and Staphylococcus aureus in vitro.

Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens.

Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth. Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.

Spore coat is altered in modB glycosylation mutants of Dictyostelium discoideum.

The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation-defective glycoprotein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which are specific because they are distinctive from changes observed in another glycosylation mutant which affects a different epitope.

Formation of the Dictyostelium spore coat.

The spore coat forms as a rigid extracellular wall around each spore cell during culmination. Coats purified from germinated spores contain multiple protein species and an approximately equal mass of polysaccharide, consisting mostly of cellulose and a galactose/N-acetylgalactosamine polysaccharide (GPS). All but the cellulose are prepackaged during prespore cell differentiation in a regulated secretory compartment, the prespore vesicle. The morphology of this compartment resembles an anastomosing, tubular network rather than a spherical vesicle. The molecules of the prespore vesicles are not uniformly mixed but are segregated into partially overlapping domains. Although lysosomal enzymes have been found in the prespore vesicle, this compartment does not function as a lysosome because it is not acidic, and a common antigen associated with acid hydrolases is found in another, acidic vesicle population. All the prespore vesicle profiles disappear at the time of appearance of their contents outside of the cell; this constitutes an early stage in spore coat formation, which can be detected both by microscopy and flow cytometry. As an electron-dense layer, the future outer layer of the coat, condenses, cellulose can be found and is located immediately beneath this outer layer. Certain proteins and the GPS become associated with either the outer or inner layers surrounding this middle cellulose layer. Assembly of the inner and outer layers occurs in part from a pool of glycoproteins that is shared between spores, and unincorporated molecules loosely reside in the interspore matrix, a location from which they can be easily washed away. When the glycosylation of several major protein species is disrupted by mutation, the coat is assembled, but differences are found in its porosity and the extractibility of certain proteins. In addition, the retention or loss of proteolytic fragments in the mutants indicates regions of spore coat proteins that are required for association with the coat. Comparative examination of the macrocyst demonstrates that patterns of molecular distributions are not conserved between the macrocyst and spore coats. Thus spore coat assembly is characterized by highly specific intermolecular interactions, leading to saturable associations of individual glycoproteins with specific layers and the exclusion of excess copies to the interspore space.

Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (I/II).

The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or I/II) has been cloned into Escherichia coli (S. F. Lee, A. Progulske-Fox, and A. S. Bleiweis, Infect. Immun. 56:2114-2119, 1988). In the present study, this gene has been disrupted in vitro by insertional inactivation with pVA981, which carries a Tcr marker, and transformed into S. mutans NG8 (serotype c) by electroporation. Upon homologous recombination, the defective spaP was integrated into the genome as demonstrated by Southern hybridization analysis. One Tcr mutant, designated 834, selected by its nonreactivity with anti-P1 monoclonal antibodies, was found to lack the cell surface fuzzy layer which was clearly present on the parent cells. Analysis of extracellular fluids, sodium dodecyl sulfate-solubilized membranes, and cytoplasmic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 834 had protein profiles identical to the parent. However, a 185-kilodalton protein which reacts with anti-P1 antibodies was missing from the wall of 834, suggesting that spaP has been specifically inactivated. This mutant displayed levels of glucosyltransferase and fructosyltransferase activities similar to those of the parent. It was much less hydrophobic than the parent. S. mutans NG8 aggregated readily in the presence of clarified whole saliva or a high-molecular-weight salivary agglutinin. This strain also adhered to agglutinin-coated hydroxyapatite. The P1-negative mutants, however, did not display these two properties, suggesting that P1 may play a role in saliva-mediated aggregation and adherence.

The spore coat of a fucosylation mutant in Dictyostelium discoideum.

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.

Enzyme-gold affinity labelling of cellulose.

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)