Peripheral Granules Research Articles

177 Collection of mouse immature oocytes using cilostazol, a phosphodiesterase 3A inhibitor treatment

Administration of cilostazol in mice and swine has resulted in the ovulation of immature oocytes at the germinal vesicle (GV) or metaphase I (MI) stages. The present study aimed to define oocyte synchronised maturation, yield, health, and ease of collection from mice treated with cilostazol. The conventional method included mice primed with pregnant mare serum gonadotrophin (PMSG) and GV oocytes isolated from preovulatory follicles 48h post-PMSG. Recovery of MI oocytes included the invitro maturation of the isolated GV oocytes into the MI stage for 6h or the superovulation of mice with PMSG and human chorionic gonadotrophin (hCG), 48h apart, and the isolation of MI oocytes from preovulatory follicles 6h post-hCG. The cilostazol method included the superovulation of mice, as described above, and oral treatment with 7.5mg of cilostazol once (at the same time as hCG) or twice (at the same time as hCG plus 6h post-hCG) to result in the ovulation of MI or GV oocytes, respectively. The cilostazol method resulted in immature oocytes that are uniform in size. For instance, the cilostazol method resulted in 98.0% (n=110) of GV oocytes with a diameter range of 60-90μm compared with only 49.5% (n=118) of GV oocytes resulting from the conventional method (P<0.0001). Similarly, 95.0% (n=93) of MI oocytes obtained from the cilostazol method were synchronised within the diameter range of 50.1-70μm compared with 60.0% (n=89) of MI oocytes obtained from the conventional method (P<0.0001). Cilostazol also resulted in immature oocytes with synchronised nuclear and cytoplasmic maturation. In this regard, the cilostazol method resulted in GV oocytes having higher levels of co-occurrence of peripheral cortical granules and surrounded nucleolus chromatin configuration compared with the conventional method (80.6% (n=124) vs. 36.6% (n=131), respectively; P<0.0001). Similarly, the co-occurrence of normally organised spindles and chromosomes and peripheral cortical granules with free domains was observed more frequently in MI oocytes obtained from the cilostazol method than in those obtained from the conventional method (82.8% (n=151) vs. 65.0% (n=100), respectively; P=0.001). The cilostazol method was more time and labour efficient (0.8±0.2 vs. 3.2±0.2 min; P<0.001) and resulted in higher oocyte yields (22.7±1.8 vs. 7.9±0.7 oocytes; P<0.0001) and normal morphology (94±1.5% vs. 80.1±3.3%; P=0.02) than did the conventional method (P<0.001). Finally, compared with the conventional method, the cilostazol method improved the blastocyst production rate of GV and MI oocytes from 39.6% (n=188) to 65.0% (n=169) and from 44.2% to 75.3%, respectively (P<0.001). The presented method provides not only oocytes with uniform size and synchronised developmental maturation but also a technique of oocyte collection that is efficient and resourceful. It is possible that not all of the immature oocytes resulting from the conventional method are from preovulatory follicles, and they do not necessarily represent the cohort of oocytes that would develop adequately and consequently ovulate as opposed to the presented method.

Structural Analysis of Mouse Platelets using Serial Block-Face Scanning Electron Microscopy

Platelets are small, anucleate cells in mammalian blood that maintain hemostasis and aggregate to seal leaks at sites of vascular injury, a vital element of wound healing. Upon detecting vascular lesions, blood platelets become activated and release components, such as the Von Willebrand factor, from at least three different storage compartments: alpha-granules, dense granules, and lysosomes. Abnormalities in platelet functions can contribute to thrombosis, atherosclerosis and other diseases. Obtaining cellular structure is critical for gaining better understanding of platelet physiology. We used Serial Block-Face Scanning Electron Microscope (SBF-SEM) to elucidate the 3-D ultrastructure of mouse blood platelets in both the activated and inactivated (resting) states. In SBF-SEM an in-situ ultramicrotome cuts slices of heavy-atom stained cells that are embedded in a resin block. The top surface of the block is then imaged using a back-scattered electron detector, and the process of slicing and imaging is repeated until the desired volume of cells is obtained. A stack of images can then be aligned, and ultrastructure visualized using segmentation software. Based on the segmentation and measurements on images from 4 different platelet activation states, we observed that rapid swelling of alpha-granules is accompanied by matrix decondensation that occurs first in peripheral granules, whereas condensed granules were situated more centrally. Decondensed granules appeared almost adjacent or connected to the plasma membrane. Based on these findings, we propose that the granule matrix organization is an important factor for platelet function, i.e., for timed-release properties of alpha-granule cargo, which could be physiologically important to thrombus formation, wound healing and angiogenesis. This work was supported by the Intramural Research Program of the National Institutes of Biomedical Imaging and Bioengineering, NIH (RDL) and NIH grants R01 HL119393 and R56 HL119393 (BS).

Lichen planus-like keratosis: clinical applicability of in vivo reflectance confocal microscopy for an indeterminate cutaneous lesion.

Lichen planus-like keratosis (LPLK) is an involuting cutaneous lesion often presenting between the fifth and seventh decades of life. These lesions typically appear abruptly as a solitary macule, papule, or plaque that continuously evolves as it undergoes regression. Clinical and dermoscopic features of LPLK can mimic both benign and malignant lesions, often prompting biopsy for accurate diagnosis. We describe a case of LPLK developing in a patient with a history of multiple skin cancers, including melanoma. Dermoscopy revealed peripheral granules and a central area with pinkish-brown pigmentation and a disorganized pattern with shiny white structures and rosettes. Handheld reflectance confocal microscopy (RCM) showed a typical honeycomb pattern with millia-like cysts and comedo-like openings, and lacked pagetoid and dendritic cells. Based on the benign features seen with RCM, the lesion was followed until complete regression was observed. In conclusion, we describe a case of LPLK with clinically and dermoscopically indeterminate features that was successfully monitored with RCM. We intend to highlight the utility of RCM as a diagnostic aid in equivocal lesions in order to prevent unnecessary excisional procedures.

Spo20p‐GFP, a probe that allows visualization of the dynamic of phosphatidic acid during regulated exocytosis.

Several reports have recently proposed phospholipase D (PLD) as a component of the molecular machinery underlying calcium‐regulated exocytosis. In consequence it is expected that PLD should be activated and phosphatidic acid (PA) produced at the site of exocytosis, which would be at the plasma membrane and/or on the membrane of arriving secretory granules. Among bioactive lipids PA is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To assess the dynamic location and actual function of PA in secretory cells, we attempted to visualize the intracellular location of PA production during the process of secretion in neuroendocrine cells, using a Green Fluorescent Protein (GFP) sensor that specifically binds to PA. PC12 cells were transfected with a chimeric protein consisting of EGFP‐fused to the PA‐binding domain (PABD) of Spo20p, a yeast homolog of SNAP25. We were able to show a transient accumulation of the PA sensor at the cell periphery during exocytosis. Using a silencing RNA strategy, we further demonstrate that PLD1 is responsible for PA accumulation at the plasma membrane in secretagogue‐stimulated cells. Finally using immuno‐gold electron microscopy, we observed PA in‐between peripheral granules and the plasma membrane in stimulated cells, suggesting that PA is generated at the granule docking sites.

Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic

The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND-1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.

Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits.

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.

Ca2+ influx does not trigger glucose-induced traffic of the insulin granules and alteration of their distribution.

This study investigated mechanisms by which glucose increases readily releasable secretory granules via acting on preexocytotic steps, i.e., intracellular granule movement and granule access to the plasma membrane using a pancreatic beta-cell line, MIN6. Glucose-induced activation of the movement occurred at a substimulatory concentration with regard to insulin output. Glucose activation of the movement was inhibited by pretreatment with thapsigargin plus acetylcholine to suppress intracellular Ca2+ mobilization. Inhibitors of calmodulin and myosin light chain kinase also suppressed glucose activation of the movement. Simultaneous addition of glucose with Ca2+ channel blockers or the ATP-sensitive K+ channel opener diazoxide failed to suppress the traffic activation, and addition of these substances on top of glucose stimulation resulted in a further increase. Although stimulatory glucose had minimal changes in the intracellular granule distribution, inhibition of Ca2+ influx revealed increases by glucose of the granules in the cell periphery. In contrast, high K+ depolarization decreased the peripheral granules. Glucose-induced granule margination was abolished when the protein kinase C activity was downregulated. These findings indicate that preexocytotic control of insulin release is regulated by distinct mechanisms from Ca2+ influx, which triggers insulin exocytosis. The nature of the regulation by glucose may explain a part of potentiating effects of the hexose independent of the closure of the ATP-sensitive K+ channel.

Regulated von Willebrand Factor Secretion Is Associated With Agonist-Specific Patterns of Cytoskeletal Remodeling in Cultured Endothelial Cells

von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, is stored and released from endothelial secretory granules called Weibel-Palade bodies. Regulated secretion occurs in reaction either to [Ca(2+)](i)-raising agents (histamine or thrombin) or to cAMP-raising agents (epinephrine, adenosine, or forskolin). We investigated the pattern of release and the cytoskeletal requirements for secretion in response to these 2 classes of agonists. Secretion induced by [Ca(2+)](i)-raising agents involves peripheral and central granules and is inhibited by colchicine-induced microtubule disruption. It is accompanied by Rho-dependent stress fiber formation and cell retraction. Secretion and remodeling occur in the same individual cells. However, secretion is potentiated by cytochalasin E and C3 toxin, indicating that stress fiber formation antagonizes vWF secretion. In contrast, vWF secretion induced by cAMP-raising agents involves the release of only peripheral granules (implying less vWF release on a per cell basis) and is not inhibited by microtubule disruption. cAMP-mediated secretion is accompanied by disruption of stress fibers, strengthening of the cortical actin rim, and preservation of cell-cell contacts. It is unaffected by cytochalasins or C3 toxin. In contrast to [Ca(2+)](i)-raising agents, cAMP-raising agents induce secretion without cell retraction/intercellular gap formation. Thus, they are likely to play a physiological role in the regulation of endothelial vWF secretion and, therefore, of plasma vWF levels.

Phagocytic and lysosomal activity in Langerhans cells from sensitized guinea pigs

Guinea pig skins were sensitized and challenged with horseradish peroxidase. Langerhans cell granules contained the horseradish peroxidase. The cytoplasmic locations of horseradish peroxidase in Langerhans cells changed with the passage of time: The antigen was found initially in peripheral Langerhans cell granules and subsequently in those located near the nucleus. The number of lysosome-like dense bodies, or multivesicular bodies increased with time. After 24 hr the antigen was absent from the Langerhans cell granules and seen only in lysosome-like dense bodies, or multivesicular bodies. When acid phosphatase activity was examined, the Langerhans cell granules attached to plasma membranes and rodshaped granules were negative. It was positive in lysosome-like dense bodies, multivesicular bodies, and the bulb part of the Langerhans cell granules. A similar sequence of observations was made when guinea pig skins were sensitized and challenged with ferritin and 2,4-dinitro-1-chlorobenzene. It is concluded that Langerhans cell granules can take up antigens and assume an aspect of the lysosome.

Morphometric analysis of atrial natriuretic peptide-containing granules in atriocytes of rats with experimental congestive heart failure

The morphometric characteristics of atrial natriuretic peptide-containing granules were studied in atrial myoendocrine cells of rats with aorto-caval fistula, an experimental model of congestive heart failure. A total of 6680 granules of control and aorto-caval rats were analyzed by a computerized image analysis system that evaluated the number and sectioned surface area of granules and their subcellular location. Compared with control animals, rats with congestive heart failure displayed a slight increase in the number of peripheral granules, adjacent to the sarcolemma, but not centrally located in the Golgi areas. The mean sectioned surface area of granules in rats with congestive heart failure was about 50% of that in controls, both in the right and left atria. Rats with aorto-caval fistula had a higher percent of small granules and lower percent of large granules compared with controls. The data demonstrate different morphometric characteristics in atrial natriuretic peptide-containing granules in atriocytes in rats with experimental congestive heart failure; this may reflect the enhanced synthesis and release of atrial natriuretic peptide in heart failure.

Characterization of a brown Nostoc species from Java that is resistant to high light intensity and UV

A brown Nostoc species was isolated from a soil sample collected in Java, Indonesia. It has three physiological types of light-absorbing compounds: the photosynthetic pigments (chlorophyll a, carotenoids, phycocyanin, phycoerythrin and allophycocyanin), a brown pigment(s) and three UV-absorbing compounds. The organism undergoes complementary chromatic adaptation by varying its phycobilin content. It is resistant to photobleaching at high intensities of white light, and also to UV-C radiation, under which conditions a green-coloured Nostoc species (ATCC 27895) was killed. Synthesis of the brown pigment and UV-absorbing compounds was not observed at low oxygen levels. A brown pigment was released suddenly from cells during exponential growth, resulting in a deep brown-coloured medium. In addition, the absorption spectrum of the medium after pigment release had maxima in the UV region, at 256 nm, 314 nm and 400 nm. The appearance of the UV-absorbing compounds coincided with the brown pigment release and with the disappearance from the vegetative cells of peripheral granules. The Nostoc species reduced acetylene, a measure of nitrogen fixation, in both the light and the dark periods when grown on a 12/12 h light/dark cycle. On a 4/20 h light/dark cycle fixation occurred predominantly in the light period. When grown on this restricted light regime, or at very low light intensity, the cells did not produce the brown pigment or the UV-absorbing compounds.

Dendritic development of dentate granule cells in the absence of their specific extrinsic afferents

Dendrites and spines are postsynaptic structures that develop in association with presynaptic fibers. Recent studies have shown that granule cells of the fascia dentata survive in slice cultures and differentiate in a manner known from in situ studies. However, all extrinsic afferent fibers are absent under culture conditions. In the present study, we study whether dendrites and spines of granule cells in slice cultures differentiate normally, although they are not contacted by their normal layer-specific afferents. Slices of hippocampus were prepared from rat pups at the day of birth. After 5, 10, 15, and 20 days of incubation, granule cells in these cultures were Golgi impregnated. For comparison, perfusion-fixed hippocampal sections of 5-, 10-, 15-, and 20-day-old rats were impregnated the same way. Our results show that the total density of spines on granule cell dendrites in culture increased as in perfusion-fixed animals. However, after 20 days of incubation, the absolute number of dendritic spines on cultured neurons was reduced because of a reduction of peripheral dendrites. This reduction was accompanied by an increase in the number of stem dendrites originating from the perikaryon. The density of spines on these proximal dendrites was larger in cultured granule cells than in controls. Our results suggest that the lack of major extrinsic (entorhinal) afferents that normally terminate on peripheral granule cell dendrites causes retraction of these dendrites. At the same time, there is growth of proximal dendritic portions. Proximal dendrites are targets of associational fibers, which are known to sprout under these culture conditions.

Abnormal Keratin 1 and 10 Cytoskeleton in Cultured Keratinocytes from Epidermolytic Hyperkeratosis Caused by Keratin 10 Mutations

Epidermolytic hyperkeratosis is caused by mutations of the differentiation-specific keratins K1 and K10. These mutations produce a weakened cytoskeleton that is prone to collapse resulting in cell fragility and lysis. In this study we have analyzed cultured keratinocytes from EHK patients bearing 10R-to-H and 15L-to-S mutations within the 1A segment of the K10 rod domain. Keratinocytes were grown submerged in serum-free medium and induced to differentiate by growing to confluence and increasing the Ca++ concentration in the medium. Cultures were either harvested for mRNA sequence analysis or subjected to immunofluorescence microscopy. Differentiating keratinocytes from these patients were found to express these K10 mutations in their mRNA. Moreover, these cells could be distinguished from normal keratinocytes by their aberrant morphology. EHK keratinocytes frequently exhibited a collapsed perinuclear network of K1/K10 filaments and sometimes peripheral granules of K1 and K10 aggregates, reminiscent of the cells of the suprabasal layers in these patients. This report documents the expression of mutant keratin 10 in cultured EHK keratinocytes.

Distribution of GABA A and GABA B binding sites in the cochlear nucleus of the guinea pig

We compared the distribution of GABA A and GABA B binding sites in the cochlear nucleus using quantitative receptor autoradiography with [ 3H]GABA. To visualize GABA A binding sites, GABA B binding sites were blocked with±baclofen. To visualize GABA B binding sites, isoguvacine was used to block GABA A binding sites. GABA A binding sites predominated over GABA B, although there were marked regional differences in the distribution of binding. In the ventral cochlear nucleus, GABA A and GABA B binding sites were concentrated in the peripheral granule cell cap, with low binding levels in the central region. In the dorsal cochlear nucleus, binding was concentrated in the superficial (fusiform and molecular) layers, with a distinct laminar pattern. GABA A binding sites predominated in the fusiform cell layer. The molecular layer contained the highest level of GABA B binding sites in the entire cochlear nucleus. These results suggest that GABAergic inhibition in the cochlear nucleus is mediated both by GABA A and GABA B receptors, particularly in the dorsal cochlear nucleus. However, low levels of binding in areas such as the magnocellular regions of the ventral cochlear nucleus, known to contain abundant GABAergic synapses, suggest heterogeneity of GABA receptors in this auditory nucleus.

Evidence of a progressive degranulation of basophil in subjects operated on without adverse reaction

We have studied the effect of surgery under general anaesthesia on whole blood histamine (WBH) and on basophil morphology in patients operated on without adverse reaction. In 10 patients administered a muscle relaxant and other anaesthetic drugs, WBH decreased significantly the day after surgery and returned to normal within 5 days. Basophils were examined after Percoll gradient concentration: after surgery, the granules were paler and located at the periphery of the cell in a great number of cell. Twentyfour hours later, the number of basophils was reduced and they often did not stain. The morphology returned to normal within 5 days. The variations of WBH were significantly related to the variations of the staining intensity and inversely related to the percent of basophils with peripheral granules. These findings indicate that surgery and/or general anaesthesia leads to a progressive release of mediators by basophils.

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.

The development and zoospore ultrastructure of a polycentric chytridiomycete gut fungus, Orpinomyces joyonii comb.nov.

Orpinomyces bovis, a polycentric gut fungus isolated from a steer, was examined with both light and electron microscopy and renamed Orpinomyces joyonii comb.nov. on the basis of its general morphology and zoospore ultrastructure. The multinucleate rhizomycelium is extensively branched, and sporangia form exogenously on branched or unbranched sporangiophores. The organelles in the zoospores have a distribution pattern typical of other gut fungi, i.e., anterior ribosomal aggregates, central nuclei, and posterior presumptive hydrogenosomes. The perikinetosomal apparatus in O. joyonii is comparable to that in monocentric gut fungi but with minor variations. New details of the posterior dome are described. It contains highly ordered specialized lamellae, peripheral granules, and megatubules. Microtubules intersect the dome predominantly at approximately right angles to its surface; this differs from monocentric gut fungi, in which microtubules form a posterior fan running parallel to the dome. We suggest that both monocentric and polycentric gut fungi are monophyletic, since both have a similar, distinctive perikinetosomal apparatus, posterior dome, and organelle distribution pattern. Key words: Orpinomyces joyonii, gut fungi, ultrastructure, posterior dome, perikinetosomal apparatus.

Eimeria apsheronica in the goat: Endogenous development and host cellular response

Most first generation schizonts of Eimeria apsheronica developed in the jejunum; others were distributed throughout the small intestine and occasionally in the caecum. Some were also found in the mesenteric lymph nodes, which were oedematous and haemorrhagic. In the intestine, haemorrhage and congestion were seen before parasites were detected, and continued throughout all later stages. Schizonts occurred in the lamina propria and occasionally in the submucosa, where they sometimes caused a cellular inflammatory response. Schizonts were first seen at 8 days post-infection (DPI); they had poorly defined nuclei and were enclosed in a capsule-like wall. At 16 DPI, many had matured, had a mean size of 125 × 82 μm, and were filled with numerous spindle-shaped merozoites, which were in ranks and loops. At 18 and 20 DPI, when small white lesions (1–3 mm in diameter) were observed in the jejunum and elsewhere in the small intestine, a second generation of schizonts, macrogametes, microgametocytes and maturing oocysts were seen, in the epithelial cells of the small intestine and caecum. Their mean sizes, respectively, were: 26.2 × 18.9, 24.7 × 18.5, 30.2 × 21.7and 26.6 × 19.3 μm. Macrogametes contained basophilic central and eosinophilic peripheral granules. The sexual stages were associated with a generalized cellular inflammatory response.

Compound versus multigranular exocytosis in peritoneal mast cells.

We have used the whole-cell patch-pipette technique to measure the step increases in the cell membrane capacitance (equivalent to the membrane area) caused by the fusion of secretory granules in degranulating murine mast cells. We have observed that up to 30% of the total membrane expansion caused by degranulation results from large fusion events that cannot be explained by the fusion of single secretory granules. These large events are observed mainly in the initial phase of a degranulation. We have developed a simple mathematical model for a mast cell to test whether these large events are caused by a stimulus-induced, granule-to-granule fusion that occurs before their exocytosis (multigranular exocytosis). Our results suggest that the large fusion events are caused by the exocytosis of granule aggregates that existed before stimulation and that are located at the cell's periphery. We propose a novel mechanism by which granule aggregates can be formed at the periphery of the cell. This mechanism relies on the ability of a transiently fused granule ("flicker") to fuse with more internally located granules in a sequential manner. This pattern may result in the formation of larger peripheral granules that later on can fuse with the membrane. The formation of peripheral granule aggregates may potentiate a subsequent secretory response.