Dense Vesicles Research Articles

Taxol inhibits endosomal-lysosomal membrane trafficking at two distinct steps in CV-1 cells.

Although taxol inhibits membrane trafficking, the nature of this inhibition has not been well defined. In this study, we define the effects of taxol on endocytosis in CV-1 cells using density gradient centrifugation of membranes over sorbitol density gradients. After taxol treatment, resident endosomal enzymes and the epidermal growth factor (EGF) receptor (EGFR) showed significant (P </= 0.05) enrichment in membranes with properties of early endosomes (fractions 4 and 5); the EGFR and Na+-K+-ATPase were also significantly (P </= 0.05) depleted in lysosomal fractions (fractions 10 and 11). The suggestion that taxol specifically reduces movement of endosomal constituents to lysosomes was supported by fluorescence microscopy studies revealing restriction of EGF to the peripheries of taxol-treated cells, in contrast to the perinuclear lysosomal-like distribution of EGF seen in controls. Kinetic studies with 125I-labeled EGF were also consistent with a taxol-induced block in traffic from endosomes and lysosomes after 15 min of uptake but also suggested an additional taxol-sensitive step in trafficking that involved redistribution of 125I-EGF within high-density compartments after 150 min. Related changes in cytoplasmic dynein distribution were observed within high-density compartments from taxol-treated cells, suggesting that this motor might participate in this later taxol-sensitive trafficking event. Electron microscopic examination of high-density membranes (fraction 12) showed that taxol increased the numbers of small (<500 nm) dense vesicles, with a relative depletion of the larger (>500 nm) vesicles found in controls. These data demonstrate that disruption of endocytic events by taxol includes the early accumulation of protein and endocytic markers in endosomes and the later accumulation in a dense compartment that we propose is a subdomain of the lysosomes.

Neurosecretion Competence, an Independently Regulated Trait of the Neurosecretory Cell Phenotype

Neurosecretion competence is intended as the ability of neurosecretory cells to express dense and clear vesicles discharged by regulated exocytosis (neurotransmitter release). Such a property, which so far has never been studied independently, is investigated here by a heterotypic cell fusion approach, using a clone of rat pheochromocytoma PC12 cells totally incompetent for neurosecretion that still largely maintains its typical molecular and cellular phenotype. When fused with wild-type partners of various species (rat, human) and specialization (PC12, neuroblastoma SH-SY5Y, HeLa), the defective cells reacquire their competence as revealed by the expression of their secretion-specific proteins. Fused wild-type cells therefore appear able to complement defective cells by providing them with factor(s) inducing the reactivation of their secretory program. The mechanism of action of these factors may consist not in a coordinate unblocking of transcription but in the prevention of a rapid post-transcriptional degradation of the mRNAs for secretion-specific genes.

Role of sialic acid in the endocytosis of prosaposin by the nonciliated cells of the rat efferent ducts.

The present study examines the mechanism of endocytosis of testicular prosaposin by the nonciliated cells of the efferent ducts. Testicular prosaposin is secreted by Sertoli cells into the lumen of the seminiferous tubules as a 70 kDa isomer where it binds to the tail of spermatozoa. In the efferent ducts, after dissociating from the plasma membrane of the spermatozoa, prosaposin is endocytosed by the nonciliated cells, presumably by receptor-mediated endocytosis. The initial step of receptor-mediated endocytosis usually results from the binding of a ligand's terminal oligosaccharide to a receptor on the cell surface. Thus, in the present study, several monosaccharides were injected in the lumen of the efferent ducts to compete with the binding and endocytosis of prosaposin. A quantitative electron microscopic approach was utilized and the number of gold particles, indicating anti-prosaposin immunoreactive sites, were scored over the various cell compartments including the plasma membrane, endocytic vesicles, early endosomes, and late endosomes. The length of the plasma membrane and the areas of endocytic vesicles, early endosomes, and late endosomes were measured with an image analyzer and the number of grains expressed per microm (plasma membrane) and microm2 (endocytic vesicles/endosomes) respectively. The quantitative analysis was performed in untreated animals (controls) and animals treated with various sugars (i.e., glucose, galactose, mannose, mannose 6-phosphate, N-acetylglucosamine and N-acetylgalactosamine) injected into the lumen of the efferent ducts at a concentration of 20 mM. Sialic acid caused the greatest decrease in the labeling density of the endocytic elements. Mannose 6-phosphate also caused a decrease in labeling but to a lesser extent. Various amounts of sialic acid (0.02 mM, 0.2 mM, 2 mM, 20 mM, and 200 mM) showed that most of these concentrations produced a significant decrease in the labeling density of endocytic vesicles and endosomes. Moreover, Western blots of prosaposin isolated from seminiferous tubular fluids followed by glycan analysis with Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), revealed that this protein has sialic acid residues that are terminally linked to galactose and/or N-acetylgalactosamine (alpha-NeuNAc-[2->6]-Gal and alpha-NeuNAc-[2->6]-GalNAc). These data indicate that testicular prosaposin is removed from the lumen of the efferent ducts by the noncialiated cells via a receptor that recognizes prosaposin's terminal sialic acid residues.

TrkB and TrkC Signaling Are Required for Maturation and Synaptogenesis of Hippocampal Connections

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trkB (-/-) and trkC (-/-) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10-P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21-49%) and decreased densities of axonal varicosities (8-17%) in both trkB (-/-) and trkC (-/-) mice. In addition, electron microscopic analyses showed that trkB (-/-) and trkC (-/-) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (-/-) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

Presence of heterogeneous peroxisomal populations in the rat nervous tissue

Peroxisomes were purified from the nervous tissue of 14-day-old rats by means of a Nycodenz gradient. Peroxisomal enzymes exhibited different sedimentation patterns: dihydroxyacetone phosphate acyl-transferase equilibrates at 1.142 g/ml together with the first peak of catalase; palmitoyl-CoA oxidase and d-amino acid oxidase activities are mainly recovered at 1.154 g/ml; the second peak of catalase is found at 1.175 g/ml. Morphological and semi-quantitative analyses of immunogold-labelled peroxisomes reveal profound heterogeneity of the particles. Very small (=0.2 μm diameter), electron dense vesicles containing catalase or thiolase, but devoid of other tested enzymes, are preferentially found in the light region, together with larger (>0.2 <0.3 μm) and less electron dense palmitoyl-CoA oxidase-positive peroxisomes. At intermediate density (1.154 g/ml) peroxisomes of more uniform size (0.25–0.27 μm), containing palmitoyl-CoA oxidase or thiolase with or without catalase are preferentially found. This population extends toward the densest region of the gradient, where very large d-amino acid oxidase-containing peroxisomes are also found. In this region, smaller peroxisomes, often polymorphic, which are catalase- and thiolase-positive and d-amino acid oxidase/palmitoyl-CoA oxidase-negative, are also observed. The possibility that the heterogeneity of neural peroxisomes may reflect both cellular heterogeneity and ongoing peroxisomal biogenesis is discussed.

The plant Golgi apparatus

The plant Golgi apparatus has an important role in protein glycosylation and sorting, but is also a major biosynthetic organelle that synthesises large quantities of cell wall polysaccharides. This is reflected in the organisation of the Golgi apparatus as numerous individual stacks of cisternae that are dispersed through the cell. Each stack is polarised: the shape of the cisternae and the staining of the membranes change in a cis to trans direction, and the cisternae on the trans side contain more polysaccharides. Numerous glycosyltransferases are required for the synthesis of the complex cell wall polysaccharides. Microscopy and biochemical fractionation studies suggest that these enzymes are compartmentalised within the stack. Although there is no obvious cis Golgi network, the trans-most cisterna or trans Golgi network often buds clathrin-coated and sometimes smooth dense vesicles as well. Here, vacuolar proteins are sorted from the secreted proteins and polysaccharides. This review highlights unique aspects of the organisation and function of the plant Golgi apparatus. Fundamentally similar processes probably underlie Golgi organisation in all organisms, and consideration of the plant Golgi specialisations can therefore be generally informative, as well as being of central importance to plant cell biology.

Scanning electron microscopic studies on the associations of vesicular arbuscular mycorrhizae in some Indian ferns

SEM studies were conducted on VAM associations in three ferns. Dense intracellular mycelia, terminal and intercalary vesicles were observed in cortical cells of Christella. VAM invasion in the stelar region of Microsorium and Nephrolepis is being reported for the first time.

BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.

The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS, PNS). In addition, exogenous application of neurotrophins has recently been found to modulate synaptic transmission in the rodent CNS. However, to provide evidence for a role of neurotophins as endogenous fast acting modulators of synaptic transmission, the synaptic localization and secretion of neurotrophins needs to be shown. We have now constructed a fusion protein consisting of N-terminal BDNF (the most abundant neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localization of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS-7 cells revealed that the cellular localization in the trans-Golgi network (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from the properties of untagged BDNF. Upon transient transfection of primary rat cortical neurons, BDNF-GFP was found in secretory granules of the regulated pathway of secretion, as indicated by colocalization with the secretory granule marker secretogranin II. BDNF-GFP vesicles were found in the neurites of transfected neurons with a pattern reminiscent of the localization of endogenous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were found predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immunocytochemical staining of synaptic terminals with synapsin I antibodies revealed that the density of BDNF-GFP vesicles is elevated in the vicinity of synaptic junctions, indicating that BDNF is localized appropriately to function as an acute modulator of synaptic transmission. These data suggest that BDNF-GFP will be a useful tool to investigate synaptic release of BDNF during physiological synaptic stimulation, and will thereby allow us to elucidate the participation of neurotrophin release in activity dependent synaptic plasticity.

Defect in synaptic vesicle precursor transport and neuronal cell death in KIF1A motor protein-deficient mice.

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron- specific microtubule plus end-directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769-780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.

Synchronized Spontaneous Ca2+ Transients in Acute Anterior Pituitary Slices

We investigated the organization of spontaneous rises in cytosolic free Ca2+ concentration ([Ca2+]i) due to electrical activity in acute pituitary slices. Real time confocal imaging revealed that 73% of the cells generated fast peaking spontaneous [Ca2+]i transients. Strikingly, groups of apposing cells enhanced their [Ca2+]i in synchrony with a speed of coactivation >1,000 microm/s. Single-cell injection of Neurobiotin or Lucifer yellow labeled clusters of cells, which corresponded to coactive cells. Halothane, a gap junction blocker, markedly reduced the spread of tracers. Coupling between excitable cells was mainly homologous in nature, with a prevalence of growth hormone-containing cells. We conclude that spontaneously active endocrine cells are either single units or arranged in synchronized gap junction-coupled assemblies scattered throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells may help shape the patterns of basal secretion.

Internalization of Iscom-Borne Antigens and Presentation under MHC Class I or Class II Restriction

Exogenous nonreplicating antigens (Ag) incorporated into immunostimulating complexes (iscoms) induce CTL responses under MHC class I restriction. A requirement for inducing CTL responses is that the Ag is delivered to the cytosol of antigen-presenting cells (APC), a route restricted to endogenously produced Ag. To investigate the mechanisms by which iscoms elicit MHC class I-restricted responses, the intracellular distribution of influenza virus envelope proteins incorporated in iscoms (flu-iscoms) or in micelles (flu-micelles) was studiedin vitrousing murine peritoneal cells (PEC). Ultrathin sections of cells pulsed with biotinylated flu-iscoms or flu-micelles were analyzed by electron microscopy after detection of the biotin label by reaction with streptavidin–gold. PEC pulsed with flu-iscoms showed a pattern of scattered gold particles distributed in clear and dense vesicles as well as in the intracellular space but not associated with organelles. In cells pulsed with flu-micelles, Ag was also detected in most cellular compartments but at a considerably lower concentration. The intracellular distribution of particulate Ag in iscom or micelle form was confirmed by lysis and differential centrifugation of Ag-pulsed APC. Furthermore, P815 cells pulsed with flu-iscoms were lysed by specific immune effectors showing that the iscom-Ag was processed and presented by class I-expressing APC. Flu-iscoms were internalized about 50-fold more efficiently than ovalbumin iscoms (ova-iscoms) suggesting that the nature of the protein and/or the presence of cellular receptors are important factors influencing the capacity of APC to take up iscom-borne proteins. PEC accounted for the most active internalization of iscom-borne Ag, although splenic dendritic cells and B cells also took up flu-iscoms with remarkable efficiency.

Parasympathetic varicosity proliferation and synaptogenesis in rat eyelid smooth muscle after sympathectomy

Parasympathetic innervation to eyelid smooth muscle inhibits sympathetic neurotransmission pre-junctionally without appreciable direct post-junctional effects. However, 5 weeks after sympathectomy, parasympathetic stimulation elicits substantial cholinergically mediated contractions. This study examined ultrastructural changes accompanying the conversion to parasympathetic excitation. In intact muscles, 64±9 nerve varicosities were encountered per 10 4 μm 2. Most were close to muscle cells and not fully enclosed by supporting cells. Axo–axonal synapses were observed occasionally. Two days following sympathectomy, varicosity numbers were reduced by 97% and, relative to controls, remaining varicosities were farther from muscle cells and more frequently fully enclosed by supporting cells, but contained greater numbers of small spherical and large dense vesicles. By 6 weeks post-sympathectomy, numbers of varicosities per unit muscle volume increased to 14% of controls. These varicosities differed from those at 2 days in being closer to smooth muscle cells, less frequently enclosed, and having fewer small vesicles. These findings indicate that intact eyelid smooth muscle varicosities are predominantly sympathetic, but a small number of parasympathetic varicosities are present, some of which may form pre-junctional synapses with sympathetic nerves. Between 2 days and 6 weeks post-sympathectomy, varicosities increased in number and established appositions with smooth muscle cells. This suggests that parasympathetic nerves are capable of re-innervating an atypical smooth muscle target after sympathectomy, and that parasympathetic synaptogenesis is likely to contribute to conversion from pre-junctional inhibition to post-junctional excitation after sympathectomy.

Presynaptic localization of G protein isoforms in the efferent nerve terminals of the mammalian cochlea

Heterotrimeric guanine nucleotide binding proteins (G proteins) are known to be involved in receptor-mediated synaptic activity. In order to determine which G protein isoforms, if any, are involved in synaptic regulation in the organ of Corti, we performed an extensive immunocytochemical screening. We localized a Gα q/11 isoform to the efferent nerve terminals using antibodies specific against the α subunit of these proteins. The label was observed in the efferent boutons contacting either the outer hair cells or the afferent fibers at the inner spiral bundle. We compared the localization of this isoform to that of the presynaptic protein SNAP-25 in double labeling experiments. Gα q/11 immunoreactivity was present predominantly in the cytoplasm of the presynaptic boutons in a region of high density of synaptic vesicles, while SNAP-25 was localized predominantly in the plasma membrane of the boutons. No label for these proteins was found at the afferent synapses, including the presynaptic terminals on hair cells. These results suggest that an isoform of the G q subfamily of the G proteins might be involved in presynaptic modulation of neurotransmitter release at the cochlear efferents.

Vesicle transfer of storage proteins to the vacuole: The role of the Golgi apparatus and multivesicular bodies

Summary The storage proteins vicilin and legumin pass through the Golgi apparatus (GApp) on their way to the protein storage vacuole (PSV). Upon entering the GApp vicilin and legumin are efficiendy segregated to the periphery of the cisternae where they begin to assemble into electron-dense aggregates. These form the nuclei of so-called dense vesicles (DV), which, presumably by cisternal progression through the stack, eventually reach the trans face of the GApp. By this time their contents have become even more condensed and many have obtained a partial clathrin coat on their cytosolic surface. Clathrin coated vesicles (CCV), which appear to bud from the DV may represent a mechanism for the retrieval of proteins missorted into the DV. The DV do not transfer their contents direct to the PSV. Instead, as is the case in yeasts and mammalian cells, an intermediate compartment is implicated. In pea cotyledon cells this takes the form of large (at least 1 μm diameter), multivesicular bodies (MVB). Double immunogold labeling with globulin antisera suggests that the MVB fuse with the PSV leading to a stratification of storage proteins in deposits at the tonoplast. Experiments with monensin lead to a depletion of the contents of the MVB and a redirection of globulin deposition to the cell surface. CCV, DV and MVB have been probed with antibodies against the vacuolar sorting receptor BP-80. CCV and MVB react positively, whereas there is no signal with the DV.

Structure and Function of Gustatory Neurons in the Nucleus of the Solitary Tract. III. Classification of Terminals Using Cluster Analysis

In sensory systems, insight into synaptic arrangements on cells of known physiological response properties has helped our understanding of the structural basis for these properties. To carry out these types of studies, however, synaptic types in the region of interest must be defined. Unfortunately, defining synaptic types in the brainstem has proved to be a challenging enterprise. Our study was done to classify synapses in the gustatory part of the nucleus solitarius using objective quantitative criteria and a cluster analysis procedure. Cluster analysis allows classification of a population of objects, such as synaptic terminals, into groups that exhibit similar characteristics. Six terminal types were identified using cluster analysis and subsequent analyses of variance and post hoc tests. Unlike classification schemes used for the cerebral cortex, where synaptic apposition density thickness and shape of vesicles is useful (Gray's Type I and II synapses), the concentration of vesicles in a terminal was a more useful measurement with which to classify terminals in the nucleus solitarius. To validate that vesicle density (vesicles/μm2) is a useful defining characteristic to classify terminals in the nucleus solitarius, terminals of a known type were used. GABAergic terminals were identified using postembedding immunohistochemical techniques, and their vesicle density was determined. GABAergic terminals fall into the range of two of the terminal types defined by the cluster analysis and, based on vesicle density, two types of GABAergic terminals were identified. We conclude that vesicle density is a helpful means to identify synapses in this brainstem nucleus.

ＤＨＡと脂質膜表面構造変化による神経機能調節

Rats were fed either linoleate-rich safflower oil or α-linolenate-rich perilla oil diets. The brightness-discrimination learning performance was significantly higher in the perilla oil group than in the safflower oil group. In synapses of hippocampus CA1, the density of synaptic vesicles was significantly higher in the perilla oil-fed rats than the safflower oil-fed rats. The number of terminals with low density of synaptic vesicles was increased in the safflower oil group after the learning. Fourier transform infrared spectroscopy of rat brain microsomes revealed that the second derivative spectrum at 1050-1250 cm-1showed difference between perilla and safflower oil groups after the learning. The infrared band at 1727 cm-1 in the safflower oil group moved to the higher wavenumber position at 1731 cm-1in the perilla oil group after the learning. The results suggested that the hydrogen bond of fatty acid ester (sn-2) with water was weakened and moved inward of the membrane, and resulted in a lowered hydration. This was confirmed by the weakened reactivity of the membrane phospholipids to phospholipase A2 in the perilla oil group after the learning. These results suggested that n-3 polyunsaturated fatty acid-deficiency may affect the turnover of synaptic vesicles in hippocampus with loss of learning ability.

Parafusin is a membrane and vesicle associated protein that cycles at exocytosis

In the unicellular eukaryote Paramecium tetraurelia, stimulation of exocytosis leads to Ca2+ activation of an alpha Glc-1-phosphodiesterase that dephosphoglucosylates the phosphoglycoprotein parafusin (PFUS). This process fails in exo mutant nd9 and also in the absence of Ca2+ influx upon stimulation suggesting that PFUS dephosphoglucosylation may be causally related to exocytosis. To further corroborate the hypothesis that PFUS is involved in the molecular events in exocytosis, we used laser confocal scanning microscopy and a PFUS specific peptide antibody to perform localization studies of PFUS in wild type (wt) and mutant Paramecium. In unstimulated wt cells, PFUS was associated both with the exocytic site of the cell membrane and with the membrane of the dense core secretory vesicles. Localization at these two sites was shown to be independent of each other since the exocytosis mutant (exo-) tam8, in which docking of its vesicles is blocked, still showed cell membrane staining. Immunofluorescence and immunoblotting of isolated intact secretory vesicles also revealed PFUS association. Upon stimulation of exocytosis, PFUS dissociated from both the dense core secretory vesicles and the cell membrane in a Ca(2+)-dependent manner. During recovery of exocytic capacity, PFUS reassociated with the newly formed secretory vesicles in the cytoplasm prior to their docking at the exocytic sites. Immunoblot analysis of PFUS during this time showed no changes in levels of the protein. Stimulation of exocytosis in wt in Mg2+ buffer or in the exo- temperature sensitive mutant (nd9) at the non-permissive temperature did not lead to dissociation of the PFUS. We conclude that PFUS is a novel critical component whose cycling probably participates in the molecular exocytic fusion machinery in these cells.

Haplosporidiumsp. (Haplosporidia) in Hatchery-Reared Pearl Oysters,Pinctada maxima(Jameson, 1901), in north Western Australia

A species ofHaplosporidiumis described from 6 of 105 pearl oyster (Pinctada maxima) spat from a hatchery at Carnarvon in north Western Australia. Presporulation and sporulation stages occurred in the connective tissue surrounding the digestive gland and were common in mantle connective tissue, but only light to moderate infections were observed in the heart, gills, foot, and adductor muscle. No stages were observed in epithelia, and the gut and diverticulae remained intact. Ultrastructurally, sporoblasts were binucleate with the two nuclei either closely apposed to form a diplokaryon or separated, with the cytoplasm containing round to pyriform haplosporosomes. In spores the sporoplasm contained a basal, or sometimes equatorial, nucleus, dense ovoid vesicles that appeared to develop to ovoid haplosporosomes with an internal axehead-shaped membrane, and spherical haplosporosomes. Apparently senescent spores with a dense content were also present and they, and normal spores, were ornamented with filaments placing the parasite in the genusHaplosporidium.

Early organogenesis and cell contacts in the proliferating hypophysis of the developing mouse.

The mouse foetal hypophysis which contains the diencephalic downgrowth (Dd) and Rathke's pouch (Rp) was examined using morphological and immunocyto/histo-chemical methods to study the cell-cell and cell-extracellular matrix (ECM) interactions that occur during early organogenesis. While many studies have described that the differentiation of ACTH cells precedes the proliferation of other endocrine cells in the Rp, light and conventional transmission electron microscopic (TEM) investigations have failed to differentiate between the various endocrine cell types in the proliferating distal lobe at the late mid-foetal stage. Conversely, TEM studies have shown that the occurrence of dense secretory vesicles in glandular cells of the presumptive pars intermedia in close apposition with the presumptive neural lobe by a basal lamina, is the earliest sign of endocrine activity in the foetal hypophysis. However, a confocal laser scanning microscopy (CLSM) and fine structure study did not observe direct cell-to-cell contacts between the Dd, Rp and their associated mesenchyme at the late mid-foetal stage. Using CLSM and TEM, we detected active cell turnover with programmed cell death in the proliferating Rp, Dd and their associated cephalic mesenchyme. Morphological findings indicated that apoptosis in the cephalic mesenchyme subsequently brings neurohypophyseal pituicytes and adenohypophyseal precursor stem cells into closer proximity, and alternations in the boundary cell surfaces might initiate signal transduction mediated via the intervening ECM at the proliferation stage.

Morphometric assessment of epidermal and mucous-biofilm changes caused by exposure of trout to chloramine-T or formalin treatment

Juvenile rainbow trout were exposed to therapeutic concentrations of formalin or chloramine-T to assess the effects of these chemicals on the morphology of the piscine epidermis and its mucous coat. Repeated treatment, once weekly for 4 weeks, with either chemical did not affect the mucous coat of the epithelium or the degree of folding of the basal lamina. However, treated fish had increased numbers of highly dense vesicles within the apical portions of epithelial cells. The epidermal mucous cells of chloramine-T-treated fish were significantly smaller than in controls. This effect was not noted in formalin-treated fish. Treatment with either chemical resulted in a significantly thinned epidermis. It is concluded that although chloramine-T and formalin may continue to be useful in the aquaculture industry they cause potentially harmful alterations to fish skin.