Cytochemical Studies Research Articles

On the origin of nuclear vacuoles in spermatozoa a fine structural and cytochemical study in mice.

Morphological and cytochemical changes in nucleoli of spermatids of the mouse during spermiogenesis were examined. Thus, it could be shown that the nucleolar derivatives of the elongation-phase, clusters of coiled fibers that are bordered with spheroidal bodies, show comparable chemical properties as the precursor structure, the fibrillar component, which is known for its transcriptional activity. With progressing development, these nucleolar derivatives continually decrease in size and density, preserving their chemical properties. They persist far beyond into the maturation-phase and will decrease at the time chromatin condensation appears finished. There only remain the spaces in which they formerly existed, now called nuclear vacuoles. A possible function of nuclear vacuoles with respect to chromatin decondensation during the process of fertilization is mentioned.

Cytochrome Oxidase and Lactate Dehydrogenase Activities in Peroxidized Rabbit Epididymal Spermatozoa/Cytochromoxydase- und Lactatdehydrogenase-Aktivität bei peroxydierten Kaninchen Spermatozoen aus dem Nebenhoden

We report a quantitative cytochemical study on cytochrome oxidase and lactate dehydrogenase activities on rabbit epididymal spermatozoa during spontaneous lipid peroxidation. Our data show that during aerobic incubation both in NTP and KTP media the sperm cytochrome oxidase activity undergoes a significant decrease. The lactate dehydrogenase activity shows different cytochemical patterns in comparison between the two media considered. Such activity significantly increases in rabbit spermatozoa suspended in NTP medium from the first until the sixteenth hour of incubation time. At the following times the lactate dehydrogenase activity significantly declines showing yet until the later times of incubation integrated optical density values fairly high. During the whole period of the aerobic incubation, the spermatozoa suspended in medium KTP show lactate dehydrogenase integrated optical density values which not significantly differ from those of the control in spite of an initial enhancement from the first until the thirteenth hour of the experimental treatment.

An Ultrastructural and Cytochemical Study of Candida aIbicans after in Vitro Treatment with Imidazoles

Summary The ultrastructural dianges in Candida albicans after exposure in vitro to different doses of clotrimazole and miconazole have been investigated. The changes obtained with low dose levels of both drugs concerned preferentially the cell periphery with eventual hyperactivity of the central vacuolar system. With high dose levels, on the other hand, the morphologic changes differed markedly between the two drugs with respect to the number of cells involved, the intensity of the lesions and their subcellular location. This discrepancy in response of C. albicans towards high doses of the imidazoles, shed some doubts on the previously propagated mode of action of these drugs. Additionally to the morphological description of the involutionary processes in C. albicans, the behavior of some oxidative and peroxidative enzymes has been investigated after miconazole treatment. In the light of these findings, an alternate hypothesis is formulated proposing hydrogen peroxide intoxication as the cause of cell death. Zusammenfassung Die ultrastrukturellen Veränderungen bei Candida albicans nach Behandlung in vitro mit verschiedenen Dosen von Clotrimazol und Miconazol wurden untersucht. Die bei niedrigen Dosen beider Arzneimittel entstandenen Veränderungen beziehen sich vorzugsweise auf die Zellperipherie, mit möglicher Hyperalttivität des zentralen Vakuolensystems. Andererseits zeigten beide Arzneimittel bei hohen Dosen cine stark verschiedene morphologische Wirkung in Hinsicht auf die beteiligte Zellzahl, den Läsionsgrad und ihre subzelluläre Lokalisierung. Infolge dieser Reaktionsverschiedenheit von C. albicans gegenüber hohen Dosen der Imidazole kommt die früher verbreitete Wirkungsweise dieser Arzneimittel wieder in Frage. Außer der morphologischen Beschreibung der Zurüdrbildungsprozesse bei C. albicans wurde das Verhalten einiger oxydativer und peroxydativer Enzyme nach Mico-nazol-Behandlung geprüfk. In Anbetracht dieser Befunde wird eine neue Hypothese formuliert, nach der der Zelltod auf Wasserstoffperoxydvergiftung zurückgehen würde.

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.

Cytochemical and biochemical study of the cellulose of Sium latifolium leaves under the influence of a water deficit

The cellulose distribution in the epidermal and mesophyll cells of Sium latifolium leaves at the flowering and seedling phases is studied with the use of confocal laser scanning microscopy. Pascal is used to determine that the relative content of cellulose in cell walls is dependent on the leaf tissue type, the plant ontogenesis phase, and the growing conditions. It is revealed through the use of biochemical techniques that a moderate water deficit decreases the amorphous cellulose content and increases the crystalline cellulose content during plant growth.

On the nucleolar and cytoplasmic RNA density during "cell dedifferentiation" represented by blastic transformation of human mature T lymphocytes - a cytochemical study.

The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimens stained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolar and cytoplasmic RNA's were visualized using a simple cytochemical method followed by computer assisted densitometry and size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blastic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells were characterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratio of the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably in the G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenomenon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA densitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at the single cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was much larger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly to their size.

Cytochemical study of the distribution of RNA and DNA in the synaptonemal complex of guinea-pig and rat spermatocytes.

The distribution of DNA and RNA in the synaptonemal complex and related structures, was studied using high resolution cytochemical methods and in situ hybridization, in guinea pig and rat testis. Serial sectioning demonstrates that frequently the formation of the synaptonemal complex (SC) occurs without a previous development of isolated chromosomal axes. The lateral elements of the forming SC are in continuity with pairs of DNA-containing thin filaments. These chromatin filaments fold in numerous short loops just before incorporating to the lateral elements. Some of these loops are included in the ribbon-like structure of the lateral elements of the mature SC. We propose that these short loops contain the DNA attachment sequences associated with the proteins of the LE. During the formation of the SC one of the two chromatin filaments incorporates at the central surface of the forming lateral element (LE) and the other is located at the external side of the LE. This unexpected distribution does not correspond to the pair of thick filaments previously discerned in structure of the LE. The presence of RNA associated with the DNA-containing thin filaments, as well as with the axial chromatin elements of the forming SC, may be related with the transcription occurring during meiotic prophase, specially during zygotene stage. We propose that RNA is involved in a still uncharacterized process essential for pairing.

Assembly of the subcellular parts of Bryopsis hypnoides Lamouroux into new protoplasts

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20A mu mol/(m(2) s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation.

Cytochemical studies on tumor cells. III. Two types of tumor cells as revealed by the acid hematein test.

Cytochemical studies on tumor cells. III. Two types of tumor cells as revealed by the acid hematein test.

Haematology, cytochemical and ultrastructural characteristics of blood cells in leopard (Panthera pardus)

The leopard (Panthera pandus) is an endangered Asian big cat found in Thailand and also listed in the CITES, Appendix 1. Blood samples from 17 leopards (six males and 11 females) were collected from the cephalic vein for haematology, cytochemical and ultrastructural studies. Red blood cells (RBCs) were slightly anisocytosis, ranged 5–6.5 μm with 5.6 μm mean diameter. They were easy to form rouleaux, blunt end crenation and some RBCs contained Heinz bodies. The male leopards had a significantly higher packed cell volume (39.3 ± 7.5%) values and absolute eosinophil number (1.658 ± 1.483 × 109/l) than the females (30.7 ± 3.2%; 0.965 ± 0.611 × 109/l). The cytochemical results were: sudanophilia and myeloperoxidase in neutrophils and some monocytes; nonspecific esterase (alpha-naphthyl acetate esterase, ANAE) in the granules of eosinophils, some lymphocytes, some monocytes and platelets; beta-glucuronidase (βG) in granules of basophils, monocytes and some lymphocytes. The ANAE and βG reaction were detected inter-granular of eosinophils. More detailed morphological aspects of all blood cells were observed by means of scanning electron microscope and transmission electron microscope (TEM). The many round granules and homogeneous content under TEM were characteristics of leopard eosinophils. The engulfed RBCs by neutrophils were detected under TEM in one male leopard. This study provides a guide for the haematology, identification of the morphology, cytochemistry and ultrastructure of blood cells in leopards that is useful for zoological veterinarians in leopard conservation.

Metabolic DNA as the Origin of Spontaneously Released DNA?

A DNA fraction is spontaneously released from living, but not dead or dying, human, other mammalian, avian, amphibian, plant, and prokaryote cells. The spontaneously released DNA fraction has been shown to be (a) present in both actively dividing and nondividing, differentiated cell populations; (b) labile; (c) associated with DNA-dependent RNA or DNA polymerase; (d) associated with an RNA fraction; and to have (e) a lower molecular weight than the typical genetic DNA fraction; and (f) Alu repeat sequences in increased proportions compared to a unique gene in plasma/serum. On the other hand, early autoradiographic and biochemical and quantitative cytochemical and cytophysical studies on DNA permitted the identification of a DNA fraction which was (1) present in both actively dividing and nondividing, differentiated cell populations; (2) labile; and (3) had a lower molecular weight than the typical genetic DNA fraction. This DNA fraction was termed metabolic DNA (m-DNA) and was proposed as possibly forming extra gene copies for the rapid production of m-RNA, to be destroyed subsequently. Therefore, we suggest that the metabolic DNA fraction might represent the precursor to the formation of the spontaneously released DNA fraction.

Fat body cells of female reproductive castes of Attini ants (Hymenoptera: Formicidae): An ultrastructural and chemical analysis

The ultrastructural study on the fat body of gynes (virgin queens) of the basal ant species Cyphomyrmex rimosus and Mycetarotes parallelus and the derived Acromyrmex disciger and Atta laevigata queens showed vesicular rough endoplasmic reticulum, Golgi complex, and mitochondria in trophocytes, suggesting the involvement of these cells in protein synthesis, in addition to digestive vacuoles associated with the digestion of endocytosed compounds or rejected cell organelles. Oenocytes, another cell type present in the fat body of these species exhibit mitochondria, digestive vacuoles, and vesicles, indicating a mobilization of compounds by these cells. In A. laevigata, oenocytes also exhibited large storage sites of glycogen, in addition to a well-developed vesicular rough endoplasmic reticulum, suggesting an intensive participation of these cells in protein synthesis. The ultrastructural cytochemistry study also revealed electrodense granules of basic proteins present throughout the cytoplasm of trophocytes. The same was observed in oenocytes, although with smaller amounts of proteins. In the cytoplasm of trophocytes and oenocytes were also found droplets or electrodense granules of lipids. In oenocytes of A. disciger and in trophocytes of A. laevigata, lipids were observed in mitochondria, suggesting that this organelle might be a site of synthesis of these compounds. The chemical analysis of lipids revealed that in gynes, the main compounds present in fat body cells were saturated fatty acids, while in queens, saturated as well as unsaturated fatty acids were found. In conclusion, the present study showed that the fat body cells of gynes and queens, in general, maintained the same compounds and original features through the evolution process of the Attini tribe.

A simple technique to minimize heat damage to specimens during thermal polymerization of LR White in plastic and gelatin capsules

London Resin (LR) White is a commonly used resin for embedding specimens to be used for immuno- and/or cytochemical studies. In some instances, due to either the properties of the specimen or the availability of various reagents and equipment, it becomes necessary and/or more convenient to polymerize LR White using heat rather than chemical accelerators or UV light. It is known, however, that heat can reduce or even eliminate the anti genicity of the tissue being embedded. It is therefore desirable to polymerize specimens at the lowest temperature possible and to remove the specimens from the oven as soon as polymerization is complete. We have developed a technique that provides a visual marker that allows the exothermic polymerization of LR White to be monitored, thus minimizing the amount of time a specimen must stay in the oven while excluding oxygen from capsules of polymerizing LR White.

The herbicide flamprop‐M‐methyl has a new antimicrotubule mechanism of action

The herbicidal mode of action of flamprop-M-methyl [methyl N-benzoyl-N-(3-chloro-4-fluorophenyl)-D-alaninate] was investigated. For initial characterization, a series of bioassays was used, which indicated a mode of action similar to that of mitotic disrupter herbicides. Cytochemical fluorescence studies, which included monoclonal antibodies against polymerized tubulin, were applied to elucidate effects on mitosis and microtubule assembly in maize roots. When seedlings were root treated with 50 microM of flamprop-M-methyl, cell division activity in meristematic root tip cells ceased within 4 h. The compound severely disturbed the orientation of spindle and phragmoblast microtubules, leading to defective spindle and phragmoblast structures. Cortical microtubules were only slightly affected. In late anaphase and early telophase cells, phragmoblast microtubules were disorganized in multiple arrays that hampered regular cell plate deposition in cytokinesis. Microtubules of the spindle apparatus were found attached to chromosomal kinetochores, but did not show regular organization associated with a zone of microtubule-organizing centres at the opposite ends of the cell. On account of this loss of spindle organization, chromosomes remained in a condensed state of prometaphase or metaphase. Unlike known microtubule disrupter herbicides, flamprop-M-methyl and its biologically active metabolite flamprop did not inhibit soybean tubulin polymerization to microtubules in vitro at 50 microM. In contrast, soybean plants responded sensitively to the compounds. The results indicate that flamprop-M-methyl is a mitotic disrupter herbicide with a new antimicrotubule mechanism of action that affects orientation of spindle and phragmoblast microtubules, possibly by minus-end microtubule disassembly.

Thematic Review Series: Sphingolipids. Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.

Chemical detection of the proteins and lipids in the fat body cells from workers of Attini ants (Hymenoptera: Formicidae)

The ultrastructural cytochemistry study and the chemical analysis carried out to examine the fat body of workers of the basal Attini Cyphomyrmex rimosus and Mycetarotes parallelus and the derived Acromyrmex disciger and Atta laevigata revealed very electrondense protein granules of varied sizes and shapes distributed throughout the cytoplasm of parietal and perivisceral trophocytes and oenocytes. Lipids were present in the cytoplasm of trophocytes as very electrondense granules. In M. parallelus, A. disciger, and A. laevigata, lipids were present as droplets. In parietal as well as perivisceral oenocytes, lipid granules were very electrondense. In A. laevigata, lipids were present as droplets. The chemical analysis using gas chromatography showed that the main lipid compounds present in trophocytes and oenocytes were saturated (myristic, palmitic, and stearic) and unsaturated fatty acids (oleic and linoleic). Also, in basal species, lipid compounds consisted of mainly saturated fatty acids, while in derived species saturated as well as unsaturated fatty acids were observed.

Structural, biochemical, and physiological characterization of photosynthesis in two C4 subspecies of Tecticornia indica and the C3 species Tecticornia pergranulata (Chenopodiaceae)

Among dicotyledon families, Chenopodiaceae has the most C(4) species and the greatest diversity in structural forms of C(4). In subfamily Salicornioideae, C(4) photosynthesis has, so far, only been found in the genus Halosarcia which is now included in the broadly circumscribed Tecticornia. Comparative anatomical, cytochemical, and physiological studies on these taxa, which have near-aphyllous photosynthetic shoots, show that T. pergranulata is C(3), and that two subspecies of T. indica (bidens and indica) are C(4) (Kranz-tecticornoid type). In T. pergranulata, the stems have two layers of chlorenchyma cells surrounding the centrally located water storage tissue. The two subspecies of T. indica have Kranz anatomy in reduced leaves and in the fleshy stem cortex. They are NAD-malic enzyme-type C(4) species, with mesophyll chloroplasts having reduced grana, characteristic of this subtype. The Kranz-tecticornoid-type anatomy is unique among C(4) types in the family in having groups of chlorenchymatous cells separated by a network of large colourless cells (which may provide mechanical support or optimize the distribution of radiation in the tissue), and in having peripheral vascular bundles with the phloem side facing the bundle sheath cells. Also, the bundle sheath cells have chloroplasts in a centrifugal position, which is atypical for C(4) dicots. Fluorescence analyses in fresh sections indicate that all non-lignified cell walls have ferulic acid, a cell wall cross-linker. Structural-functional relationships of C(4) photosynthesis in T. indica are discussed. Recent molecular studies show that the C(4) taxa in Tecticornia form a monophyletic group, with incorporation of the Australian endemic genera of Salicornioideae, including Halosarcia, Pachycornia, Sclerostegia, and Tegicornia, into Tecticornia.

Host plant resistance against broomrapes (Orobanche spp.): defence reactions and mechanisms of resistance

AbstractOver 4000 species of angiosperms are able to directly invade and parasitise other plants, but only very few are weedy and parasitise cultivated plants. Together with the witchweeds (Striga spp.) and dodders (Cuscuta spp.), the broomrapes (Orobanche spp.) affect important crops causing complete yield loses with severe infestations. Genetic resistance to parasitisation remains as one of the most desirable components in an integrated control strategy. However, breeding for resistance is a difficult task and many aspects of the host/parasite interaction remain unknown. In the present work, we review the cytological and cytochemical studies investigating the mechanisms of resistance involved in the process leading to an incompatible host–parasite interaction. We have attempted to identify the main gaps and problems related to such studies with parasitic plants and present an in‐depth review of histochemical techniques used to assess mechanisms of resistance against these parasitic plants. Furthermore, we discuss future research directions and novel techniques and finally, how such techniques can be applied and incorporated within a breeding programme.

Ultrastructure of Enteromyxum leei (Diamant, Lom, &amp; Dyková, 1994) (Myxozoa), an Enteric Parasite Infecting Gilthead Sea Bream (Sparus aurata) and Sharpsnout Sea Bream (Diplodus puntazzo)

The ultrastructure of the developmental stages of the myxozoan Enteromyxum leei parasitizing gilthead seabream (Sparus aurata) intestine and sharpsnout sea bream (Diplodus puntazzo) intestine and gallbladder are described. The earliest stage observed was a small dense trophozoite located among enterocytes. Proliferative stages, observed intercellularly in the epithelium of the intestine and gallbladder as well as in the lumen, possessed the typical cell-in-cell configuration throughout their development. Secondary cells were seen undergoing division within a common vacuolar membrane that also encompassed pairs of tertiary cells. Cytochemical studies showed that primary cells stored mainly lipids whereas secondary cells stored abundant beta-glycogen granules. Sporogonic development resembled that described for other disporous myxozoans. Within sporogonic stages, nonsporogonic secondary cells were observed accompanying two developing spores. Mature spores had a binucleated sporoplasm in which glycogen stores were abundant and no sporoplasmosomes were found. Our observations are discussed in relation to our knowledge on other myxozoans of the genus Enteromyxum.

Ultrastructural and cytochemical studies on the infection process of Sclerotinia sclerotiorum in oilseed rape

Stem rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is one of the major diseases of oilseed rape worldwide. The infection process of S. sclerotiorum in leaves and stems and the alterations of cell wall components in the infected host tissues were examined by electron microscopy and cytochemical labelling techniques. One day after inoculating (dai) leaves and stems with fungal grown agar disks, dense mycelial networks were usually formed on the inoculated tissues. Then, the infection cushions of different size were developed. Mucilage produced by the pathogen covered mycelium and infection cushions. Hyphae forming infection cushions were often flattened and increased in diameter. After removing the infection cushions, the diameters of the numerous pores through which penetration pegs had entered the cuticle of leaves and stems were small and had almost the same diameter. Penetration of leaves and stems occurred 2 and 3 dai, respectively. Small changes in cuticle were observed. After penetration, hyphae of the pathogen extended between the cuticle and epidermal cell walls as well as inside the epidermal cell walls. Then inter- and intra-cellularly spreading hyphae were observed in the hemi- and ultra-thin sections by light- and transmission electron microscopy, 5 dai. Hyphae also colonized xylem and phloem. During colonization marked alterations in the host tissues were detected, including disorganization of cytoplasm, cell organelles, disintegration of cell walls and collapse of host cells. The enzyme- and immunogold-labelling investigations showed obvious degradation of cellulose, xylan and pectin in the host cell walls of infected tissues. The degradation of cell wall components suggests that the pathogen may secrete cell wall degrading enzymes (Cwdes) such as cellulases, xylanases and pectinases during infection and spreading in the oilseed rape tissues.