Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

An automated technique for double staining mouse fetal and neonatal skeletal specimens to differentiate bone and cartilage

Historically, some fetuses for regulatory developmental toxicity studies have been stained with alizarin red S and cleared with glycerol to visualize the ossified portion of their skeletons. Interest in examining cartilage arose owing to its inclusion in some regulatory guidelines. Methods for double staining rat skeletons have been published previously. The method described here for staining mouse skeletons is fully automated and uses alizarin red S to stain bone and Alcian blue to stain cartilage. Pregnant mice (Crl:CD1) were euthanized on gestation day 18 to obtain fetal specimens. Day 0 post-partum mouse pups also were stained. Our method was developed using the Shandon Pathcentre , which is a fully enclosed automated staining system that allows staining to be carried out at 30° C with a final clearing at 35° C. Our method uses the same solutions as for fetal rat processing, but with reduced time periods for the smaller size of mice vs. rat specimens. Staining, maceration and clearing of the specimens requires approximately 2 days. The time required of laboratory personnel, however, is minimal, because all solutions are changed automatically and the specimens do not require examination or removal from the processor until processing is complete. After processing, the specimens are suitable for immediate assessment of bone and cartilage. A mouse developmental toxicity study using 20 animals/group and approximately 10 fetuses/animal could be processed in only three runs using one machine.     

High-throughput staining for the evaluation of fetal skeletal development in rats and rabbits

Typical developmental toxicity studies require the assessment of fetal skeletal development. Regulatory guidelines require the assessment of bone ossification and indicate preferences for an assessment of both ossified bone as well as cartilaginous elements. Current manual methods to process fetuses for skeletal examination, whether single or double staining, are laborious and time consuming, and ultimately extend the time before study interpretations. There is a definite need for a quick and efficient, yet reliable, procedure to generate stained fetal skeletons for analysis. A non-automated high-throughput method for single and double staining rat and rabbit fetuses for skeletal evaluations is described, which results in excellent quality specimens ready for evaluations in approximately 3 days for rats and 7 days for rabbits.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Ultrastructure of the basement membrane and its precursor in developing rat submandibular gland as shown by alcian blue staining

Summary The ultrastructure of the epithelial basement membrane and membrane precursor was studied in rat submandibular rudiment and a model system of the reconstructed basement membrane, by transmission electron microscopy following alcian blue staining. Directly beneath the epithelial plasma membrane, a meshwork layer was found to consist of anastomosing thin fibers arranged as a three-dimensional meshwork (100–400 nm in thickness). Straight strands (5–10 nm in diameter) could sometimes be seen to pass through the meshwork. Adjacent to this layer, a coarse network composed of threads (20–40 nm in diameter) connected the meshwork layer with collagen fibers of the underlying connective tissue. The earliest precursors recognized in the reconstruction-model system were part of the fine-meshwork structure, and showed this structure to be a fundamental component of the basement membrane.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

The protein,lipid and carbohydrate composition of protein bodies from Lupinus angustifolius seeds

Protein bodies isolated from dehulled seeds of Lupinus angustifolius (cv New Zealand Bitter Blue) contained 73 % protein, of which 78 % were globul     

α-D-mannosidase and α-D-galactosidase from protein bodies of Lupinus angustifolius cotyledons

Two forms of p-nitrophenyl α-D-mannosidase and p-nitrophenyl α-D-galactosidase were purified from the protein bodies of mature Lupinus angustifolius seeds. A MW of 300 000 was calculated for both α-mannosidase A and B with K_m = 1.92 and 2.70 mM and activation energies of 10.9 and 10.8 kcal/mol, respectively. α-Galactosidase I and II had MWs of 70800 and 17000 with K_m = 0.282 and 0.556 mM and activation energies 17.7 and 11.5 kcal/mol, respectively. The enzymes had acid pH optima and were inhibited by various metal ions, carbohydrates and glycoproteins. They were able to release free sugar from several putative natural substrate oligosaccharides and the Lupinus storage glycoprotein, α-conglutin.     

The polypeptide composition of highly purified prolamellar bodies and prothylakoids from wheat (Triticum aestivum) as revealed by silver staining

The inner membranes from wheat ( Triticum aestivum L. cv. Walde, Weibull) etioplasts were separated by density centrifugation. The etioplasts were broken by osmotic shock and the inner membranes were split by the sheering forces when pressed through a syringe needle. Membrane fractions representative of prolamellar bodies and prothylakoids, respectively, were achieved by separation on a 20–50% continuous sucrose density gradient followed by different purification procedures. The membrane contents of the isolated fractions were characterized by low temperature fluorescence spectra, sodium dodecyl sulphate polyacrylamide gel electrophoresis and electron micrographs. The prolamellar body and the prothylakoid fractions had a fluorescence emission ratio 657/633 nm of 18 and 0.9, respectively. The main part of the total amount of PChlide was found in the prolamellar body fraction. The electrophoretograms stained with Coomassie Blue showed the presence of mainly two polypeptides. The NADPH-protochlorophyllide oxidoreductase was the dominating polypeptide in the prolamellar body fraction, and the α and β subunits of the coupling factor 1 of chloroplast ATP synthase the dominating polypeptides in the prothylakoid fraction. Silver staining revealed at least 4 additional prominent bands with molecular weights of 86, 66, 34 and 28 kDa. The polypeptide composition of the prolamellar body is thus more complex than earlier judged after Coomassie Blue staining. The function of these polypeptides is unknown, but the knowledge of their presence is important in understanding the formation and function of the prolamellar body.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Nucleolus-like bodies in the pineal gland of the Djungarian hamster (Phodopus sungorus)

Summary Light- and electron-microscopic observations on the pineal gland of Phodopus sungorus revealed intracytoplasmic inclusions resembling nucleolus-like bodies similar to those found in other regions of the central nervous system. Bernhard's EDTA method was used to confirm that these inclusions were nucleolus-like bodies. These structures were rarely found in pinealocytes of sexually active longday animals, whereas large numbers of them were observed in pinealocytes of sexually quiescent short-day animals. Nucleolus-like bodies may therefore be involved in pineal secretion.     

Efficient solubilization of inclusion bodies

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.     

Histochemical characterization of the lead intranuclear inclusion bodies

A battery of histological and histochemical techniques was applied on the lead intranuclear bodies that have resuted in the kidneys of adult Wistar male rats receiving lead acetate in their diet to determine their nature. The intranuclear inclusion bodies have stained strongly with xanthene, anthraquinone, and trisulfonated basophilic dyes and weakly with dyes containing both positive and negative radicals, and they have responded negatively to acidophilic cationic dyes. They have also reacted positively to proteins and lead stains, but weakly to lipid stains, and negatively to Feulgen and methyl green pyronin techniques. The intranuclear bodies proved to be lead lipoprotein complexes containing sulfyhydryl groups and are basic in nature with orthochromatic, eosinophilic, argyrophilic, osmophilic, fuchsinophilic, and sudanophilic characteristics.     

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or α1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.     

水稻籽粒胚乳的石蜡切片方法改良及其结构发育观察

以杂交水稻保持系‘岗46B’授粉后不同天数的籽粒为材料,利用PAS反应原理,通过对传统石蜡切片技术中材料固定、染色、脱水、浸蜡等环节的改进,即采用FAA和FPA相结合方式固定材料,先用热配法席夫(Schiff)试剂进行整染,再以稀释和氨水蓝化后的埃利希氏(Ehrlich)苏木精复染,中间以70%、83%、95%质量分数梯度乙醇进行脱水和复水处理,渐进式浸蜡与把握时间等实验环节,对水稻胚乳结构发育进行观察研究,结果授粉后第2~10天籽粒胚乳的石蜡切片完整,且染色分明,结构显示完整清晰。观察发现,随着授粉后天数推移,胚乳内部逐渐由游离胚乳核期向细胞化过渡,细胞层数增加并趋向充满子房,淀粉粒开始累积,胚乳细胞进一步增大。授粉后第5~10天是胚乳细胞淀粉粒从开始到大量累积的重要时期,同时胚乳细胞向无核化发展,不同部位胚乳细胞和淀粉粒的排列分布也逐渐表现出一定的差异。表明改良后的石蜡切片方法适合于授粉后第0~10天的水稻胚乳结构发育研究。     

High-throughput automated refolding screening of inclusion bodies

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.