Resin Sections Research Articles

Adherens junction breakdown in the periderm following cadmium administration in the chick embryo: distribution of cadherins and associated molecules

Background The teratogenic metal cadmium (Cd) has been found to cause ventral body wall defects similar to human omphalocele when administered to post-gastrulation chick embryos prior to body wall folding. From 4 h after Cd, affected embryos demonstrate varying degrees of cell junction breakdown and desquamation in the periderm. We examined the effect of Cd on tissue and cell distribution of cadherins and their intracellular associates. Methods Chicks were explanted and given 50 μl of 50 μM Cd solution at 60 h incubation (Hamburger-Hamilton stage 16–17). To examine peridermal junctions, embryos were processed into resin and ultra-thin sections examined by transmission electron microscopy (TEM). Tissue was processed into paraffin and 6 μm sections treated according to standard protocols for immunohistochemical detection of L-CAM, pan-cadherin, β-catenin, α-1 and α-2 catenin. To examine actin distribution, frozen sections were cut at 10–20 μm, stained with oragon green phalloidin and nuclei counter-stained with propidium iodide. Results The overall tissue distribution of L-CAM, pan-cadherin and the α-catenins did not appear to be altered following Cd. However, β-catenin changed from its normal sub-membranous location to a more general cytoplasmic distribution, with translocation to the nucleus in both peridermal and ectodermal cells. Similarly, actin distribution in the periderm in embryos demonstrating cell junction breakdown was markedly altered, with clumping and disorganization after 4 h. Conclusions Although L-CAM is distributed normally after Cd, post-translational modification may occur causing breakdown of its normal association with the catenins and actin, and allowing β-catenin to translocate to the nucleus in peri-ectodermal tissue, mimicking the canonical Wnt pathway.

Inhibition of Inflammatory Lymphangiogenesis by Integrin α5 Blockade

The interaction between endothelial cells and extracellular matrix proteins plays an important role in (hem)angiogenesis. Integrins are able to mediate the outgrowth of newly formed blood vessels. In contrast, the role of integrins in lymphangiogenesis, ie, the outgrowth of new from pre-existing lymphatic vessels, has so far been unclear. Here, expression and functional relevance of integrins on lymphatic endothelium in vivo was investigated using the mouse model of combined inflammatory corneal hemangiogenesis and lymphangiogenesis. Immunohistochemistry revealed novel expression of both integrin alpha5 and alphav on both resting and activated lymphatic vessels in vivo. Integrin alpha5-inhibiting small molecules significantly blocked the outgrowth of new lymphatic vessels into the cornea in a dose-dependent manner. The outgrowth of blood vessels was less significantly affected by this treatment, thus allowing for selective inhibition of lymphangiogenesis at lower dosages. Combined inhibition of integrin alpha5 and alphav using inhibiting molecules did not significantly increase the anti-lymphangiogenic effect in vivo, thus suggesting an important functional role of integrin alpha5 in lymphangiogenesis. In summary, our findings demonstrate novel expression of specific integrins on growing lymphatic endothelial cells in vivo and reveal their functional role during lymphangiogenesis. This opens new treatment options for selective inhibition of lymphangiogenesis, eg, in oncology and transplant immunology.

High-Resolution Immunohistochemistry on Improved Glycol Methacrylate—Resin Sections

Glycol methacrylate (GMA) embedding resins can be im­proved by the use of self-prepared mixtures. By the use of 99% 2-hydroxyethyl-methacrylate (i.e., GMA), the dibenzoyl peroxide content of solution A can be reduced and in addition the proportion of accelerator solution that is necessary for polymerization. The resulting lower polymerization temperature is advantageous in maintaining the tissue structure and also antigenic activity. Most immunohistochemistry (IHC) reactions can be made successfully without retrieval treatment if a suitable tissue fixative has been used. The required high sensitivity needed for IHC on GMA-resin embedded tissue sections can be achieved with SuperPic-Ture™ horseradish peroxidase polymer conjugates (Zymed) and visualised with antihorseradish peroxidase fluorochrome conjugates or Movat silver-intensified DAB or immunogold-silver staining. Outstandingly clean and clear results can be obtained by using phosphate-buffered saline/Tris buffer mixtures with 0.1% Extran for rinsing. In this work, we detail instructions for the 99% GMA resin embedding and step-by-step procedures for IHC on GMA-resin sections for fluorescence and bright-field microscopy

Embryo abortion and histological features in the interspecific cross between Phaseolus vulgaris L. and P. coccineus L

In this paper, the causes of early embryo abortion in the reciprocal crosses between Phaseolus vulgaris L. (a cultivar) and Phaseolus coccineus L. (a wild form) were studied. Methacrylate resin sections, 3–5 μm thick, of 3 to 14 day-old seeds were used to examine the embryo developmental stages and the state of seed tissue. It was observed that, embryos aborted at different developmental stages (globular to early cotyledon) depending on the maternal parent. The use of P. coccineus cytoplasm resulted in a higher number of abortion than in reciprocal crosses. Many of them took place between 5 and 6 days after pollination (DAP). Histological analyses permitted to observe that the embryo development was slower in the cross between P. coccineus and P. vulgaris, compared to parental seeds. It would be related to a deficient endosperm development in reciprocal crosses and, in some extent, hypertrophy of the suspensor might be the main cause of early embryo abortion. Then, it would be practical to overcome this incompatibility by rescuing the embryo at the globular stage of development.

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

The intensity of immunogold labeling of deplasticized acrylic sections compared to deplasticized epoxy sections—Theoretical deductions and experimental data

The purpose of this study was to compare the level of immunogold labeling of deplasticized acrylic sections and deplasticized epoxy sections. Pure protein gels of IgG, albumin and thyroglobulin were produced by glutaraldehyde fixation and embedded in non-crosslinked acrylic resin (Technovit 9100) and epoxy resin (Epon 812), respectively. Ultrathin sections of acrylic and epoxy resin were separately deplasticized in 2-methoxyethyl acetate (MEA) and sodium ethoxide. Quantitative immunogold labeling was performed with anti-IgG, anti-albumin and anti-thyroglobulin antibodies on sections of the corresponding protein gels. For all antibodies tested, the intensity of labeling for deplasticized acrylic sections was significantly higher (two to four times) than for the corresponding deplasticized epoxy sections. The results fit with a theoretically deduced relation: the quotient of the labeling of two deplasticized sections of different resins is equivalent to the square root of the quotient of the labeling of the similar sections not exposed to any kind of pre-treatment. The practical significance of the results is that immunolabeling of deplasticized non-crosslinked acrylic resin results in more intense immunogold labeling than deplasticized epoxy sections. Deplasticizing is most useful when the requirements for ultrastructural preservation according to conventional criteria are moderate. Our theoretically deduced results also indicate that deplasticized Technovit (or other non-crosslinked acrylic resins) sections will be significantly better suited for immunolabeling at the light microscopic level than deplasticized epoxy sections.

Fragmentation of the gastrodermis and detachment of zooxanthellae in symbiotic cnidarians: a role for hydrogen peroxide and Ca2+ in coral bleaching and algal density control

Coral bleaching involves the detachment of zooxanthellae and the simultaneous fragmentation of the gastrodermis. Results obtained with a cell permeant fluorescent probe for calcium ions (Ca2+) indicates that “thermal” bleaching is the result of a temperature related breakdown of the Ca2+ exclusion system. “Solar” bleaching, which takes place at lower temperatures and is driven by light, is the result of a build-up of photosynthetically produced hydrogen peroxide in the tissues. Gastrodermal tissue with its symbionts, scraped from between septa of corals, was observed under controlled conditions of high light and temperature. Pieces of gastrodermis round off, zooxanthellae move to the surface, protrude from the surface and after a delay, detach, surrounded by a thin layer of host cytoplasm, inclusions and plasma membrane. The higher the temperature and light level the shorter the delay and higher the rate of algal detachment. Fragmentation by the ballooning-out and detachment of small spheres of cytoplasm (bleb formation) takes place simultaneously. This is likely to be due to oxidation, by hydrogen peroxide (H2O2), of -SH groups on the cytoskeleton and its attachment to the plasma membrane. Ground, polished and stained thin acrylic resin sections reveal similar processes taking place in artificially bleached corals. Isolated zooxanthellae and whole corals are shown to release H2O2 in the light. This process of algal detachment and fragmentation that takes place at normal sea temperatures may underlie the mechanism limiting algal populations in the gastrodermis and may be localized to areas with a concentration of algae near the membrane. At above-normal temperatures under the synergistic effect of light and temperature, the rate of production of H2O2 exceeds the rate at which can it be lost by diffusion or destroyed and H2O2 accumulates. This results in damage to the calcium exclusion system, detachment of zooxanthellae into the coelenteron and fragmentation of the gastrodermis. Rev. Biol. Trop. 54 (Suppl. 3): 79-96. Epub 2007 Jan. 15.

Genital system development of Williamia radiata (Gastropoda, Siphonariidae)

The adult anatomy and development of the genital system of the limpet Williamia radiata (Siphonariidae, Gastropoda) was investigated by means of 3D computer reconstruction and visualization of serial resin and paraplast light microscopical sections. As is characteristic for siphonariid species, the adult genital system consists of a single duct, the spermoviduct leading from the nidamental glandular system to the anteriorly located genital atrium with opening. The copulatory complex as well as the bursa copulatrix also open into the common genital atrium. The genital system develops from three separate anlagen. The posterior one appears first at a body length of 0.7 mm and gives rise to the ovotestis and part of the hermaphrodite duct. The nidamental glandular complex, the fertilization pouch-spermatheca complex, part of the hermaphrodite duct, the posterior part of the spermoviduct, and the bursa copulatrix develop later from the pallial anlage. Finally, the anterior anlage is formed on the right side of the head and gives rise to the genital atrium, the copulatory complex, and the anterior spermoviduct. This formation of the genital system from three, locally separated anlagen, differs strikingly from that of most other species of the Euthyneura. In both the Nudibranchia (Opisthobranchia) and the Stylommatophora (Pulmonata) development proceeds from a single site. We regarded this as a secondary condition as a result of derived features like heterochronies in development in these taxa. Comparison of development with that of other species of the Pulmonata allows conclusions on homology. The homology of the bursa copulatrix within the pulmonates is confirmed. The two separate chambers inside the spermoviduct of W. radiata correspond to oviduct and vas deferens of the freshwater Basommatophora.

Donor corneas for transplantation: a scanning electron microscopic study of the epithelium

Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem. A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy. Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2-3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells. The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

Utilization of fresh human tympanic membranes for structural analysis and cytokeratin immunocytochemistry implementing resin techniques

Conclusions. The results of this study have demonstrated for the first time that tympanic membrane (TM) structure is preserved following removal of fresh, normal tissue from patients undergoing surgery. Greater clarity has been demonstrated using resin sections than in previous studies on paraffin sections. Of particular note, cytokeratin (CK) immunocytochemistry was successfully performed on resin sections, which has not been previously reported. This may have potential applications for future work involving tissues that express CKs. Objectives. To analyse the structure of normal, fresh human TM specimens after surgical removal and to evaluate their CK immunocytochemistry using resin techniques, neither of which have been demonstrated previously. Material and methods. Seven TM specimens were removed during surgery and then preserved in a modified Karnovsky's fixative. Semi-thin and thin sections were examined by means of light and electron microscopy, respectively. For comparison purposes, paraffin block-embedded specimens were also sectioned. CK immunocytochemistry was performed on semi-thin sections using standard immunoperoxidase techniques, with expression being demonstrated using light microscopy. Results. The three-layer architecture of the TM was preserved. The morphology of the TM was vastly superior in the semi-thin resin sections than in the thicker paraffin sections. The outer, middle and inner layers were clearly demonstrated. The integrity of the outer epithelial layer was maintained, with an outer keratinizing stratum corneum and underlying stratum granulosum, stratum spinosum and stratum basale layers resting on the basal lamina. The thin inner mucosal layer was also viable, consisting of simple squamous or cuboidal cells. Preservation of the middle lamina propria was achieved, with demonstration of the outer radial and inner circular fibres. CK immunocytochemistry utilizing resin techniques provided excellent staining of CK 7 and 8 in the inner layer, with positive staining of CK 5 and 10 in the outer layer.

Immunohistochemical expression of keratins 13 and 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae

We examined the immunofluorescence of keratins 13 (K13) and 14 (K14) and differential interference contrast (DIC) images during the morphogenesis of filiform papillae and the keratinization of the lingual epithelium of rats on semi-ultrathin sections of epoxy resin-embedded samples by laser-scanning microscopy. We also examined semi-ultrathin sections of epoxy resin embedded, toluidine blue stained samples by light microscopy to obtain details of cell histology and morphology. No immunoreactivity specific for K13 and K14 was detected on the lingual epithelium of foetuses on days 13, 15 and 17 after conception (E13, E15 and E17), during which time the number of layers of cuboidal cells in the lingual epithelium increased from one to several. Immunoreactivity specific for K13 and K14 was first detected on the lingual epithelium of foetuses on E19. The immunoreactivity specific for K13 appeared in the suprabasal cells of the papillary and interpapillary cell columns and immunoreactivity specific for K14 was detected in the basal and suprabasal cells of the papillary and interpapillary cell columns. The lingual epithelium was composed of stratified squamous cells. The rudiments of filiform papillae were compactly arranged and interpapillary cell columns were very narrow. Filiform papillae developed gradually from postnatal day 0 (PO) to 21 (P21). The width of interpapillary spaces also increased during this period. Immunoreactivity specific for K13 and K14 was distinct at all postnatal stages examined. Thus, the patterns of immunoreactivity of K13 and K14 differed as the filiform papillae developed.

Quantitative immunoelectron-microscopic analysis of the type IV collagen a1-6 chains in the glomerular basement membrane in childhood thin basement membrane disease

Thin basement membrane disease (TBMD) is characterized histologically by diffuse thinning of glomerular basement membrane (GBM). Although recent genetic analysis has shown that TBMD might be included within type IV collagen disorders, conventional immunohistochemical studies demonstrated normal labeling of type IV collagen alpha chains in the GBM. We have, however, successfully used confocal laser scanning microscopy to demonstrate a significantly reduced signal of type IV collagen alpha5 chain (alpha5(IV)) along capillary walls in TBMD. In order to further understand the association of type IV collagen with TBMD, we used immunoelectron microscopy to examine renal biopsies from 6 children with TBMD and six control children with minimal change nephrotic syndrome. Ultrathin sections of LR gold resin were incubated with a rat monoclonal antibody against human alpha1(IV), alpha2(IV), alpha3(IV), alpha4(IV) alpha5(IV) or alpha6(IV) followed by colloidal gold conjugated goat anti-rat IgG. After taking electron micrographs, the labeling was quantitatively evaluated in the area occupied by the segments of basement membrane. The basement membrane was divided into three equal segments viz. subepithelial side, central portion and subendothelial side. In control subjects, the number of gold particles for alpha1(IV) or alpha2(IV) was significantly greater in the subendothelial side and central portion than in the subepithelial side of the GBM, whilst alpha3(IV), alpha4(IV) or alpha5(IV) labeling was significantly more prominent in the central portion compared to the subepithelial and subendothelial side of the GBM. TBMD samples showed a similar distribution pattern except that the subepithelial side and central portion of the GBM had a significantly reduced amount of alpha5(IV) antigen compared to control subjects. This is the first report demonstrating a diminished labeling intensity of alpha5(IV) in the central portion and subepithelial side of the GBM in renal biopsy specimens from patients with TBMD. These findings suggest that an abnormality of alpha5(IV) might possibly be associated with the pathogenesis of TBMD.

Direct Labeling of Polyphosphate at the Ultrastructural Level inSaccharomyces cerevisiaeby Using the Affinity of the Polyphosphate Binding Domain ofEscherichia coliExopolyphosphatase

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.

Immunohistochemical Staining of Epoxy Resin Sections of Peripheral Nerve

The authors describe a simple and cost-effective method of immunostaining semithin epoxy resin sections of peripheral nerve for light microscopy with antibodies to myelin protein zero, peripheral myelin protein 22, myelin basic protein, and neurofilament protein 200 using a combined technique of surface etching with sodium ethoxide and heat-induced antigen retrieval.

Cytocompatibility Studies of a Novel Bioactive Glass Coated Porous Hydroxyapatite Bioceramic for Use as a Bone Substitute

Cytotoxicity and cytofunctionality of the in-house prepared novel bioactive glass coated hydroxyapatite (BGHA) is described here as a first step in characterizing the biocompatibility of the material for orthopaedic application. Sintered hydroxyapatite was coated with bioactive glass by a sol-gel method. The BGHA granule was 2 - 4 mm in size with a pore size in the range of 50 – 150 µm. FT-IR spectrum depicted the functional groups and XRD spectrum defined the crystallinity of the material. MTT assay proved high cellular activity and morphologically the cells showed normal proliferation with the expression of actin and vimentin network in the cytoplasm. In the resin sections, cells were found located on the walls of the pores while in osteoblast-cultured granules, cellular matrix was also observed along the pore interconnections. Recrystallization on the surface of the cultured granules did not support the adhesion of cells. The electron diffraction pattern displayed the high crystallinity of the raw granule while a diffused pattern of amorphous apatite like crystallites was observed for the cultured granules. High calcium and low phosphorous concentration was observed in the medium of the cells simultaneously with ALP and osteocalcin production. This pre-clinical evaluation proved BGHA, as a promising material for bone scaffolds.

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.

119. Over-expression of activin βC in vivo reveals a role in male fertility

Introduction: Activin βC subunit antagonises the formation and bioactivity of activin A via intracellular heterodimerisation and decreases activation of the activin signalling pathway (Mellor et al. 2003). Therefore the activin βC subunit heterodimers provide a new mechanism of regulating activin levels. Vedja and colleagues over-expressed the activin βC subunit in malignant liver cell lines, which subsequently displayed inhibition of cell proliferation and induction of apoptosis (Vedja et al. 2003). Conversely, Wada et al. demonstrated that treatment with hr-activin C stimulates growth of a liver cell line (Wada et al. 2004). These recent (and contradictory) reports about the in vitro activity of activin βC have prompted us to examine the in vivo role of activin βC by creating a transgenic mouse over-expressing the βC activin subunit. Methods: The full-length human cDNA under the control of a CMV promoter was incorporated into the genome of three founder C57/B6 mice. Genotyping was performed by both Southern and PCR. Mice were monitored weekly and culled at 14–16 weeks (adult). Blood was collected by cardiac puncture, organs were weighed and a portion fixed in Bouin’s or frozen for subsequent RNA and protein extraction. Daily sperm production (DSP) was determined by standard methods. Sertoli and germ cell number will be determined using the optical disector (sic) stereological technique in Bouin’s fixed resin sections. Proliferation and apoptosis will be examined using PCNA and TUNEL respectively. Activin A was assessed by ELISA, while FSH, LH, follistatin and total inhibin were determined by RIA. Results and conclusions: Over-expression of activin-βC resulted in decreased circulating activin A (P < 0.005 TG1, P < 0.05 TG2 and P = 0.08 TG3), a progressive age-related decrease in litter sizes (9.3 WT v. 6.3 TG1, 5.8 TG2 and 4.5 TG3; P < 0.005 v. WT) and testicular DSP (P < 0.05). These data support the hypothesis that βC is a novel in vivo regulator and is the first indication of a role for activin-βC in male fertility. This novel mouse model will significantly advance our understanding of the in vivo role of activin-βC. (1)Mellor et al. (2003). Endocrinology 144, 4410–4419.(2)Vejda et al. (2003). Carcinogenesis 24, 1801–1809.(3)Wada et al. (2004). Am. J. Physiol. Endocrinol. Metab. 287, E247–E254.

Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen

Mitochondrial DNA in the male reproductive cells of rapeseed (Brassica napus L.) was monitored by fluorescence microscopy of Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide. Mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen. This result corresponds well with the maternal inheritance of mitochondria in rapeseed determined by previous genetic analyses. To better characterize the mode of inheritance of the mitochondrial linear plasmid in rapeseed, which is transmitted through pollen, we analyzed by indirect immunofluorescence microscopy the expression and localization of ORF6 protein, a putative RNA polymerase encoded by the plasmid. ORF6 protein was expressed in mature pollen and specifically localized in the cytoplasm of sperm cells in the mature pollen. This suggests that the genes encoded by the plasmid DNA are transcribed in the mature pollen by its own RNA polymerase (ORF6 protein) and that the gene expression in the generative cells may be needed for transmission of plasmid DNA through the pollen.

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica

Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4',6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.

Clinical and histopathological progression of lesions in lupus-prone (NZB×NZW) F 1 mice

This study was conducted to review the progression with age of renal and other changes in (NZB×NZW) F 1 females obtained from a commercial colony, housed under SPF conditions. Sixty-two mice were given food and water ad libitum and sacrificed at intervals up to 47 weeks of age. Routine haematology and urinalysis were performed at regular intervals. The serum levels of IgA, IgG, IgM and antinuclear antibody levels were monitored along with urinary protein. Major organs and tissues from all animals were preserved and examined. Special attention was paid to the kidneys: in addition to routine 4-μm paraffin sections, 1-μm resin sections were prepared and examined. Positive levels of urinary protein (>1 g/L) were only found in 50% of the animals, even at 32 weeks of age. Histopathologically, the expected type of glomerular damage was present; but the incidence and severity of the change were low, with only 33% of the mice affected at 32 weeks of age. Most mice had fatty vacuolation in the liver. At 24 weeks of age, 50% of the mice sacrificed had haemorrhagic degeneration of the liver. This was not seen at later time points. Only two mice survived until 47 weeks of age. In summary, these findings were of the expected type, but at a much lower incidence and severity than formerly reported. The background of the strain and the experimental conditions are of great importance in the progress of these renal lesions. The lack of homogeneity of the renal changes would make it difficult to determine any influence of xenobiotics on the progress of autoimmune diseases.