A time-lapse video image intensification analysis of cytoplasmic organelle movements during endosome translocation

Vital fluorescence staining has been used in conjunction with time- lapse video image intensification microscopy to analyze the distribution and movement of endosomes, lysosomes, and mitochondria in cultured rat ovarian granulosa cells. Exposure of 5-d granulosa cell cultures to pyrene-concanavalin A (P-Con A) or 3,3'- dioctadecylindocarbocyanine-labeled low-density lipoprotein (dil-LDL) at 4 degrees C results in the formation of randomly distributed endosomes 10 min after warming to 37 degrees C that exhibit saltatory motion for 20 min. If granulosa cells are labeled at 4 degrees C with both P-Con A and dil-LDL and warmed to 37 degrees C, both ligands are found within the same endosomes which migrate centripetally to the cell center where label accumulates within phase-dense structures by 60 min. The initial endosome saltations occur over short distances (mean distance = 4.6 micron) with a mean velocity of 0.03 micron/s. Endosome saltations then cease and are followed by a gradual centriptal migration of endosomes to the cell center where they accumulate and fuse with phase-dense structures. The second phase of movement involves a continuous, unidirectional migration of endosomes over distances ranging from 5 to 40 micron at a mean velocity of 0.05 micron/s. Lysosomes were simultaneously visualized as acridine orange-staining, phase-dense structures in control cells and cells exposed to fluorescent ligands. In untreated cells, lysosomes are dispersed throughout the cytoplasm and undergo bidirectional saltations covering a mean distance of 8.7 micron with a mean velocity of 0.3 micron/s. Lysosomes redistribute centripetally to the perinuclear region of the cell by saltatory movement within 20 min of exposure to ligand. Mitochondria were visualized with the fluorescent dye rhodamine 123 in granulosa cells labeled with P-Con A and were found to redistribute to the cell center coincident with endosomes. The microtubule-disrupting agent nocodazole was found to inhibit lysosome saltations and all phases of endosome movement. Taxol, a microtubule-stabilizing agent, partially impaired lysosome movement and led to a redistribution of lysosomes into linear aggregates surrounding the nucleus. Taxol was also found to inhibit endosome movement. The data indicate that (a) endosome movement proceeds initially by saltation and later by a nonsaltatory centripetal migration in association with mitochondria, that (b) lysosomes and endosomes undergo a temporally distinct but spatially similar change in cytoplasmic distribution, and that (c) microtubules are required for the directed translocation of endosomes and lysosomes towards the cell center.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

Releaser and primer pheromones in Collembola

In Collembola, pheromones appear to be present in the faecal pellets. Pheromone release after cessation of faeces production points to the digestive tract as a possible site of biosynthesis.During the pre-moulting periods Collembola do not react to pheromones, possibly due to their low activity at that time, whereas the production of the pheromones continues.Starvation periods of up to 14 days diminish pheromone release but do not cause complete cessation. Production per animal seems to decrease at increasing densities.The effect of pheromones on the reproductive efficiency of Collembola is discussed in the context of their physiological and behavioural ecology.     

Vitamin C increases the prostacyclin production and decreases the vascular lesions in experimental atherosclerosis in rabbits

Feeding a cholesterol-rich diet (0.3%) to rabbits resulted in an intimal thickening and lipid infiltration of the aorta. The prostacyclin production by the vascular endothelium was significantly decreased, after a transient increase after 2 weeks of diet. The arachidonic acid metabolism in platelets was hardly changed. Addition of a low dose vitamin C (150 mg/day) to the cholesterol rich diet resulted in decreased lipid infiltration and intimal thickening and the transient increase of the prostacyclin production was postponed to the 4th week. Although this dose of vitamin C could not restore the decreased prostacyclin production observed after 6 weeks diet, a higher dose of vitamin C (600 mg/day), besides its beneficial effect on the lipid infiltration and the intimal thickening in the thoracic aorta, kept the intimal prostacyclin production at normal levels for at least 8 weeks.     

Mechanism of complement-induced stimulation of prostacyclin production by isolated rabbit peritoneum

The interaction between the complement system and prostaglandin synthesis has not thoroughly been explored, although both mediators are known to be involved in inflammatory reactions and endotoxic shock. When rabbit peritoneum, a rich source of prostacyclin forming activity was incubated in serum in which the complement system was activated (CVF, LPS, zymosan), the tissue produced significantly more PGI2, when compared with appropriate controls, indicating that by activation of the complement, factors were generated that stimulated PGI2 biosynthesis. Further results indicated that tryptic cleavage products of complement factor C3 and C5 also led to the appearance of PGI2 releasing principles with a molecular weight of about 7000-11000. The stimulation of PGI2 biosynthesis was explained by enhanced release of AA, and not due to increased activity of cyclo-oxygenase or PGI2 synthetase. Our results suggest that complement-derived products may promote the supply of prostaglandins at the site of inflammation.     

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.     

Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody,YBM/42

A rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS-II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25-fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU-E). In the most enriched fraction, 20% of the cells are day 2 CFU-E. Day 7 CFC can therefore be well separated from day 2 CFU-E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G-CFC), macrophage (M-CFC), mixed granulocyte/macrophage (GM-CFC), pure erythroid (E), or mixed erythroid (E_mix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM-CFC and M-CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM-CFC and M-CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.