Ganglioside patterns and cholera toxin--peroxidase labeling of aggregating cells from the chick optic tectum.

2The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. GD3 and GD1b were the predominant gangliosides early in development, while GD1a and several other multisialogangliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin--peroxidase binding site GM1 showed conjugate binding correlates with increasing levels of GM1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of GM1 in the synaptic cleft region.     

Ganglioside patterns and cholera toxin-peroxidase labeling of aggregating cells from the chick optic tectum

The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. G_D and G_D1b were the predominant gangliosides early in development, while G_D1a and several other multisialogan gliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin-peroxidase binding site G_M1 showed conjugate binding correlates with increasing levels of G_M1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of G_M1 in the synaptic cleft region.     

The tonoplast, a specific marker of embryogenic microspores of Datura cultured in vitro

Microspores of Datura metel L. have a thin tonoplast which, upon culture in vitro, develops a more or less uniform coating or deposit (as early as 12 h). This response was specific for embryogenic microspores, thereby making it possible to distinguish them from the nonembryogenic microspores. Cytochemical tests indicated the presence of tannins in this deposit which persists only until the early globular stage of embryogenesis. Thus tannin-coated tonoplasts can be used as a reliable and specific cytological marker for microspores following the embryogenic pathway in vitro.     

Histochemical studies on reserve substances and enzymes in female gametophyte of Zea mays.

Cytochemical changes during the early development of maize caryopsis are reported. Changes in the localization of different reserve substances (e.g. polysaccharides, proteins, nucleic acids and lipids) and enzymes (acid phosphatase, esterase, lipase, phosphorylase, succinate dehydrogenase, cytochrome oxidase and peroxidase) have been studied in unfertilized and fertilized ovules. Before pollination very feeble enzyme activity (acid phosphatase, succinate dehydrogenase, cytochrome oxidase and peroxidase) was observed. Reserve substances were present in low amounts before pollination. Pollination stimulated the accumulation of several substances and enzymes in the tip of the nucellus, micropylar zone. Just prior to, during and after fertilization, the cells in the micropylar zone had strong reaction for several enzymes indicating temporary enhancement of metabolic activity in the micropylar zone. The role of antipodals in the storage of reserve food products and nutrition of embryo and early stages of endosperm development is discussed. The pattern of enzymatic changes within the embryo sac reflected the biochemical changes operative during quiescent and active stages. The nucellus of Zea mays contains many enzymes required for hydrolysis of reserved food substances. A role of acid phosphatase in autolysis of nucellar cells, after fertilization is suggested. Post-fertilization increase in the activity of enzymes and accumulation of reserve materials is interpreted as reflecting a presumed increase in the metabolic rate relative to growth and differentiation.     

Effect of cyclic AMP on human blood platelets: induction of pseudopod formation and protein phosphorylation

A variety of techniques, including immunofluorescence, electron microscopy and biochemical analysis, were used to examine shape changes and cytoskeletal reorganization of human blood platelets during treatment with N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and agent known to elevate the intracellular level of cyclic AMP (cAMP). Cytochemical analysis shows that the unstimulated platelets have a discoid shape with no obvious membrane projections. Platelets treated with dbcAMP produce pseudopod-like structures containing cytoskeletal proteins such as actin and microtubules. Biochemical analysis reveals that a 125,000 dalton phosphoprotein (P-125) is preferentially recruited into cytoskeletal fractions of platelets treated with dbcAMP. This protein, which is one of the substrates for cAMP-dependent kinase(s) and/or is closely associated with the cytoskeleton, may play an important role in regulating the shape changes and cytoskeletal reorganization that occur during the early stages of platelet activation.     

Peroxisomes in disease.

Cytochemical, biochemical and morphological changes in peroxisomes have been described in human metabolic disorders, in experimental models of disease and in response to drugs and toxins. These include the cerebrohepatorenal syndromes, in which peroxisomes can not be detected and mitochondrial respiration is inhibited, atherosclerosis, alcoholic cardiomyopathy, and tolerance to oxygen toxicity. Although information on the role of peroxisomes in disease is limited, increased awareness of their widespread distribution and the availability of an improved cytochemical procedure for staining peroxisomes in human specimens should provide new insights into their function.     

Ultrastructural changes of renal tubules in the riboflavin deficient mouse

Ultrastructural changes of the tubular epithelium in the mouse kidney produced by dietary riboflavin deficiency were studied by electron microscopy and cytochemistry. In riboflavin deficient mouse kidney, the ultrastructural changes are localized to the pars recta of the proximal tubule. They comprise so called vacuolar degeneration on light microscopy, which consists of the formation of giant mitochondria and vacuoles. During the development of riboflavin deficiency, mitochondria decrease in number and enlarge in size through fusion. Sometimes they are larger than nuclei in size. The vacuoles observed in tubular epithelia are divided into two different groups according to their morphological characteristics and origins. One is derived from proliferated peroxisomes, and another from increased cytoplasmic bodies termed cytosomes and cytosegresomes. These increased vacuoles occupy almost all of cytoplasm. Cytochemical studies also reveal that these vacuoles are peroxisomes and lysosomes. These changes are reversible on supplementation with riboflavin.     

Effect of coniophage decomposition products on the biological activity of fibroblasts]

Cytochemical studies showed that destroyed coinophages, which phagocytized cytoxic and fibrogenic quartz dust, activated the synthetic function of rat fibroblasts when added to the culture medium. Inert talcum dust used under similar experimental conditions failed to produce any noticable changes in comparison with control studies.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

Observations on mechanisms of attachment in the green algaUlva mutabilis Føyn

The development of the rhizoid cells of the green alga Ulva mutabilis was investigated at the ultrastructural level paying special attention to the mechanism of attachment of the plant. Cytochemical data concerning the initial settling of the early zygote are also given. On the basis of histochemical staining and enzyme treatment it is concluded that the adhesive material secreted by the rhizoid cells is chemically different from that secreted by the zygote during the initial settling of the alga.     

Occurrence, characterization and development of two different types of microbodies in the nematophagous fungus Arthrobotrys oligospora

Abstract The occurrence of microbodies in different cells of the nematophagous fungus Arthrobotrys oligospora has been investigated. In the predacious phase this organism forms complex 3-dimensional network traps. Mature trap cells generally were crowded with "special" microbodies which possessed an electron dense matrix and were surrounded by a membrane of approx. 9 nm. These organelles developed during the early stages of trap formation and were derived from specialized regions of the endoplasmic reticulum. Cytochemical staining experiments revealed that the electron-dense microbodies contained catalase and d -amino acid oxidase and thus must be considered peroxisomal in nature. Electron-dense bodies were absent in normal vegetative cells of the fungus. These cells contained "normal" microbodies which developed from each other by the separation of small organelles from mature ones. As in yeasts, the metabolic function of these latter organelles was dependent upon environmental conditions.     

The role of cytochemistry in human genetic research

The role of cytochemistry in human genetics is reviewed. In basic research, autoradiography and cytochemical staining procedures for DNA, RNA, proteins and other cell constituents have contributed to the understanding of the way DNA is localized, duplicated and translated. The development of new "banding techniques" for the identification of human chromosomes and parts of these together with somatic cell hybridization procedures have significantly contributed to the mapping of the human genome. Cytochemical methods have also been of great help in the elucidation of the responsible molecular defects in Mendelian disorders based on a single gene mutation. The combination of immunological methods and electron-microscopical cytochemistry now enables different posttranslational processing defects to be related to various subcellular compartments. Cytochemistry is also likely to be of importance for the direct demonstration of gene mutations using recombinant DNA technology. Examples are given of contributions of cytochemical methods to the early diagnosis and prevention of congenital disorders. The main examples are the early diagnosis of patients with a chromosomal aberration and of carriers with a balanced translocation. Early genetic counseling of couples at risk forms the basis for prevention of subsequent affected children. Cytochemistry also contributes to the early detection of heterozygotes of X-linked mutations. Finally, autoradiography and ultramicrochemical procedures have been of great help in improving the prenatal diagnosis of genetic metabolic diseases.     

Occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes.

Evidence of the presence of polysaccharide polymers is shown in Streptomyces sp. for the first time. Cytochemical methods revealed the occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes. Onset of the sporogenesis coincided with the appearance of the granules, which reached a maximum number during the early stages of maturation. The later stages of maturation showed a decrease of these granules, and in mature spores no granules were observed.     

Ultrastructural differentiation of glandular stomach (proventriculus) in chick embryo

Cytochemical characterization of mucosubstances of chick glanular stomach (proventriculus) changes from 15 days of development to postnatal and adult stages was studied. To corroborate these data cytochemical, ultrastructural and ultracytochemical study of chick embryo proventriculus from 7 to 20 days of development was performed. At the 7th day several layers of undifferentiated cells formed an epithelium which covered the walls of the glandular stomach. Mocosubstances were not found. Between the 9th and 5th day a single layer of cylindrical cells was encountered forming invaginations which originated deep glands. Three types of cells were separated from the above mentioned layer, dark, clear and undifferentiated. The dark cells had organelles which are involved in protein synthesis and the clear ones were rich in mitochondria. Argentaffine cells appeared at 15th day instead mucosubstances formed a thin coat on the epithelium at 9th day which increased at the end of development in the apical cytoplasm and gland cells. These observations demonstrate that proventriculus of chick embryo has ultrastructurally differentiated cells involved with enzymatic and hydrochloric acid secretion after the 9th day. These progressive events are correlated with the digestion process of yolk during embryogenesis. At the end of development the proventriculus has completely organized the glandular layer.     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE MYOTOME OF THE EMBRYONIC RABBIT : An Electron Microscope Cytochemical and Biochemical Study

Acetylcholinesterase (AChE) activity has been studied in the myoblast of skeletal muscle of the 9–13 day fetal rabbit. Cytochemical activity is present in the nuclear envelope and the endoplasmic reticulum, including its derivatives the subsurface reticulum and the sarcoplasmic reticulum. End product is also found in the Golgi complex of the more differentiated myoblasts. The formation of reticulum-bound acetylcholinesterase in the myoblast appears to be independent of nerve-muscle contact, since the enzyme is present before the outgrowth of the spinal nerve. The nerve lacks cytochemical end product until the myoblast is well differentiated. Possible mechanisms of spontaneous muscle contraction have been discussed. A second type of myotomal cell, which exhibits a poorly localized end product of AChE activity, has been described. The ready solubility of the enzyme or diffusibility of its end product suggests that the enzyme may be a lyoesterase. This cell may be the precursor of the morphologically undifferentiated cell which is closely apposed to the myotubes in later stages of skeletal muscle development. Biochemical studies show a significant increase in AChE activity in the dermomyotome by day 12, when many of the myoblasts are well differentiated and the second type of myotomal cell is prominent. Cytochemical studies have indicated that many of the cells in the sample lack reaction product of enzymic activity, whereas others are very active. Biochemical values, therefore, reflect the amount of enzyme in the dermomyotome as a whole, but give little information on the enzymic content of individual cells.     

The oligosaccharidic component of the glycoconjugates in lichen planus, granuloma annulare, seborrheic keratosis and plamoplantar keratoderma: lectin histochemical study.

It is well known that cell surface glycoconjugates play an important role in cell proliferation, adhesion and differentiation. The aim of this investigation was to define the changes of the glycoconjugate saccharidic moieties in the epidermis and derma of patients affected by several skin pathologies such as seborrheic keratosis, lichen planus, granuloma annulare and palmoplantaris keratoderma. Bioptical specimens from skin lesions as well as from normal skin were fixed in Carnoy's fluid and routinely processed. The sections were treated with HRP-lectins (PNA, DBA, SBA, WGA, ConA, LTA and UEAI). Cytochemical controls were performed for specificity of lectin-sugar reaction. Some sections were pre-treated with neuraminidase prior to staining with HRP lectins. In comparison with normal human skin, epidermal lectin binding pattern in the considered diseases showed considerable qualitative and quantitative variations. In general, in all the considered pathologies, a lack and/or a decrease in lectin binding at the epidermal layers was observed; among the various diseases, differences in cellular localisation of the sugar residues were also noted. In such respect, an exception was represented by seborrheic keratosis, where the cells of the basal layer showed PNA reactivity, which was absent in the basal layer of the normal skin. Although seborrheic keratosis and lichen planus have been studied by others authors, our findings are not in total accordance concerning lectin binding; this is probably due to the different fixatives employed. Our findings seem to reveal significant changes in keratinocyte glycoconjugate oligosaccharides in the previously mentioned diseases, providing clues to their pathogenesis.     

The protein composition of nuclei during spermiogenesis in the leopard frog,Rana pipiens

Cytochemical studies of the basic and non-basic protein composition of nuclei in succeeding stages of spermiogenesis of the leopard frog are described. The histones which occur in nuclei of each stage, including the mature sperm, are of the somatic type. Nuclei of early stages contain nonhistone proteins. As chromatin condensation occurs in mid spermiogenesis, nonhistone proteins are detected where DNA and histones are distributed diffusely but not where DNA and histones are concentrated. In the uniformly condensed nuclei of late stages, nonhistone proteins are absent.