Electron-lucent Vacuoles Research Articles

Obesity worsens mitochondrial quality control and does not protect against skeletal muscle wasting in murine cancer cachexia.

More than 650 million people are obese (BMI>30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. Male C57BL/6 mice were given a purified high fat or standard diet for 16weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P=0.0001) but had reduced survival (33.3% vs. 6.67%, χ2 =10.04, P=0.0182). Obese LLC mice had reduced muscle weights (-24%, P<0.0354) and mCSA (-16%, P=0.0004) with similar activation of muscle p65 (P=0.0337), and p38 (P=0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P=0.0072) consistent with reductions in volitional cage activity (-39%, P<0.0001) and grip strength (-41%, P<0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P=0.0009; SS: -21%, P=0.0101) and LLC (IMF: -40%, P=0.0019; SS: -27%, P=0.0383) mice. Obese LLC mice had increased pAMPK (T172; P=0.0103) and reduced FIS1 (P=0.0029) and DRP1 (P<0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P=0.0395; SS: 138%, P=0.0174) in concert with an accumulation of p62 (P=0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P=0.0260; SS: 392%, P=0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.

A lower motor neuron disease in takahē (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease

Aims: To investigate the pathogenesis of a disease in takahē with intracytoplasmic inclusion bodies in lower motor neurons. Methods: Four cases aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the bird with spinal dysfunction were examined by electron microscopy. Results: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D so they were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method. A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. Conclusions: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Ultrastructure of the intestinal system in unfed larvae of Limnesia maculata (O.F. Müller, 1776) (Acariformes, Limnesiidae)

The intestinal system of unfed fresh-water mite larvae Limnesia maculata (O.F. Müller, 1776) (Acariformes, Limnesiidae) has been studied with transmission electron microscopy. The intestinal system is composed of the foregut, including the pharynx and the esophagus, the sac-like blind midgut and the excretory organ. The pharynx begins with the mouth covered by the labrum. The pharynx runs along the bottom of the gnathosoma and shows ventral dilators. No valves are expressed between the pharynx and the esophagus. The esophagus possesses strongly plicate walls and, before entering the midgut, passes through the brain. The sac-like midgut does not reveal a well-pronounced lumen, developed epithelium and separate lobes. It consists of the two cell types of endoderm origin mixed in the midgut volume. The first type – the vacuolated cells – does not possess Golgi bodies and lysosomal apparatus and shows electron-lucent vacuoles with a granular inclusion inside. These cells apparently do not take part in digestion of the embryonic yolk. The second type – the non-vacuolated cells – shows both, a well developed Golgi complex and large heterolysosomes, and obviously digests the embryonic yolk. Consequently, they may be attributed as specialized vitellophages. Nevertheless, both cell types may take part in formation of the definitive midgut epithelium. The sac-like thin-walled excretory organ is strongly dilated and contains the embryonic wastes in the form of electron-dense globules and birefringent particles. No muscle envelope surrounds the excretory organ. The embryonic wastes together with wastes accumulated during feeding may be evacuated from the organ only after completion of feeding. The excretory canal on this developmental stage is not connected with the excretory organ. It opens to the outside with a simple slit-like excretory pore. Before feeding, larvae have to pass the process of the post-molt development before their midgut would be ready to receive nutrients.

Ultrastructure of the prosomal gland complex in unfed larvae of the fresh-water mite Limnesia maculata (Müller, 1776) (Acariformes, Limnesiidae)

The salivary and coxal glands in unfed larvae of the fresh-water mite Limnesia maculata (Müller, 1776) (Acariformes, Limnesiidae) were studied using light and electron microscopy. Three pairs of the salivary and one pair of the coxal glands constitute the prosomal gland complex. The salivary glands pertain to simple alveolar glands consisting of the single acinus with prismatic cells arranged around the intra-alveolar lumen. Owing to their spatial distribution, the salivary glands may be termed as medial, ventral and lateral. While the ventral and lateral glands are arranged symmetrically, the medial glands are disposed one after another at the frontal wall of the synganglion (brain). The medial and the ventral glands possess mostly electron-dense secretory granules supposedly of protein nature. The lateral glands show variable organization mainly having electron-lucent vacuoles. No well pronounced rough endoplasmic reticulum and Golgi bodies were discernible in the salivary glands. The coxal glands are well developed and composed of (i) the main portion (the so-called labyrinth) including at least two tightly opposed loops and extending backward along the brain, and (ii) the very short anterior portion/end piece transforming anteriorly into the terminal sac/bladder. The cells of the main portion are provided with apical microvilli, whereas the basal infolding/basal labyrinth is lacking. The cells of the anterior portion do not show the apical microvilli and the basal infoldings. The terminal sac is lined by a cuticle and has an ectodermal origin. The ducts of the salivary glands come together at the dorsal aspect of the bladder, to which they open forming the common podocephalic duct anteriorly. The latter opens on each side into the subcheliceral space. The well-developed podocephalic system suggests its active role in the living processes such as feeding and ion-water balance.

Antifungal Activity of the Biphosphinic Cyclopalladate C7a against Candida albicans Yeast Forms In Vitro and In Vivo.

Vulvovaginal and invasive candidiasis are frequent conditions in immunosuppressed individuals caused by Candida albicans and non-albicans Candida spp. Fluconazole and Amphotericin B are the main drugs used to fight the infection. However, resistance to fluconazole and other azole antifungal drugs is an important clinical problem that encourages the search for new therapeutic alternatives. In this work, we evaluate the antifungal activity of the biphosphinic cyclopalladate C7a in the in vitro and in vivo model. Our results showed fungicidal activity, with low values of minimal inhibitory concentrations and minimum fungicidal concentrations, even for fluconazole and/or miconazole resistant Candida isolates. Fluorescence microscopy and transmission electron microscopy revealed that the compound was able to inhibit the formation of hyphae/pseudohyphae and, moreover, promoted morphological alterations in cellular organelles and structures, such as disruption of cell wall, apparent mitochondrial swelling, chromatin marginalization into the nuclei and increased numbers of electron-lucent vacuoles. C7a significantly decreased the biofilm formation and reduced the viability of yeast cells in mature biofilms when tested against a virulent C. albicans strain. In vivo assays demonstrated a significant decrease of fungal burden in local (vaginal canal) and disseminated (kidneys) infection. In addition, we observed a significant increase in the survival of the systemically infected animals treated with C7a. Our results suggest C7a as a novel therapeutic agent for vaginal and disseminated candidiasis, and an alternative for conventional drug-resistant Candida.

Miltefosine is fungicidal to Paracoccidioides spp. yeast cells but subinhibitory concentrations induce melanisation

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungi Paracoccidioides spp. The duration of antifungal treatment ranges from months to years and relapses may nevertheless occur despite protracted therapy. Thus, there remains an urgent need for new therapeutic options. Miltefosine (MLT), an analogue of alkylphospholipids, has antifungal activity against species of yeast and filamentous fungi. The aim of this study was to evaluate the antifungal effects of MLT on the yeast forms of Paracoccidioides brasiliensis and Paracoccidioides lutzii. MLT demonstrated inhibitory activity from 0.12 to 1 µg/mL, which was similar to amphotericin B or the combination trimethoprim/sulfamethoxazole but was not more effective than itraconazole. The fungicidal activity of MLT occurred at concentrations ≥1 µg/mL. Ultrastructural alterations were observed following exposure of the fungus to a subinhibitory concentration of MLT, such as cytoplasmic membrane alteration, mitochondrial swelling, electron-lucent vacuole accumulation and increasing melanosome-like structures. Melanin production by yeasts following MLT exposure was confirmed by labelling with antibodies to melanin. In addition, the combination of a subinhibitory concentration of MLT and tricyclazole, an inhibitor of DHN-melanin biosynthesis, drastically reduced yeast viability. In conclusion, MLT had a fungicidal effect against both Paracoccidioides spp., and a subinhibitory concentration impacted melanogenesis. These findings suggest that additional investigations should be pursued to establish a role for MLT in the treatment of PCM.

Imidacloprid impairs the post-embryonic development of the midgut in the yellow fever mosquito Stegomyia aegypti (=Aedes aegypti).

The mosquito Stegomyia aegypti (=Aedes aegypti) (Diptera: Culicidae) is a vector for the dengue and yellow fever viruses. As blood digestion occurs in the midgut, this organ constitutes the route of entry of many pathogens. The effects of the insecticide imidacloprid on the survival of St. aegypti were investigated and the sub-lethal effects of the insecticide on midgut development were determined. Third instar larvae were exposed to different concentrations of imidacloprid (0.15, 1.5, 3.0, 6.0 and 15.0 p.p.m.) and survival was monitored every 24 h for 10 days. Midguts from imidacloprid-treated insects at different stages of development were dissected and processed for analyses by transmission electron microscopy, immunofluorescence microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays. Imidacloprid concentrations of 3.0 and 15.0 p.p.m. were found to affect midgut development similarly. Digestive cells of the fourth instar larvae (L4) midgut exposed to imidacloprid had more multilamellar bodies, abundantly found in the cell apex, and more electron-lucent vacuoles in the basal region compared with those from untreated insects. Moreover, imidacloprid interfered with the differentiation of regenerative cells, dramatically reducing the number of digestive and endocrine cells and leading to malformation of the midgut epithelium in adults. The data demonstrate that imidacloprid can reduce the survival of mosquitoes and thus indicate its potentially high efficacy in the control of St. aegypti populations.

Polycystic kidneys and GM2 gangliosidosis-like disease in neonatal springboks (Antidorcas marsupialis).

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 μm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 μm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies.

Ultrastructure of the Male Cloacal Glands of the Eastern Red-backed Salamander, Plethodon cinereus

Abstract: This paper represents the first ultrastructural descriptions of the cloacal glands of a male salamander. All male salamanders in the suborder Salamandroidea possess these glands, which are required for internal fertilization. These glands are responsible for producing spermatophores and, in some species, certain glands produce female-attracting pheromones. Both scanning and transmission electron microscopy provided higher resolution and magnification of the cytology of cloacal glands than possible in previous studies limited to light microscopy. The glands are hypertrophied in October and April samples, and much reduced in size and secretory activity in June and August samples. Each of the cloacal glands, when active, has secretory vacuoles that are unique in appearance and that secrete carbohydrates and/or proteins. Kingsbury's glands are modified mucous glands that have secretory vacuoles filled with a flocculent substance. Active pelvic glands have uniform electron-lucent secretory vacuoles ...

Extreme Signet Ring Cell Change in a Large B-Cell Lymphoma of Follicular Origin

We report a large B-cell lymphoma of follicular origin with extreme signet ring cell differentiation. Initially classified as follicular lymphoma on a fine needle core biopsy, the presence of cohesive sheets of extrafollicular signet ring cells triggered an excisional biopsy for further characterization. The excised lymph node revealed focal follicular hyperplasia, follicular lymphoma, and a neoplasm composed of vague nodules and sheets of large atypical cells, all of which virtually exhibited large clear intracytoplasmic vacuoles with peripheral displacement of nuclei. The tumor cells were negative for mucin and lacked immunoreactivity with pancytokeratin, but were strongly immunoreactive with CD20, BCL-2, BCL-6, and CD10 antibodies. Electron microscopy studies revealed electron-lucent vacuoles with no particular internal structure. This case is unique in that extreme signet ring cell differentiation somewhat obscured the true cytological identity of the interfollicular lymphoma and suggested alternative diagnoses.

Hematodinium sp. and its bacteria-like endosymbiont in European brown shrimp (Crangon crangon)

BackgroundParasitic dinoflagellates of the genus Hematodinium are significant pathogens affecting the global decapod crustacean fishery. Despite this, considerable knowledge gaps exist regarding the life history of the pathogen in vivo, and the role of free living life stages in transmission to naïve hosts.ResultsIn this study, we describe a novel disease in European brown shrimp (Crangon crangon) caused by infection with a parasitic dinoflagellate of the genus Hematodinium. This is the second example host within the Infraorder Caridea (shrimp) and significantly, the first description within the superfamily Crangonoidea. Based upon analysis of the rRNA gene (SSU) and spacers (ITS1), the parasite in C. crangon is the same as that previously described infecting Nephrops norvegicus and Cancer pagurus from European seas, and to the parasite infecting several other commercially important crab species in the Northern Hemisphere. The parasite is however distinct from the type species, H. perezi, found infecting type hosts (Carcinus maenas and Liocarcinus depurator) from nearby sites within Europe. Despite these similarities, the current study has also described for the first time, a bacteria-like endosymbiont within dinospore stages of the parasite infecting shrimp. The endosymbionts were either contained individually within electron lucent vacuoles within the parasite cell cytoplasm, or remained in direct contact with the parasite cytoplasm or in some cases, the nucleoplasm. In all of these cases, no apparent detrimental effects of colonization were observed within the parasite cell.ConclusionsThe presence of bacteria-like endosymbionts within dinospore life stages presumes that the relationship between the dinoflagellate and the bacteria is extended beyond the period of liberation of spores from the infected host shrimp. In this context, a potential role of endosymbiosis in the survival of free-living stages of the parasite is possible. The finding offers a further intriguing insight into the life history of this enigmatic pathogen of marine crustacean hosts and highlights a potential for mixotrophy in the parasitic dinoflagellates contained within the genus Hematodinium.

Abstract B37: F14512, a polyamine-vectorized anticancer drug, triggers senescence cell death mechanism.

Abstract Drugs specifically vectorized to cancer cells may offer a reinforced activity, resulting in an improved therapeutic index. In this context, F14512 exploits the Polyamines Transport System (PTS) to accumulate into cells and exhibits an enhanced anti-proliferative activity on a large panel of tumor cell lines as compared to etoposide. At the molecular level, the spermine tail of F14512 contributes to enhance the water solubility of the drug, and reinforces the activity of the drug toward its primary molecular target, topoisomerase II. This observation has encouraged the set up of a phase 1 clinical study with F14512 in patients with relapsed or refractory acute myeloid leukemia (AML). In order to determine how these molecular properties translate at the cellular level, we have compared the kinetic of cell cycle modulation associated with the inhibition of cell proliferation. F14512 proved to be &amp;gt;30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent, nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased -galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 as compared to etoposide, and this difference may account for their distinct pharmacological profiles and the superior activity of F14512 in vivo. The identification of such response markers for senescence are currently pursued on in vivo MX-1 models by following a transcriptomic analysis focused on cell death cascades including apoptosis and senescence. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor response, applicable in clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B37.

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.

Trypanosoma cruzi: Altered parasites after in vitro treatment with gangliosides, a therapeutic agent in experimental Chagas’ disease

Biochemical and structural modifications were investigated in axenic cultured Trypanosoma cruzi after treatment with gangliosides. Fluorescence anisotropy showed dose dependent increments in parasite membranes of ganglioside treated epimastigotes. NADP-GDH activity increased in parasites treated at day 4 (13%), 7 (137.2%), and 14 (28.50%) while NAD-MDH but decreased from day 7 to 21 (−5.74%, −32.22%, −27.92%). Treated parasites presented electron-lucent vacuoles opposite to the cytostoma, multilamellar bodies and dilated mitochondrion cristae, disorganized kinetoplast and altered heterochromatin structure. Gangliosides inhibited fusogenic ability (80%) and PLA2 activity (>75%) from the parasite. The same occurred with anti-PLA2 antibodies. Trypomastigotes suffered loss of cytoplasmic material and organelles when GM1 was present in culture medium. We propose that exogenous gangliosides produced: altered lipid order, inhibited membrane enzymes, the parasite energy source shifted from glucose to amino acids, ending on a structural transformation which signals parasite cell death.

A Case of Familial Lecithin-cholesterol Acyltransferase (LCAT) Deficiency

Purpose: To report a case of a familial lecithin cholesterol acyltransferase (LCAT) deficiency patient with bilateral corneal opacities. Case summary: A 26-year-old man with bilateral corneal opacities visited our hospital. We took slit lamp examination, corneal thickness measurement, corneal endothelial cell counts and fundus examination. Blood and urine tests were included. Kidney biopsy was done. The tissues were observed by a light microscopy and an electron microscopy. Hemolytic anemia, proteinuria, hematuria, hypertriglyceridemia, decreased HDL cholesterol level, and lecithin cholesterol acyltransferase (LCAT) deficiency were found. At kidney biopsy, electron-lucent vacuoles and lamellar inclusion body were found. Conclusions: Bilateral corneal opacities can be an imporant clinical sign of systemic disease which is caused by abnormal lipid metabolism like the familial lecithin cholesterol acyltransferase (LCAT) deficiency.

The ring nerve of the box jellyfish Tripedalia cystophora

Box jellyfish have the most elaborate sensory system and behavioural repertoire of all cnidarians. Sensory input largely comes from 24 eyes situated on four club-shaped sensory structures, the rhopalia, and behaviour includes obstacle avoidance, light shaft attractance and mating. To process the sensory input and convert it into the appropriate behaviour, the box jellyfish have a central nervous system (CNS) but this is still poorly understood. The CNS has two major components: the rhopalial nervous system and the ring nerve. The rhopalial nervous system is situated within the rhopalia in close connection with the eyes, whereas the ring nerve encircles the bell. We describe the morphology of the ring nerve of the box jellyfish Tripedalia cystophora as ascertained by normal histological techniques, immunohistochemistry and transmission electron microscopy. By light microscopy, we have estimated the number of cells in the ring nerve by counting their nuclei. In cross sections at the ultrastructural level, the ring nerve appears to have three types of neurites: (1) small "normal"-looking neurites, (2) medium-sized neurites almost completely filled by electron-lucent vacuoles and (3) giant neurites. In general, only one giant neurite is seen on each section; this type displays the most synapses. Epithelial cells divide the ring nerve into compartments, each having a tendency to contain neurites of similar morphology. The number and arrangement of the compartments vary along the length of the ring nerve.

Ultrastructural Changes of Schwann Cells during Nerve Regeneration Following a Crush Injury of the Sural Nerve in Rats

Abstract Twenty-four male albino rats (200 to 250 g in weight) were used in the present study. The left sural nerve of 18 rats was subjected to crush injury while the sural nerves of 6 animals were used as control. After one week of the crush injury the Schwann cells showed multiple cytoplasmic processes. Those with long cytoplasmic processes wrapped only one axon while those wrapping multiple unmyelinated axons showed shorter processes. Some of these processes wrapped or surrounded collagen bundles “collagen pockets” and degenerated myelin, and some contained electronlucent vacuoles. Schwann cell cytoplasm showed asymmetric hypertrophy and contained dilated rough endoplasmic reticulum and ribosomes. Also, electronlucent vacuoles and whorls of degenerated myelin were seen in the cytoplasm of some Schwann cells. Schwann cells were surrounded by basal laminae which may be redundant. Two weeks post-crush, the number of regenerating Schwann cells increased and the myelin sheaths covering the myelinated axons were thicker. Schwann cells possessed long cytoplasmic processes that wrapped unmyelinated axons. After the third week of the crush injury, the Schwann cells wrapped shrunken myelinated axons with degeneration of the myelin of such axons. The number of myelinated axons increased, together with the thickness of their myelin sheath. It could be concluded that the Schwann cells play a phagocytic role during regeneration of peripheral nerves which is indicated by the presence of cytoplasmic vacuoles and degenerated myelin. Such phagocytic process might be performed by the use of their cytoplasmic processes.

Effects of tricyclazole (5-methyl-1,2,4-triazol[3,4] benzothiazole), a specific DHN–melanin inhibitor, on the morphology ofFonsecaea pedrosoi conidia and sclerotic cells

The influence of tricyclazole (5-methyl-1,2,4-triazol[3,4]benzothiazole), a specific DHN-melanin inhibitor, on the cell walls and intracellular structures of Fonsecaea pedrosoi conidia and sclerotic cells was analyzed by transmission electron microscopy (TEM), deep-etching, and field emission scanning electron microscopy. The treatment of the fungus with 16 microg mL(-1) of tricyclazole (TC) did not significantly affect fungal viability, but electron microscopy observations showed several important morphological differences between TC-treated and non-TC treated cells. Control sclerotic cells presented patched granules, with an average diameter of 47 nm, on the cell surface, which were absent in TC-treated cells. Also, TC-treated sclerotic cells showed an undulated relief. TC treatment leads to an accumulation of electron lucent vacuoles in the fungal cytoplasm of both conidia and sclerotic cells, and treated conidia observed by deep etching showed a relevant thickening of the fungal cell wall. Together, these observations support the previous data of our group that F. pedrosoi synthesizes melanin in intracellular organelles. In addition, we suggest that melanin is not only an extracellular constituent but could also be dispersing all over the cell walls and could have an effective role in cross-linking different cell wall compounds that help maintain the regular shape of the cell wall.

An ultrastructural investigation of the testes and spermiogenesis in Myobia murismusculi (Schrank) (Acari, Actinedida: Myobiidae)

Summary The stages of spermiogenesis in Myobia murismusculi were investigated on the basis of ultrastructural analysis of both the testes and the female organs: receptaculum seminis and seminal duct. The walls of the testes consist of a thin epithelial layer. Germ and secretory cells lie free in the lumen of the testes. In the early stages of differentiation, both cell types represent clusters of sister cells joined by intercellular bridges. Each secretory cell contains prominent RER and Golgi complex, which produce single dense granule. Growing gradually the granule fills the whole volume of the cell's cytoplasm. Mature secretory cells disintegrate and the secretory product discharges into the testicular lumen. The germ cells are represented by the early, the intermediate and the late spermatids as well as the immature sperm (prospermia). Neither spermatogonia nor meiotic figures were observed in adult males. As spermiogenesis starts, numerous narrow invaginations of the outer membrane (peripheral channels) develop on the cell surface. They form a wide circumferential network connected to pinocytotic vesicles. Owing to the secretory activity of the Golgi complex, a large acrosomal granule is formed in the early spermatids. A long acrosomal filament runs along the intranuclear canal. Nuclear material condenses and forms two spherical bodies of different electron density. The lighter one can be observed until the stage of the late spermatids, when the nuclear envelope almost completely disappears. The electron-dense nuclear body transforms into a definite chromatin body, which is observed in the mature sperm as a cup-shaped structure. The late spermatids are characterized by the presence of a large electronlucent vacuole, which seems to be unique for the process of spermiogenesis in Actinedida. After the spermia enter the female genital tract, the peripheral channels disappear as well as the vacuole. The cells form long amoeboid arms with a special microtubular layer underneath the plasma membrane. The chromatin body is encircled by a large acrosomal granule of complex shape provided by long extensions running deep into the cytoplasm. The cytoplasm contains no organelles except for a group of unmodified mitochondria in the post-nuclear region. The main characteristics of the Myobia spermiogenesis are discussed with regard to other actinedid mites.

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.