Myelin-like Figures Research Articles

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50=28.5±2.0μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.

English

Control of leishmaniasis remains a challenge due the high toxicity of the chemotherapeutic drugs presently available. The ongoing search for better leishmanicidal compounds has brought herbal drugs into the limelight as safe and effective substitutes to conventional therapies which have various drawbacks. The current study was designed to evaluate the antileishmanial effect of Quassia amara against Leishmania amazonensis and Leishmania infantum. Different fractions (Hexane (Q1), dichloromethane (Q2), ethyl acetate (Q3) and butanol (Q4)) obtained from the liquid-liquid partition of the crude Q. amara methanol extract were tested against Leishmania promastigote forms. After 120 h of treatment with Q. amara fractions (1 to 500 µg/ml), the minimal inhibitory concentration (MIC) was determined using resazurin. The most active fraction (Q2) was analyzed using thin layer chromatography (TLC) and high performance liquid chromatography-diode array detector (HPLC-DAD) techniques. The effects of Q2 on parasite ultrastructure were investigated by transmission electron microscopy. In addition, Leishmania-infected macrophages were treated with Q2 in order to evaluate the anti-amastigote effect. Among the fractions tested, Q2 showed the highest activity against L. amazonensis and L. infantum promastigotes MIC values of 62.5 and 31.25 µg/ml, respectively. There were ultrastructural alterations, such as nuclear chromatin condensation, intense vacuolization and autophagic and myelin-like figures in parasites treated with Q2 (62.5 µg/ml). Macrophages previously infected with L. amazonensis and L. infantum promastigotes showed a drastic reduction in the number of parasites recovered in the supernatant after Q2 treatment at MIC values. The TLC and HPLC fingerprints of Q2 showed that alkaloids were the main chemical constituents in this fraction. The results presented herein showed that the alkaloid-rich fraction Q2 is a promising source of antileishmanial agents. Further investigation will be necessary in order to isolate and test the substance(s) responsible for the bioactivity. Key words: Antileishmanial activity, Quassia genus, alkaloids, macrophage infection, nitric oxide.

Physalin F, a seco-steroid from Physalis angulata L., has immunosuppressive activity in peripheral blood mononuclear cells from patients with HTLV1-associated myelopathy.

Human T-lymphotropic virus type 1 (HTLV-1) induces a strong activation of the immune system, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Physalin F is a secosteroid with potent anti-inflammatory and immunomodulatory activities. The present study aimed to investigate the effects of physalin F on peripheral blood mononuclear cells (PBMC) of HAM/TSP subjects. A concentration-dependent inhibition of spontaneous proliferation of PBMC from HAM/TSP subjects was observed in the presence of physalin F, as evaluated by (3)H-thymidine uptake. The IC50 for physalin F was 0.97 ± 0.11 μM. Flow cytometry analysis using Cytometric Bead Array (CBA) showed that physalin F (10 μM) significantly reduced the levels of IL-2, IL-6, IL-10, TNF-α and IFN-γ, but not IL-17A, in supernatants of PBMC cultures. Next, apoptosis induction was addressed by using flow cytometry to evaluate annexin V expression. Treatment with physalin F (10 μM) increased the apoptotic population of PBMC in HAM/TSP subjects. Transmission electron microscopy analysis of PBMC showed that physalin F induced ultrastructural changes, such as pyknotic nuclei, damaged mitochondria, enhanced autophagic vacuole formation, and the presence of myelin-like figures. In conclusion, physalin F induces apoptosis of PBMC, decreasing the spontaneous proliferation and cytokine production caused by HTLV-1 infection.

Malpighian Tubules in Larvae of Diatraea saccharalis (Lepidoptera; Crambidae): A Morphological Comparison between Non-Parasitized and Parasitized by Cotesia flavipes (Hymenoptera; Braconidae)

In Diatraea saccharalis larvae, the Malpighian tubules are found along the digestive tube, extending from the middle mesenteric region to the end of the posterior intestine, where they come in contact with the rectum to form the cryptonephridium. Scanning and transmission electron microscopy of non-parasitized and parasitized larvae by Cotesia flavipes have indicated that the tubules consist of secretory and reabsorption cells. In parasitized larvae, the occurrence of hemocytes and teratocytes around the tubules is indicative of their role in immunological defense; however, they were not observed in non-parasitized larvae. At day 9 of parasitism, the mitochondria-containing vacuoles and myelin-like figures show signs of degeneration. The results of this study have confirmed that C. flavipes manipulates the physiology and biochemistry of D. saccharalis because the Malpighian tubules of the parasitized larvae remain active until the parasitoid completes its pupal stage and is released from the host organism.

Physalins B and F,seco-steroids isolated fromPhysalis angulataL., strongly inhibit proliferation, ultrastructure and infectivity ofTrypanosoma cruzi

We previously observed that physalins have immunomodulatory properties, as well as antileishmanial and antiplasmodial activities. Here, we investigated the anti-Trypanosoma cruzi activity of physalins B, D, F and G. We found that physalins B and F were the most potent compounds against trypomastigote and epimastigote forms of T. cruzi. Electron microscopy of trypomastigotes incubated with physalin B showed disruption of kinetoplast, alterations in Golgi apparatus and endoplasmic reticulum, followed by the formation of myelin-like figures, which were stained with MDC to confirm their autophagic vacuole identity. Physalin B-mediated alteration in Golgi apparatus was likely due to T. cruzi protease perturbation; however physalins did not inhibit activity of the trypanosomal protease cruzain. Flow cytometry examination showed that cell death is mainly caused by necrosis. Treatment with physalins reduced the invasion process, as well as intracellular parasite development in macrophage cell culture, with a potency similar to benznidazole. We observed that a combination of physalins and benznidazole has a greater anti-T. cruzi activity than when compounds were used alone. These results indicate that physalins, specifically B and F, are potent and selective trypanocidal agents. They cause structural alterations and induce autophagy, which ultimately lead to parasite cell death by a necrotic process.

Immunoreactive Endothelin-1 and Endothelin A Receptor in Basilar Artery Perivascular Nerves of Young and Adult Capybaras

The purpose of this qualitative morphological study was the immunocytochemical and ultrastructural comparison of perivascular nerves of the basilar artery (BA) of young (6-month-old) and adult (12-month-old) capybaras - adult capybaras showed regression of the internal carotid artery (ICA). The study focused on immunolabeling for the vasoactive peptide endothelin-1 (ET-1) and endothelin A receptor (ET_A) as well as for the synapse marker synaptophysin (SYP). In the BA of young capybaras, immunoreactivity for ET-1, ET_A receptor and SYP was detected in perivascular nerve varicosities and/or intervaricosities. Immunoreactivity for ET-1 and ET_A receptor was also displayed by some Schwann cells, which accompanied perivascular nerves. In addition to the presence of the above-described perivascular nerve characteristics, the BA of adult animals also revealed structurally altered perivascular nerves, where axon profiles were irregular in shape with dense axoplasm, while the cytoplasm of Schwann cells was vacuolated and contained myelin-like figures. These structurally altered perivascular nerves displayed immunoreactivity for ET-1, ET_A receptor and SYP. These results show that the ET-1 system is present in some of the BA perivascular nerves and it is likely that this system is affected during animal maturation when ICA regression takes place. The role of ET-1 in cerebrovascular nerves is still unclear but its involvement in neural (sensory) control of cerebral blood flow and nerve function is possible.

Activity of antimalarial drugs in vitro and in a murine model of cutaneous leishmaniasis

The currently used treatments for leishmaniasis, a neglected parasitic disease, are associated with several side effects, high cost and resistance of the Leishmania parasites. Here we evaluated in vitro and in vivo the antileishmanial activity of five antimalarial drugs against Leishmania amazonensis. Mefloquine was effective against promastigotes in axenic cultures and showed an IC50 (concentration giving half-maximal inhibition) value of 8.4±0.7 µM. In addition, mefloquine, chloroquine and hydroxychloroquine were active against intracellular amastigotes in macrophage-infected cultures, presenting IC50 values of 1.56±0.19 µM, 0.78±0.08 µM and 0.67±0.12 µM, respectively. The ultrastructural analysis of chloroquine- or mefloquine-treated amastigotes showed an accumulation of multivesicular bodies in the cytoplasm of the parasite, suggesting endocytic pathway impairment, in addition to the formation of myelin-like figures and enlargement of the Golgi cisternae. CBA mice were infected with L. amazonensis in the ear dermis, and treated by oral and/or topical routes with chloroquine and mefloquine. Treatment of L. amazonensis-infected mice with chloroquine by the oral route reduced lesion size, which was associated with a decrease in the number of parasites in the ear, as well as the parasite burden in the draining lymph nodes. In contrast, mefloquine administration by both routes decreased the lesion size in infected mice without causing a reduction in parasite burden. Our results revealed a promising antileishmanial effect of chloroquine and suggest its use in cutaneous leishmaniasis treatment.

Effect of thiazolidine LPSF SF29 on the growth and morphology of Trypanosoma cruzi

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic illness in Latin America. Efforts have been made by several groups to develop new effective and safe anti-T. cruzi drugs. In the present work, we show that thiazolidine LPSF SF29 inhibited growth of the epimastigote and amastigote forms and caused lysis in the trypomastigote form of T. cruzi, leading to death of the protozoan. Mitochondrial dysfunction was also observed. The thiazolidine induced ultrastructural alterations such as detachment of the flagellar membrane, intense mitochondrial swelling, formation of myelin-like figures and the appearance of autophagosomes. Taken together, these results suggest that this new thiazolidine is active against T. cruzi and constitutes a promising drug for the therapy of Chagas disease.

Trypanocidal activity of guaianolide obtained from Tanacetum parthenium (L.) Schultz-Bip. and its combinational effect with benznidazole

In the present study, we evaluated the in vitro antiprotozoal activity of a guaianolide (11,13-dehydrocompressanolide) isolated from Tanacetum parthenium against Trypanosoma cruzi and investigated the possible combinational effect of guaianolide and benznidazole. The isolated compound was shown to be effective against T. cruzi, with IC50 values of 18.1±0.8 and 66.6±1.3μM against the multiplicative epimastigote and amastigote forms, respectively. The best results were obtained against trypomastigotes, with an EC50 of 5.7±0.7μM. The guaianolide presented no toxicity in LLCMK2 cells (CC50 of 93.5μM) and was 16.4-fold more selective for trypomastigotes. The study of the combinational effect of benznidazole and guaianolide revealed the presence of a synergistic effect against the epimastigote form and marginal additive effect against the trypomastigote form. Striking morphological changes were observed in epimastigotes treated with guaianolide, such as thinning and stretching of the cell body and flagellum and changes in the format of the cell body with apparent leakage of the cytoplasmic content in trypomastigote forms. The ultrastructural analysis of epimastigotes revealed the presence of membranes that involved organelles and formation of myelin-like figures. Flow cytometry revealed a cell volume reduction and decrease in mitochondrial membrane potential. However, no major changes in cell membrane integrity were found in the epimastigote form treated with guaianolide.

Muscle Biopsy as a Diagnostic Tool in Pompe Disease

Today, myopathological, biochemical and genetic characteristics of Pompe disease are well established, and many reports have been published following the first very precisely documented myopatholgical description of adultonset Pompe disease in 1968. The clinical awareness and need for diagnostic precision of Pompe disease has increased since enzyme replacement therapy (ERT) has become widely available. Primary characteristic myopathological findings of Pompe disease are rounded vacuoles that are irregular in shape and size. All infantile biopsies show pronounced vacuolisation of almost all fibres. In juvenile biopsies, the percentage of vacuolated fibres decreases to 75%, and in most adult patients only 10–50% of the fibres reveal vacuolisation. In infantile cases, both fibre types are equally affected, while in juvenile and adult cases a predominant vacuolisation of type 2 fibres occurs. The second myopathological hallmark is periodic acid-Schiff (PAS)– positive material packed in these vacuoles, sometimes lost due to fixation procedures and therefore sometimes only notable as optically empty vacuolar spaces. Thirdly, acid phosphates histochemistry reveals positive staining in vacuolated fibres, indicating lysosomal dysfunction. In addition to these classic findings, autophagic vacuoles, sometimes containing heterogeneous material including cytoplasmic degradation products and myeloid structures, are seen at all ages, especially in type 2 fibres. Ultra-structurally, these autophagic vacuoles contained particulate glycogen and/or autophagic debris, including myelin-like figures. Almost all samples show lipofuscin accumulation in the area of lysosomal residual bodies and rarely lipid droplets as a sign of lysosomal ageing. Infrequently, single rimmed vacuoles are notable. Fibre splitting is an uncommon aspect. Due to excessive fibre vacuolisation, a subsarcolemmal packing of mitochondria is notable in most cases. Single fibre necrosis, basophilia and phagocytosis may be present in biopsies of all ages. Furthermore, mild fibrosis and fatty degeneration may be found in about 10% of the biopsies. Immunohistochemistry does not normally present signs of an inflammatory myopathy. In selected biopsies, some macrophages may be noticed, and vacuoles may contain caveolin-3 and dysferlin positive membrane debris. Although all classic hallmarks of Pompe disease are well documented over several decades, Pompe disease in adult-onset patients may still be missed histologically, especially when a clinically unaffected muscle is chosen for biopsy. Consequently, in modern diagnostics, dry blood spot (DBS) testing followed by -glucosidase (GAA) gene nalysis helps to overcome this muscle biopsy neglect. However, in some adult patients presenting with a blurred imb girdle weakness and atrophy, combined muscle MRI, light and electron microscopy, and biochemical analysis ay be still essential for making an accurate diagnosis of Pompe disease.

Antileishmanial activity of an essential oil from the leaves and flowers ofAchilleamillefolium

An essential oil was recently extracted from the leaves and flowers of yarrow (Achillea millefolium) and tested for in-vitro activity against Leishmania amazonensis and murine macrophages (i.e. the J774G8 cell line). The median inhibitory concentration (IC(50)) against L. amazonensis promastigotes was 7.8 μg/ml whereas the survival of amastigotes of this pathogen, within peritoneal murine macrophages, was halved by treatment with the oil at 6.5 μg/ml. The mean value for the median cytotoxic concentration of the oil, measured against adherent (uninfected) J774G8 macrophages, was 72.0 μg/ml (i.e. 9.2 and 11.0 times higher, respectively, than the IC(50) against the promastigotes and intracellular amastigotes). Scanning electron microscopy revealed that the oil caused morphological changes in the treated parasites, including alterations in their shape and size. In transmission electron microscopy, promastigotes treated with the oil (at the IC(50) of 7.8 μg/ml) showed various ultrastructural alterations, including changes in the flagellar membrane, abnormal membrane structures, rupture of the plasma membrane, atypical vacuoles, myelin-like figures, and vesicles that resembled autophagic vacuoles.

Evaluation of antileishmanial activity of eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens

Infection with Leishmania spp. causes a disease with multifaceted clinical manifestations in humans. The treatment for leishmaniasis is dependent on a limited range of drugs. Here we investigated the antileishmanial activity of eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. We showed that eupomatenoid-5 had a dose-dependent activity during 72 h of treatment, exhibiting IC 50 of 9.0 µg/mL and 13.0 µg/mL for promastigote and axenic amastigote forms, respectively, and IC 50 of 5.0 µg/mL for intracellular amastigote forms of Leishmania amazonensis. When L. amazonensis was treated with eupomatenoid-5, it underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Among the alterations was the appearance of intense exocytic activity in the region of the flagellar pocket, myelin-like figures, and vacuoles in the cytoplasm of parasites treated with 9.0 µg/mL. Cells treated with 25.0 µg/mL showed a very large structure, apparently an extension of the endoplasmic reticulum. Also, mitochondrial swelling was detected at this concentration, indicating damage and significant change in this organelle. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa and it is not toxic to macrophages. Our studies indicated that eupomatenoid-5 might be a potential new drug for the treatment of leishmaniasis, because this compound displays interesting antileishmanial activity in vitro against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis.

Histopathological and Ultrastructural Studies on the Effects of Electromagnetic Fields on the Liver of Preincubated White Leghorn Chicken Embryo

There are several reports indicating a linkage between exposures to 50–60 Hz electromagnetic fields and abnormalities in the early stages of embryonic development of chicken embryos. The present study was designed to demonstrate whether electromagnetic fields could be an environmental factor invoking histopathological and ultra-structural changes in livers of preincubated chicken embryos exposed to EMFs. Following other researchers and our previous results from different groups of Developmental Biology at the Animal Sciences, Faculty of Biological Sciences, Shahid-Beheshti University, effects of most effective intensities (1.33, 2.66, 5.52, and 7.32 mT) of electromagnetic fields (EMFs, 50 Hz ) on livers of pre-incubated white leghorn chicken embryos were investigated . 150 healthy, fresh, and fertilized eggs (55–65 gr) were divided into 6 groups of experimental(1–4, n = 30), control (n = 60), and sham (n = 50). Experimental eggs (inside coil) were exposed to 4 different intensities (1.33, 2.66, 5.52, and 7.32 mT). Sham groups were located inside same coil, with no exposure, for 24 h before incubation. Control, sham, and experimental groups (1–4) were then incubated in an incubator (38 ± 0.5°C, 60% humidity) for 17 days. At the end of this period, livers of experimental, sham, and control groups were processed for light and transmission electrom microscopes (TEM and SEM) studies. So, livers of 17-day old chicken embryos were removed by C-sections, fixed in formalin 10%, stained with H&E and reticulin, and studied under light microscope. Others were prepared for electron microscopes (TEM and SEM) investigations. Morphological observations indicated exencephalic embryos, embryos with asymmetrical faces, crossed beak, shorter upper beak, deformed hind limbs, gastroschesis, anophthalmia, and microphthalmia. H&E and reticulin stainings, TEMS, and SEMs studies indicated EMFs would create hepato-cytes with fibrotic bands, severe steatohepatitis, vacuolizations, swollen and extremely electron-dense mitochondria, reduced invisible cristae, crystalized mitochondria with degenerated cristae, myelin-like figures, macrophages engulfing adjacent cells, dentated nuclei, nuclei with irregular envelopes, degenerated hepatocytes, abnormal lipid accumulations, lipid droplets pushing hepatocytes' nuclei to the corner of the cells, abundant cellular infiltrations cellular infiltrations inside sinusoid and around central veins, disrupted reticulin plexus, and release of chromatin into cytosol,, with partially regular water layers. An elevated oxyradical generation and, subsequentely, cell membrane disruptions were the reasons for electromagnetic fields inducing cell damages.

Erythrophagosomal haemolytic degradative pathway in rat brown adipocytes induced by hyperinsulinaemia: an ultrastructural study

The phagocytosis and degradation of erythrocytes were studied in brown adipose tissue of experimentally hyperinsulinaemic rats. We found that insulin induces intensive erythrophagocytosis by brown adipocytes and their degradation by haemolytic pathway. Ultrastuctural study revealed that haemolytic degradation of erythrophagosomes was characterized by progressive and uniform decrease of erythrocyte matrix density. At the beginning of the degradative process small, clear vesicles resembling primary lysosomes were visible inside the erythrophagosome. With time, the erythrocyte structure totally disappeared and transformed into a fine, granular material within the erythrophagosomal vacuole. Finally, the erythrocyte membrane detached from the phagosomal and clumped into the vacuolar space forming one or several small myelin-like figures. In conclusion, brown adipocytes are capable of performing intensive erythrophagocytic activity when brown adipose tissue is stimulated and blood flow is enhanced. The molecular basis for favouring a haemolytic instead of more common granular erythrophagosomal degradative pathway remains unknown.

Anti-Trypanosoma cruzi activity of Pterodon pubescens seed oil: geranylgeraniol as the major bioactive component

In the search for new therapeutic agents for Chagas' disease, we screened extracts obtained from the Brazilian plant Pterodon pubescens found commercially in the medicinal flora market. We investigated the potential trypanocidal effect of the oleaginous ethanolic extract of P. pubescens seeds and its fractions (PF1, PF1.1, PF1.2, and PF1.3) and of geranylgeraniol (GG-OH), the sole component of the hexane fraction (PF1.2). In experiments with bloodstream trypomastigotes of Trypanosoma cruzi, performed at 37 degrees C in culture medium, PF1.2 and GG-OH showed similar potency, while the oleaginous extract from P. pubescens seeds and the other fractions were about three times less active. GG-OH inhibited the proliferation of intracellular amastigotes, at concentrations which do not affect the mammalian host cell. Transmission electron microscopy and flow cytometry analysis indicate the mitochondrion, an organelle that plays a central role in apoptosis, of both epimastigotes and of trypomastigotes as the major target of GG-OH. On the other hand, the ultrastructural images of the endoplasmic reticulum profiles, myelin-like figures, and concentric membranous arrangements inside damaged mitochondrion are suggestive of an autophagic pathway leading to parasite death. Because the different forms of cell death share some morphological features such as mitochondrial collapse, further studies are needed to disclose the trypanocidal action of GG-OH.

Effect of Resveratrol and Melatonin on Oxidative Stress Enzymes, Regeneration, and Hepatocyte Ultrastructure in Rats Subjected to 70% Partial Hepatectomy

Aim We sought to compare the antioxidant effects of resveratrol (R) and melatonin (M) after 70% partial hepatectomy (PH) as evidenced by ultrastructural alterations and effects on hepatocyte proliferation and apoptosis. Methods Twenty-six male Wistar albino rats were randomized into four groups: group A ( n = 8) resveratrol (R); group B ( n = 8) melatonin (M); group C ( n = 5) control PH; group D ( n = 5) sham operated animals. The rats that received either R or M were sacrificed a week after PH. The malondialdehyde, glutathione, glutathione S-transferase, and nitric oxide levels were estimated in liver homogenates. The morphological changes were investigated using light and electron microscopy (EM). Cell proliferation was detected by immunohistochemical staining with monoclonal antibodies to Ki-67. Apoptosis was detected by the transferase-mediated dUTP nick end-labeling method. Results PH induced hepatic LP, decreased GSH and NO, and inhibited GST activity ( P < .05). R and M completely prevented PH-induced lipid peroxidation, decreased hepatic GSH and NO levels ( P < .05). The inhibition of GST activity was prevented by R ( P < .05), but not with M ( P > .05). In the PH group EM showed severe morphological changes: mitochondrial degeneration, vacuoles, lipid droplets, and myelin-like figures. In both the R and M groups, morphological alterations repaired protective effects more prominently in the R group. Ki-67 indices (KI) were increased in the PH group and decreased in both R and M groups ( P < .001). In the M group, KI was the lowest, but the difference compared with R was not significant ( P > .05). Apoptosis was slightly increased in PH, but in either the R or M groups, apoptosis was intensively increased ( P < .001). Increased apoptosis was greatest in the M group and the difference compared with the R group was statistically significant ( P < .05). Conclusion R and M suppressed PH-induced oxidative damage, attenuated proliferation, and stimulated apoptosis. When we compared R and M, R showed more potent antioxidative effects and was morphologically more protective to hepatocytes. Antiproliferative effects of M were more potent. Because of their potent antioxidative effects, R and M can be effective for oxidative damage like ischemia-reperfusion injury; however, because of the adverse effects on proliferation and apoptosis more studies are needed in states in which regeneration is critical.

Elaiophore structure and oil secretion in flowers of Oncidium trulliferum Lindl. and Ornithophora radicans (Rchb.f.) Garay & Pabst (Oncidiinae: Orchidaceae).

Many orchid flowers have glands called elaiophores and these reward pollinating insects with oil. In contrast to other reward-producing structures such as nectaries, the anatomy of the elaiophore and the process of oil secretion have not been extensively studied. In this paper, elaiophore structure is described for two members of Oncidiinae, Oncidium trulliferum Lindl. and Ornithophora radicans (Rchb.f.) Garay & Pabst. Elaiophores of both species were examined using light microscopy, scanning electron microscopy and transmission electron microscopy. In flowers of Oncidium trulliferum and Ornithophora radicans, oil is secreted by morphologically distinct elaiophores associated with the labellar callus. However, in O. trulliferum, elaiophores also occur on the lateral lobes of the labellum. In both these species, the epithelial elaiophores are composed of a single layer of palisade-like epidermal cells and a distinct subepithelial layer. Secretory elaiophore cells may contain numerous, starchless plastids, mitochondria and smooth endoplasmic reticulum profiles. In O. trulliferum, the cytoplasm contains myelin-like figures but these are absent from O. radicans. In the former species, cavities occur in the cell wall and these presumably facilitate the passage of oil onto the elaiophore surface. In O. radicans, the accumulation of oil between the outer tangential wall and the cuticle causes the latter to become distended. Since it is probable that the full discharge of oil from the elaiophores of O. radicans occurs only when the cuticle is ruptured by a visiting insect, this may contribute towards pollinator specificity. The structure of the elaiophore in these species resembles both that found in previously investigated species of Oncidiinae and that of certain members of the Malpighiaceae.

The U95 Protein of Human Herpesvirus 6B Interacts with Human GRIM-19: Silencing of U95 Expression Reduces Viral Load and Abrogates Loss of Mitochondrial Membrane Potential

To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection. To study the functional role of the HHV-6B IE gene encoding U95, we generated a U95-Myc fusion protein and screened a pretransformed bone marrow cDNA library for U95-interacting proteins, using yeast-two hybrid analysis. The most frequently appearing U95-interacting protein identified was GRIM-19, which belongs to the family of genes associated with retinoid-interferon mortality and serves as an essential component of the oxidative phosphorylation system. This interaction was verified by both coimmunoprecipitation and confocal microscopic coimmunolocalization. Short-term HHV-6B infection of MT-4 T-lymphocytic cells induced syncytial formation, resulted in decreased mitochondrial membrane potential, and led to progressively pronounced ultrastructural changes, such as mitochondrial swelling, myelin-like figures, and a loss of cristae. Compared to controls, RNA interference against U95 effectively reduced the U95 mRNA copy number and abrogated the loss of mitochondrial membrane potential. Our results indicate that the high affinity between U95 early viral protein and GRIM-19 may be closely linked to the detrimental effect of HHV-6B infection on mitochondria. These findings may explain the alternative cell death mechanism of expiration, as opposed to apoptosis, observed in certain productively HHV-6B-infected cells. The interaction between U95 and GRIM-19 is thus functionally and metabolically significant in HHV-6B-infected cells and may be a means through which HHV-6B modulates cell death signals by interferon and retinoic acid.

STRUCTURE OF THE PHARYNX IN THE ADULT NEMATODE ANGUILLICOLOIDES CRASSUS (NEMATODA: RHABDITIDA)

The structure of the pharynx of the adult female nematode Anguillicoloides crassus (Spirurina) has been studied for the first time using light and transmission electron microscopy. The cylindrical pharynx consists of a short anterior muscular corpus and an enlarged posterior glandular and muscular postcorpus. The main cellular components of the pharynx of A. crassus include the muscle cells, the marginal cells, the nerve cells, and 1 dorsal and 2 subventral glands. New observations for nematodes include: (1) the non-contractile regions of pharyngeal musculature in the corpus have specific appearance; (2) the ventrosublateral longitudinal nerve in the pharynx has an enlarged, enucleated anterior part, with a pronounced palmate projections; and (3) abundant lysosomelike membranous bodies consisting of myelinlike figures of varied size present in marginal cells and pharyngointestinal valve. The 2 subventral glands and, apparently, the single dorsal gland, have their openings at the same level, i.e., at the border between the corpus and postcorpus. The pharyngeal-intestinal valve joins the pharynx to the intestine. Knowledge of the ultrastructure of these complex characters may be useful in understanding of functional features, and for comparative morphology as well as evolutionary considerations within the Chromadorea.

Cadmium effect on the growth, photosynthesis, ultrastructure and phytochelatin content of green microalga Scenedesmus armatus: A study at low and elevated CO 2 concentration

Short-term experiments were carried out to examine the toxicity of cadmium chloride (CdCl 2) at a concentration of 93 μM (EC 50/24) to green microalga Scenedesmus armatus, cultured at low (0.1%) and elevated (2%) concentration of CO 2. Cadmium did not affect the viability of cells cultured for 24 h in both CO 2 variants but markedly inhibited the growth of algae. This inhibition was more pronounced in cultures aerated with 0.1% (about 50% of control) than with 2% CO 2 (about 75% of control) and did not change during 72 h of culture. Cadmium inhibited the rate of oxygen evolution ( P oxy.) (70% of control) of cells cultured at 0.1% CO 2 and had no effect on P oxy. of cells cultured at 2% CO 2. The values of the chlorophyll fluorescence parameters, i.e. F M (maximum fluorescence yield), F V (variable fluorescence), F V/ F M (maximum quantum yield of PSII), Φ PSII (effective quantum yield of PSII) and qP (photochemical quenching) were reduced by cadmium treatment in algae grown at 0.1% CO 2 concentration, whereas F 0 (initial fluorescence yield) remained unaffected. In high-CO 2 grown cells only F V was significantly reduced. Cd-treated cells synthesized several thiol-containing peptides identified by HPLC as a dimer (PC 2), a trimer (PC 3) and a tetramer (PC 4) of phytochelatins (PC S). High-CO 2 grown cells produced significantly more PCs than low-CO 2 grown cells and their individual appearance depended on the time of exposure and CO 2 level. The ultrastructural analysis of low-CO 2 grown cells showed in chloroplasts an increased number of small starch grains visible around the pyrenoid. In the enhanced vacuome compartment, various types of vacuoles were clearly seen in Cd-treated cells. Vacuoles containing non-membranous, electron-opaque deposits of an undefined structure and myelin-like figures were especially observed. The results suggest that algae living in conditions of elevated CO 2 are better protected against cadmium than those at ordinary CO 2 level, and productive processes are less affected than the growth ones.