Ultrastructural Visualization of Lipids in Trypanosomatids1

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis, T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.     

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

Summary The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.     

Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

The Fine Structure of the Gregarine Lankesteria culicis Parasitic in the Yellow Fever Mosquito Aedes aegypti

SYNOPSIS. The fine structure of the eugregarine Lankesteria culicis from the larvae of the mosquito Aedes aegypti was examined by light and electron microscopy and compared with that of other gregarines. The cell organelles found in L. culicis included a nucleus, mitochondria, Golgi-complexes, endoplasmic reticulum, vesicles, droplets, granules and lipid bodies.
The surface of L. culicis was composed of a highly differentiated membrane-cortex, differing slightly from that of other eugregarines. This complex was limited by a unit membrane, the plasmelemma, and underlying cortical laminae which appeared to be composed of several layers. The homogeneous electrondense layer present in Pyxinoides balani was greatly diminished or absent in L. culicis.
A series of laminar folds supported by ground substance and longitudinal subpellicular fibrils gave the organism's surface a ridge-like appearance. Permanent cytostome-like openings in the surface, which appeared to be supported by a narrow band of thickened cortex, were present as specializations of the surfacemembrane complex. The structural composition of the parasite appeared quite striking in that it was made up almost entirely of vesicles, granules, and droplets which were absent only in the area of the protomerite. The mitochondria were usually found just beneath the surface or near the nucleus. Mitochondria were also seen in the region of demarcation between the protomerite and deutomerite.     

Electrophoretic Analysis of Endonuclease-Generated Fragments of k-DNA,of Esterase Isoenzymes,and of Surface Proteins as Aids for Species Identification of Insect Trypanosomatids1

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Effects of methylglyoxal bis(ganylhydrazone) on trypanosomatid flagellates: inhibition of growth and nucleoside incorporation in Trypanosoma brucei

Methyglyoxal bis (guanylhydrazone) (MGBG) at 0.5 mM had little effect in vitro on Blastocrithidia culicis, Crithidia oncopelti, and Leishmania spp., but completely inhibited growth of Trypanosoma brucei. Inhibition became irreversible after a 3-h exposure of T. brucei culture procyclics. Treated organisms remained motile, but failed to divide. Polyamines, spermidine, and spermine, did not reverse the anti-trypanosome action of MGBG (preloading of cells or concurrent administration). Two intraperitoneal injections of the drug at a concentration of 50 mg/kg body weight at a 1-day interval greatly reduced the parasitemia of T. brucei and T. congolense in rats. Trypanosome infections, however, relapsed and killed the animals in 6 days after treatment. It was evident from the results of tracer experiments with T brucei that MGBF significantly lowered incorporation of [3H]thymidine by culture pocyclics and of [3H]uridine by bloodstream forms; in both stages [3H]leucine incorporation was only slightly inhibited. It is suggested that MGBG interferes with nucleoside incorporation by Trypanosoma and that its mode of action is different in bloodstream and culture procyclics.     

Biological effects of lithium chloride on Herpetomonas muscarum muscarum and Blastocrithidia culicis (kinetoplastida: trypanosomatidae).

Lithium is widely used in medicine as an antidepressive drug and for myelosuppression attenuation during chemotherapy. In spite of abundant literature, questions on the biological action of lithium ions are far from being answered. We have here examined the effects of lithium (10-200 mM) on culture forms of the trypanosomatid protozoa Herpetornonas muscarum muscarum and Blastocrithidia culicis. Incubation of these parasites with LiCl inhibited cell growth in a concentration-dependent manner, but growth could be restored when the drug was removed from the medium. Furthermore, Li+ induced cell differentiation in H. m. muscarum. Light microscopy examination of cell viability, using erythrosin B staining, showed that all treated parasites remained viable with all drug concentrations used. Ultrastructural analysis by transmission electron microscopy showed that the cells presented no signs of degeneration. However, in H. m. muscarum the nuclei lost their peripheral heterochromatin and appeared filled with a homogeneous matrix, whereas in B. culicis an increased amount of lipid droplets was present in the cytoplasm. Our data show that LiCl treatment arrested the cell division process, stimulated cell differentiation, and affected the metabolism of these parasites.     

A Mixture of Paraphenylenediamine and Imidazole: Its Effect on the Extraction of Lipid Droplets during Electron Microscopy Staining

To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.     

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.     

Five trypanosomatid species of insects distinguished by isoenzymes.

Blastocrithidia culicis, Crithidia deanei, Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri and Leishmania tarentolae grown in cultures were compared by electrophoretic mobility for isoenzymes in 6 enzymes. All species were found distinct in these characteristics. Endosymbiotic C. deanei, which was identical to the aposymbiotic C. deanei in 5 enzymes, had an extra band in aspartate aminotransferase. No differences in isoenzymes were found between members of one species maintained in 2 different culture media.     

Lipid droplets in the adrenal cortex of the rat. Preservation after tannic acid—paraformaldehyde—glutaraldehyde fixation and extraction during staining

Adrenocortical tissue from the rat was fixed in glutaraldehyde-paraformaldehyde-tannic acid with or without potassium pyroantimonate. An electron opacity was observed in lipid droplets from unstained sections of tissue with or without antimonate in the fixative and is most likely attributable to inclusion of tannic acid in the fixative. The opacity was largely removed after staining with uranyl acetate in absolute methanol followed by lead citrate. Removal of the opacity is attributable to staining in lead citrate, not uranyl acetate, because highly basic solution without lead also removes the density. An electron-opaque rim is present at the interface of lipid droplet and cytoplasm, although no distinct membranous structure is observable. The rim may correspond to myelin-like structures seen sometimes in lipid droplets from adrenocortical cells fixed by routine procedures employing pre-fixation with glutaraldehyde and post-fixation with osmium tetroxide. Results of this study point to the conclusion that ultrathin sections should be examined unstained in the validation of a new regime for processing tissues in electron microscopy.     

非酒精性脂肪肝病肝组织超微结构特点

目的:阐明非酒精性脂肪肝病(NAFLD)的超微结构特点。方法:收集我校和其他单位送检的3例单纯性非酒精性脂肪肝,16例NASH患者和4例NAFLD肝硬化患者的肝穿刺组织。用2.5%戊二醛、1%锇酸双固定、Epon 812包埋,超薄切片70nm,醋酸铀和柠檬酸铅染色后,JEM-2000EX透射电镜观察。结果:单纯性脂肪肝患者主要表现为大小不等的脂滴沉积、以小脂滴为主,可互相融合。NASH患者的肝细胞都可出现大量脂滴积聚,为大小脂滴混合型、内容物主要为中等电子密度、比较均一的甘油三酯,部分脂滴周围可见磷脂成分,NASH患者肝细胞内脂滴也互相融合。肝细胞线粒体的超微结构改变包括多形性线粒体、基质颗粒增多、线粒体增大和嵴的丧失是主要的电镜异常发现,线粒体内还可见副晶格样包涵体。部分NASH患者肝细胞内可见Mallory小体。NASH患者肝细胞周围可见淋巴细胞浸润。肝血窦Kupffer细胞增生不明显,NAFLD肝硬化患者Disse间隙和肝细胞间可见胶原纤维增生。结论:NAFLD具有较为明确的超微结构改变,电镜检查有助于诊断。     

The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development.

During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.     

Presence of genes of ribosomal and transfer RNAs in kinetoplast DNA from two Crithidia species

The coding properties of kinetoplast DNA from two respresentatives of the order Kinetoplastidae--Crithidia oncopelti and C. fasciculata--were studied. Experiments on hybridization of the whole network and fraction of minicircles with labelled 23S and 16S rRNA and with tRNA isolated from kinetoplasts of C. oncopelti clearly demonstrated the presence of the genes for these RNAs in the whole network and their absence in the minicircles. It may be thus concluded that the genes of ribosomal and transfer RNAs are localized in the maxicircular molecules. Similar efficiency of hybridization of rRNAs from C. oncopelti with kDNA from C. fasciculata and C. oncopelti revealed significant conservativity of ribosomal genes in the protozoa under study.     

Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain

Nir2, like its Drosophila homolog retinal degeneration B (RdgB), contains an N-terminal phosphatidylinositol-transfer protein (PI-TP)-like domain. Previous studies have suggested that RdgB plays an important role in the fly phototransduction cascade and that its PI-transfer domain is critical for this function. In this domain, a specific mutation, T59E, induces a dominant retinal degeneration phenotype. Here we show that a similar mutation, T59E in the human Nir2 protein, targets Nir2 to spherical cytosolic structures identified as lipid droplets by the lipophilic dye Nile red. A truncated Nir2T59E mutant consisting of only the PI-transfer domain was also targeted to lipid droplets, whereas neither the wild-type Nir2 nor the Nir2T59A mutant was associated with lipid droplets under regular growth conditions. However, oleic-acid treatment caused translocation of wild-type Nir2, but not translocation of the T59A mutant, to lipid droplets. This treatment also induced partial targeting of endogenous Nir2, which is mainly associated with the Golgi apparatus, to lipid droplets. Targeting of Nir2 to lipid droplets was attributed to its enhanced threonine phosphorylation. These results suggest that a specific threonine within the PI-transfer domain of Nir2 provides a regulatory site for targeting to lipid droplets. In conjunction with the role of PI-TPs in lipid transport, this targeting may affect intracellular lipid trafficking and distribution and may provide the molecular basis underlying the dominant effect of the RdgB-T59E mutant on retinal degeneration.     

Influence of the endosymbiont of Blastocrithidia culicis and Crithidia deanei on the glycoconjugate expression and on Aedes aegypti interaction

Blastocrithidia culicis and Crithidia deanei are trypanosomatid protozoa of insects that normally contain intracellular symbiotic bacteria. The protozoa can be rid of their endosymbionts by antibiotics, producing a cured cell line. Here, we analyzed the glycoconjugate profiles of endosymbiont-harboring and cured strains of B. culicis and C. deanei by Western blotting and flow cytometry analyses using lectins that recognize specifically sialic acid and mannose-like residues. The absence of the endosymbiont increased the intensity of the lectins binding on both trypanosomatids. In addition, wild and cured strain-specific glycoconjugate bands were identified. The role of the surface saccharide residues on the interaction with explanted guts from Aedes aegypti gut was assessed. The aposymbiotic strains of B. culicis and C. deanei presented interaction rates 3.3- and 2.3-fold lower with the insect gut, respectively, when compared with the endosymbiont-bearing strains. The interaction rate of sialidase-treated cells of the wild and cured strains of B. culicis and C. deanei was reduced in at least 90% in relation to the control. The interaction of B. culicis (wild strain) with explanted guts was inhibited in the presence of mucin (56%), fetuin (62%), sialyllactose (64%) and alpha-methyl-D-mannoside (80%), while in C. deanei (wild strain) the inhibition was 53%, 56%, 79% and 34%, respectively. Collectively, our results suggest a possible involvement of sialomolecules and mannose-rich glycoconjugates in the interaction between insect trypanosomatids and the invertebrate host.     

Ⅲ型纤连蛋白组件包含蛋白5通过抑制ERK1/2磷酸化抑制C3H10T1/2细胞成脂分化

旨在探究Ⅲ型纤连蛋白组件包含蛋白5(type Ⅲ domain-containing protein5,FNDC5)对C3H10T1/2细胞成脂分化的调控作用.利用qRT-PCR和Western印迹检测FNDC5在C3H10T1/2细胞成脂分化过程中的时序性表达规律;构建慢病毒包被的过表达/干扰FNDC5载体,转染C3...     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

OSMIUM STAINING OF ENDOPLASMIC RETICULUM AND MITOCHONDRIA IN THE RAT ADRENAL CORTEX

The zona fasciculata of the rat adrenal cortex synthesizes and secretes glucocorticoids. As observed after aldehyde fixation, the cells in this zone contain an extensive endoplasmic reticulum (ER), a small Golgi apparatus, a moderate number of lipid droplets, and abundant mitochondria with tubulovesicular cristae. Numerous areas within the endoplasmic reticulum and mitochondrial cristae appear clear. In addition, a small percentage of mitochondria encompasses large, clear areas. After immersion of finely minced adrenal cortex in unbuffered 2% OsO₄ (40–48 hr at 40°C), deposits of osmium are seen within the Golgi apparatus, the entirety of the ER, and occasionally within mitochondria. In some mitochondria, the deposits are within cristae; in others, within vacuoles; in still others, in both cristae and vacuoles. These localizations correspond best to the clear areas found in aldehyde-fixed tissue. Osmium is not deposited in lipid droplets, in bar-containing inclusions, in mitochondrial matrix inclusions, or in the peripheral, outer mitochondrial spaces. Addition of zinc-iodide to OsO₄ increases the amount of Golgi apparatus and mitochondrial staining. Adrenocorticotropin (ACTH) does not affect the localization of deposits; hypophysectomy decreases mitochondrial staining. This study (a) emphasizes the necessity for electron microscopic confirmation of osmium localization when this technique is used as a Golgi apparatus stain; and (b) suggests that the ER-staining pattern may be consistent in cells actively synthesizing steroids or steroid-like compounds.