Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

Biodegradation of textile azo-dyes byPhanerochaete chrysosporium

Of 18 commercially used textile dyes, eight were degraded by the white rot fungus,Phanerochaete chrysosporium, by 40 to 73% based on decrease of colour. Both the lignin-degrading enzyme system ofP. chrysosporium and adsorption to its cell mass were involved in the degradation of the diazo dye, Reactofix Gold Yellow. Degradation was best achieved by adding the dye to the medium and then inoculating with pre-grown mycelium; inoculation with spores resulted mainly in dye adsorption.     

Some cytochemical studies on the cell surface ofAmphidinium carterae (Dinophyceae)

Summary The surface coat of the dinoflagellateAmphidinium carterae Hulburt was examined by fluorescence and transmission electron microscopy, using various fluorochromes and cationic dyes. The overall results showed cell-surface reactions typical of acid mucopolysaccharides. The cationic dye staining revealed an outer fine fibrillar layer (15–70 nm thick) overlying a dense anionic coat (40–60 nm thick) which appeared to thicken progressively with age. In general, the structure of the amphiesmal vesicles was similar to that previously described by other investigators. However, an acidic mucopolysaccharide layer was observed on the inner surface of these vesicles. Each of these structures is traversed by 1–3 pores and at least 2 types of extrusomes are formed, the spindle trichocysts and the mucocysts. Cell to cell adhesion through the surface coat was frequently observed. Evidence was also obtained for internalization of all the surface-coat markers used.This investigation forms part of a doctoral thesis submitted by the first author to the University of British Columbia, Vancouver, B.C.     

Toxic effects of 2-methyl-4-dimethylaminoazobenzene in normal and partially hepatectomized rats

In order to obtain a more precise definition of the conditions under which 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) and liver cell proliferation play a role in the initiation of hepatocarcinogenesis, the toxicity of 2-Me-DAB for normal and partially hepatectomized rats was investigated. Continuous feeding of a basal low protein, low riboflavin diet supplemented with 2-Me-DAB was found to be highly toxic for male albino rats. All animals fed on such a diet died before 200 days. Sham operation and partial hepatectomy (PH) at 30 days of 2-Me-DAB feeding reduced the median survival time from 122 days to 107 and 94 days, respectively. Transfer to the basal diet after 30 days of 2-Me-DAB feeding and PH prolonged the median survival time to 216 days while 97% of the rats returned to the normal complete diet after the same treatments survived for more than 300 days. 2-Me-DAB was not necrogenic and there was no evidence of reparative proliferation or hepatic tumor formation in any group. Feeding rats with the 2-Me-DAB containing diet for 1 month delayed and strongly inhibited the mitotic response of the liver to the stimulus of partial hepatectomy. This is the result of a blockage of the cells in G1 as revealed by the fact that only 1% of the hepatocytes became labeled when 2-Me-DAB fed animals were injected with tritiated thymidine prior to sacrifice at 24 h post-hepatectomy, as compared to 40% in rats fed the normal or the control basal diet. This inhibitory effect of 2-Me-DAB is reversible however since rats returned to the normal diet for 1 or 2 months after 2-Me-DAB feeding showed percentages of mitoses and labeling indices comparable to those of control animals following PH. The number of abnormal mitoses was high (13%) in regenerating livers of rats fed 2-Me-DAB and the lesions responsible for this effect are apparently not repaired since 2-Me-DAB fed rats partially hepatectomized after being transferred to the normal diet for 1 or 2 months showed the same number of mitotic irregularities. The present results suggest that assays with 2-Me-DAB as 'pure initiator' or agent of selective toxicity should be pursued in attempts to improve existing experimental models of hepatocarcinogenesis.     

CCD imaging of the electrical activity in the leech nervous system

A single ganglion of the nervous system of the leechHirudo medicinalis was isolated. One or both roots emerging from each side of the ganglion were sucked into suction pipettes used either for extracellular stimulation or for recording the gross electrical activity. The ganglion was stained with the fluorescence voltage sensitive dye Di-4-Anepps. The fluorescence was measured with a nitrogen cooled CCD camera. Our recording system allowed us to measure in real time slow optical signals corresponding to changes in light intensity of at least 5. These signals were caused by the direct polarization of neuronal structures, the afterhyperpolarization or the afterdischarge induced by a prolonged stimulation. When images were acquired at fixed times, several of them could be averaged and optical signals of at least 2 could be reliably measured. These optical signals originated from well identified neurons, such as T, P and N sensory neurons. By taking images at different times and at different focal planes, electrical events could be followed at a temporal resolution of 50 Hz. The three dimensional dynamics of electrical events, initiated by a specific stimulation, was imaged and the spread of excitation among leech neurons was followed. When two roots were selectively stimulated, their neuronal interactions could be imaged and the linear and non-linear terms of the interaction could be characterized.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Second-harmonic generation in monolayers of hemicyanine dyes at the air/water interface: Observation of chemical degradation

In situ studies of second-harmonic generation (SHG) in monolayers of pure hemicyanine dyes and their mixtures with arachidic acid at the air/water interface were performed. The studies revealed an unexpected chemical instability of the hemicyanine dyes involving a cleavage of the stilbene double bond when deposited onto the water surface. This reaction occurred especially rapidly in the presence of the arachidic acid. Dilution with arachidic acid did not lead to a significant enhancement of the harmonic intensity.     

Calcium-sensitive cells of the pars intermedia and osmotic balance in the eel

Summary The structure of the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia was investigated in eels kept in deionized water (DW) or fresh water (FW) supplemented with Ca²⁺ or Mg²⁺. Ca²⁺ (2mM) reduces considerably the response to DW; plasma osmolarity, Na⁺ and Ca²⁺ levels are not significantly affected. In eels adapted to DW for 21 or 28 days, showing highly stimulated Ca-s cells, an addition of CaCl₂ for 2 days inhibits the release of granules, but does not immediately block their synthesis and the mitotic activity. The nuclear area is reduced, osmolarity and plasma sodium increase, but the rise in calcium is not always significant. Magnesium, at a 10-fold greater concentration than in FW (2 mM), slightly inhibits the release of secretory granules without reducing other indicators of stimulation. In Ca-enriched FW, the Ca-s cells appear inactive. These data show that the PAS-positive cells in the pars intermedia of the eel are calcium-sensitive, similar to those of the goldfish; their role in calcium regulation is briefly discussed.     

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum

Summary The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca²⁺ transport. The absorbance and fluorescence responses of 3,3-diethyl-2,2-thiadicarbocyanine, 3,3-dimethyl-2,2-indodicarbocyanine and oxonol VI during ATP-dependent Ca²⁺ transport are influenced by the effect of accumulated Ca²⁺ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca²⁺ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca²⁺, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulumin vivo andin vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca²⁺ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca²⁺ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca²⁺ translocation. The potential generated during Ca²⁺ transport is rapidly dissipated by passive ion fluxes across the membrane.     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.