Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro.

The loss of variable antigen from Trypansoma brucei during transformation from the bloodstream to the procyclic form in vitro has been monitored by agglutination and immunofluorescence reactions using antisera against both forms. Maximum agglutination of transforming trypanosomes with anti-culture form sera was obtained in 36–48 hr coinciding with loss of the surface coat as seen by electron microscopy. Agglutination with antisera against homologous bloodstream forms, however, reached a constant minimum but still positive level after 7–9 days: absorption of such antisera with culture or heterologous bloodstream forms reduced this period of persistent agglutinability to 72–84 hr, suggesting that the sera contained antibodies to “common” (surface membrane) antigens which became exposed when the surface coat was lost during transformation. The indirect immunofluorescence reaction provided a direct correlation of loss of antigen with loss of coat. The majority of trypanosomes lost detectable variable antigen by 36–48 hr, but a few flagellates, morphologically resembling bloodstream forms, retained the coat and capacity for labeling up to 84 hr; the numbers of such persistent bloodstream forms were shown to be sufficient to give a positive agglutination reaction for the population as a whole up to this time. Variable antigen appeared to be lost by dilution over the entire trypanosome surface rather than in patches or caps and the relevance of this observation to the process of antigenic variation is discussed.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Trypanosoma brucei: Biochemical and morphological changes during in vitro transformation of bloodstreamto procyclic-trypomastigotes

Trypanosoma brucei brucei in whole rat blood inoculated into a semidefined medium undergoes complete morphological transformation (observed by light microscopy) in 72 hr. This reproducible system permits the biochemical and physiological study of transformation from bloodstream to procyclic trypomastigotes and mitochondrial biogenesis in these organisms. Infectivity for mice is lost after 6 days. Proline stimulates cell growth after transformation. High levels of glucose adversely affect the growth of transforming cells. Respiration during transformation is by an α-glycerophosphate oxidase although a cyanide-sensitive pathway is present after 24–48 hr but does not become fully functional with respect to procyclic trypomastigotes until 20–24 days in culture. The success of this system will permit the biochemical characterization of African trypanosomes as the development of the cytochrome system occurs.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

Differentiation and maturation of cultured fetal rat jejunum.

To determine whether differentiation and maturation of mammalian intestinal mucosa require influences available only in vivo or whether they can occur in vitro, fetal rat jejunum was cultured in chemically defined medium using organ culture methodology. Segments of jejunum from 18-day fetal rats were cultured in modified Liebowitz L-15 medium in room air at 37°C. Segments harvested after 24, 48, and 72 hr of culture were examined by light and electron microscopy. Uncultured jejunum from 18-day fetuses had either no or very few rudimentary villi and was lined largely by undifferentiated stratified epithelium. Goblet cells were not seen. In contrast, villi were present in the majority of 24-hr cultures, and simple columnar rather than stratified epithelium predominated. After 48 and 72 hr, villi were present in over 90% of cultured jejunal segments and stratified epithelium had disappeared. Goblet cells were seen in jejunal segments cultured 48 hr or longer in 47 of 74 fetuses. Electron microscopy further documented progressive differentiation of the epithelium during culture. Microvilli increased in number and height, a terminal web developed in the apical cytoplasm and the number of apical vesicles, mitochondria and formed elements of endoplasmic reticulum increased in absorptive cells. Jejunal lactase and alkaline phosphatase activity increased nine- and sevenfold, respectively, during 72 hr of culture, while the activity of the mitochondrial enzyme, ornithine carbamoyl transferase, increased fourfold. These observations indicate that jejunum from 18-day fetal rats can be cultured in vitro for at least 72 hr in chemically defined medium and that, during culture, maturation of the jejunal mucosa takes place with the appearance of villi, conversion of stratified to columnar epithelium and differentiation of individual epithelial cells.     

Synthesis of glycoproteins in the Golgi complex of the mouse gallbladder epithelium during fasting,refeeding, and gallstone formation

Summary The mouse gallbladder epithelium was studied with light microscopic autoradiography and quantitative electron microscopy during fasting, refeeding and experimental gallstone formation. To determine the intracellular pathway of glycoproteins, H³-galactose was injected at different time intervals into the mice. At 10, 25 and 40 min after an intraperitoneal injection the gallbladders were fixed and prepared for light microscopy. As early as 10 min after injection, label was observed in supranuclear cytoplasmic regions and at 25 min, an increased radioactivity was present throughout the apical cytoplasm. At 40 min, silver grains were mainly present at the cell surface. Autoradiographs processed 25 min after an intraperitoneal H³-galactose injection after fasting for 48 h showed decreased supranuclear and apical radioactivity. After refeeding (12 h) there was an enhanced activity in both these regions. Animals fed a lithogenic diet for one month showed a marked increase of radioactive label mainly in cells of crypts and invaginations of the mouse gallbladder mucosa.Morphometric measurements of the Golgi apparatus revealed that deprivation of food significantly diminished the volume density of the Golgi apparatus. Refeeding the animals restored the volume density values to normal levels. In the course of gallstone formation there was a further significant increase in the volume density of the Golgi complexes as compared to controls.     

Observations on the role of smooth endoplasmic reticulum in glucocorticoid-induced hepatic glycogen deposition

We have studied by quantitative electron microscopy the relationship of specific hepatic cellular organelles to glycogen synthesis using dexamethasone, a potent synthetic glucocorticoid, to induce glycogen deposition in livers of adrenalectomized rats. Chemical and ultrastructural glycogen determinations revealed that the livers of fasted adrenalectomized rats had very low glycogen levels. Dexamethasone caused a time-related increase in hepatic glycogen which was the result of increases in the number of hepatocytes depositing glycogen and the amount of glycogen in each cell. The surface density of smooth endoplasmic reticulum (SER) in centrilobular and periportal hepatocytes also increased after treatment with dexamethasone; this increase preceded glycogen deposition. The newly deposited glycogen was spatially associated with membranes of SER, and a continued increase in SER surface density was correlated temporally with the increasing glycogen volume density. In both centrilobular and periportal hepatocytes, the suface density of rough endoplasmic reticulum (RER) initially decreased after dexamethasone administration but later increased. These data support the hypothesis that dexamethasone-induced enhancement of SER is functionally associated with the increase in glycogen, and that although the initial increase in SER may occur through transformation of RER to SER, later increases in SER require synthesis of new membranes.     

A quantitative investigation of microvascular changes in the thyroid gland after infrared (IR) laser radiation.

We present an ultrastructural study of thyroid capillaries in which 50-day-old rats Wistar rats, were irradiated with an infrared (IR) laser, (total dose, 46.80 J/cm2), the tissue quantified 1 day after ending treatment and a quantitative capillary analysis carried out by light and electron microscopy. Light microscopy was used to calculate capillary volume density revealing a significant increase in the irradiated rats. The quantitative measurement of parameters by electron microscopy required a two stage analysis: Level I, Electron Microscopy (Magnification x5,000); and Level II, Electron Microscopy (Magnification x26,000). At Level I, the following parameters were measured in each capillary: capillary area, capillary diameter, luminal area, luminal diameter, endothelial area, nuclear area and mean endothelial thickness. At Level II, pinocytotic vesicle diameter and their numerical density in endothelial cells were evaluated. Electron microscopic analysis revealed an increased luminal area in the capillaries of the irradiated rats. They also presented a decrease in endothelial cell thickness and vesicular diameter and an increase in vesicle numerical density. This latter increase is indicative of presumptive changes in capillary permeability, but the possible functional significance of these morphological changes in the endothelial cells requires further investigation.     

Light microscopic morphometric analysis of peroxisomes by automatic image analysis: advantages of immunostaining over the alkaline DAB method.

The feasibility of light microscopic post-embedding immunocytochemistry for morphometry of peroxisomes using automatic image analysis was investigated and compared with the classical alkaline DAB method. Perfusion-fixed rat liver tissue was either embedded in LR White or incubated in the alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase. Sections from the LR White-embedded material were incubated with a monospecific antibody against catalase, followed by protein A-gold and silver intensification. Determination of peroxisomal volume density in sections of different thickness revealed that the values increased with section thickness in DAB-stained sections but were unaffected in immunostained preparations. Moreover, the absolute value for volume density of peroxisomes, as determined by light microscopy in immunostained sections, was quite close to the value obtained by analysis of electron microscopic preparations. Finally, morphometric analysis of bezafibrate-induced peroxisome proliferation revealed that the ratio of proliferation obtained by light microscopy in immunostained sections was very close to the results obtained by electron microscopic morphometry. The main advantage of post-embedding immunostaining for light microscopic morphometry is that it restricts the immunocytochemical reaction product to the surface of the section, thus making it independent of section thickness.     

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce''s disease (PD) of grapevine. Pierce’s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.     

Ultrastructural and immunohistological characterization of a cell culture model for the study of neuronal-glial interactions.

The differentiation of reaggregating cell cultures of dissociated cerebellar cells from 3- and 6-day-old mice was analyzed by electron microscopy of and immunofluorescence to the glial fibrillary acidic protein (GFA) at intervals between 8 hr and 20 days in vitro. Aggregates in culture for 8 hr were composed of 8–12 undifferentiated cells that were negative for the GFA protein and indistinguishable from each other by electron microscopy. Some cells with extended processes and a few immunofluorescent cells had already appeared after 24 hr in vitro, and the elaboration of a complex neural ultrastructure was observed during the subsequent days. After 10 days in vitro the interior of the aggregate was occupied principally by neurons, the majority of which were granule neurons, and regions of neuropil containing many synaptic complexes. Glial fibers with intense immunofluorescence to GFA were present throughout the aggregates but were mainly concentrated at the periphery. Large unidentified cells protruded from the surface. The subsequent days in culture evidenced a decline in the neuronal character of the aggregates with a concomitant increase in fibrous neuoglia. We suggest that factors controlling neuronal-glial interactions and fibrous gliosis are amenable to analysis in this tissue culture system.     

An electron microscopy study of the effects on dibutyryl cyclic AMP on Chinese hamster ovary cells

The effect of reverse transformation agents (dibutyryl 3′:5′ cyclic adenosine monophosphate plus synergizing compounds like testosterone) on Chinese Hamster Ovary cells has been studied by scanning and transmission electron microscopy, under conditions of low and high cell density. The disappearance of the cell surface knobs or blebs and the cell elongation previously described by light microscopy are even more striking under the higher resolving power of electron microscopy. The cell elongation is accompanied by and appears to be due to an increase in number of microtubules per unit volume of cytoplasm, and a change in their distribution from a random arrangement to an orderly alignment in parallel to each other and to the long axis of the cell. Increasing cell density, and therefore number of cell contacts in untreated cultures, causes the morphology to change in the direction to be expected if the cellular concentration of cyclic AMP is increased. Ultrastructural details of the cells in the various conditions studied are presented.     

Trypanosoma brucei: fluxes of the morphological variants in intact and X-irradiated mice

The number of dividing, slender, intermediate, and stumpy forms of Trypanosoma brucei in the blood of inbred mice changed daily. In both intact mice and mice which were exposed to whole body X-irradiation before infection, slender and dividing forms predominated during the first 72 hr of infection, and the number of intermediate and stumpy forms increased to a maximum between 72 and 140 hr. The rate at which stumpy forms accumulated in the blood and the number of these forms which eventually circulated were the same in both groups. However, the fluxes in the number of slender and dividing forms differed in intact and X-irradiated mice. In intact mice, the number of dividing forms in the blood decreased between 72 and 140 hr, and the number of slender forms decreased between 96 and 140 hr. In X-irradiated mice, the number of both these forms increased throughout infection. Electrophoresis of serum proteins and agglutination tests showed that X-irradiation severely depressed the ability of the mice to make antibody. Homogenates of spleen and bone marrow of intact mice contained many dividing forms throughout the infection. It is concluded that although host antibody does not directly induce the transformation of slender forms into stumpy forms, it may influence the morphological composition of the peripheral blood population of trypanosomes in several ways.     

A comparison between coral colonies of the genus Madracis and simulated forms

In addition to experimental studies, computational models provide valuable information about colony development in scleractinian corals. Using our simulation model, we show how environmental factors such as nutrient distribution and light availability affect growth patterns of coral colonies. To compare the simulated coral growth forms with those of real coral colonies, we quantitatively compared our modelling results with coral colonies of the morphologically variable Caribbean coral genus Madracis. Madracis species encompass a relatively large morphological variation in colony morphology and hence represent a suitable genus to compare, for the first time, simulated and real coral growth forms in three dimensions using a quantitative approach. This quantitative analysis of three-dimensional growth forms is based on a number of morphometric parameters (such as branch thickness, branch spacing, etc.). Our results show that simulated coral morphologies share several morphological features with real coral colonies (M. mirabilis, M. decactis, M. formosa and M. carmabi). A significant correlation was found between branch thickness and branch spacing for both real and simulated growth forms. Our present model is able to partly capture the morphological variation in closely related and morphologically variable coral species of the genus Madracis.     

Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi

The origin of Trypanosoma cruzi slender and broad forms found in the circulation of the mammalian host has remained obscure and, unlike what has been proposed for African trypanosomes, no precise form-function relationship has been ascribed to them. We show here that parasites circulating in the blood of infected animals display a high degree of polymorphism. Around 10% of the forms found circulating in mice during the acute phase of infection were amastigotes, and the other 90% included slender and broad trypomastigotes and intermediate forms between amastigotes and trypomastigotes. Slender trypomastigotes, from blood or cell culture, undergo extracellularly morphological rearrangements in which the parasites become gradually broader and transform into amastigotes. By scanning electron microscopy a progressive internalization of the flagellum and reorganization of the cell shape in a helical fashion were observed in parasites undergoing transformation. After 48 hr of extracellular incubation the parasite population consisted exclusively of amastigotes with a short protruding flagellum. The morphological changes were associated with the expression of different surface antigens defined by monoclonal antibodies: the trypomastigote-specific antigens Ssp-1 (a 100-120-150-Mr glycoprotein), Ssp-2 (a 70-Mr glycoprotein), Ssp-3 (undefined), and Ssp-4, an amastigote-specific surface antigen. Ssp-4 was also detected on intracellular amastigotes (in vitro and in vivo). We conclude that trypomastigotes are programmed to develop into amastigotes whether or not they enter cells, and that the differentiation can occur in the blood of the vertebrate host. These findings raise some questions regarding conventional views on the life cycle of T. cruzi.     

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.     

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.     

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.