scholarly journals Acyclic Sesquiterpenes from the Fruit Pericarp of Sapindus saponaria Induce Ultrastructural Alterations and Cell Death in Leishmania amazonensis

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Amanda Louzano Moreira ◽  
Débora Botura Scariot ◽  
Bruna Luíza Pelegrini ◽  
Greisiele Lorena Pessini ◽  
Tânia Ueda-Nakamura ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Hemolytic Activity ◽  
Solid Phase ◽  
Reversed Phase ◽  
Leishmania Amazonensis ◽  
Antileishmanial Activity ◽  
Ultrastructural Changes ◽  
Phosphatidylserine Exposure ◽  
Sapindus Saponaria

Previous studies reported antiprotozoal activities of Sapindus saponaria L. The aim of this work was the evaluation of antileishmanial activity and mechanism of action of extract and fractions of S. saponaria L. Hydroethanolic extract (EHA) obtained from fruit pericarps was fractionated using solid-phase extraction in a reversed phase, resulting in fractions enriched with saponins (SAP fraction) and acyclic sesquiterpene oligoglycosides (OGSA fraction). The activities of EHA, SAP, and OGSA were evaluated by antiproliferative assays with promastigote and intracellular amastigote forms. Cytotoxicity on macrophages and hemolytic activity were also analyzed. Morphological and ultrastructural changes in Leishmania amazonensis promastigotes were evaluated by electron microscopy. Flow cytometry was used to investigate mitochondrial dysfunction and phosphatidylserine exposure. OGSA was more selective for parasites than mammalian J774A1 macrophage cells, with selectivity indices of 3.79 and 7.35, respectively. Our results showed that only the OGSA fraction did not present hemolytic activity at its IC50 for promastigote growth. Electron microscopy revealed changes in parasite flagellum, cell body shape, and organelle size, mainly mitochondria. Flow cytometry analysis indicated mitochondrial membrane and cell membrane dysfunction. OGSA showed antileishmanial activity, resulting in several changes to protozoa cells, including mitochondrial depolarization and early phosphatidylserine exposure, suggesting a possible apoptotic induction.

scholarly journals Endlicheria bracteolata (Meisn.) Essential Oil as a Weapon Against Leishmania amazonensis: In Vitro Assay

Molecules ◽  
2019 ◽  
Vol 24 (14) ◽  
pp. 2525 ◽  
Author(s):  
Mariana Margatto Rottini ◽  
Ana Claudia Fernandes Amaral ◽  
José Luiz Pinto Ferreira ◽  
Edinilze Souza Coelho Oliveira ◽  
Jefferson Rocha de Andrade Silva ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Flow Cytometry ◽  
Essential Oil ◽  
Cutaneous Leishmaniasis ◽  
Leishmania Amazonensis ◽  
Vitro Assay ◽  
Transmission Electron ◽  
Biological Potential ◽  
Intracellular Amastigote

The difficulties encountered and the numerous side effects present in the treatment of cutaneous leishmaniasis have encouraged the research for new compounds that can complement or replace existing treatment. The growing scientific interest in the study of plants, which are already used in folk remedies, has led our group to test Endlicheria bracteolata essential oil against Leishmania amazonensis. Several species of the Lauraceae family, or their compounds, have relevant antiprotozoal activities Therefore, the biological potential on L. amazonensis forms from the essential oil of Endlicheria bracteolata leaves was verified for the first time in that work. The antileishmanial activity was evaluated against promastigotes and intracellular amastigotes, and cytotoxicity were performed with J774.G8, which were incubated with different concentrations of E. bracteolata essential oil. Transmission electron microscopy and flow cytometry were performed with E. bracteolata essential oil IC50. Promastigote forms showed E. bracteolata essential oil IC50 of 7.945 ± 1.285 µg/mL (24 h) and 6.186 ± 1.226 µg/mL (48 h), while for intracellular amastigote forms it was 3.546 ± 1.184 µg/mL (24 h). The CC50 was 15.14 ± 0.090 µg/mL showing that E. bracteolata essential oil is less toxic to macrophages than to parasites. Transmission electron microscopy showed that E. bracteolata essential oil treatment is capable of inducing mitochondrial damage to promastigote and intracellular amastigote forms, while flow cytometry showed ΔѰm disruption in treated parasites. These results could bring about new possibilities to develop products based on E. bracteolata essential oil to treat cutaneous leishmaniasis, especially for people who cannot receive the conventional therapy.

scholarly journals Copaiba Oil: An Alternative to Development of New Drugs against Leishmaniasis

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Adriana Oliveira dos Santos ◽  
Tânia Ueda-Nakamura ◽  
Benedito Prado Dias Filho ◽  
Valdir Florêncio da Veiga Junior ◽  
Celso Vataru Nakamura
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Propidium Iodide ◽  
Mitochondrial Membrane ◽  
New Drugs ◽  
Ultrastructural Changes ◽  
Plasma Membrane Permeability ◽  
Copaiba Oil

Leishmaniasis is a neglected disease that is increasing globally at an alarming rate. Glucantime has been the therapy of choice for more than 50 years. A recent study reported the antileishmanial activity of copaiba oil againstLeishmania amazonensis. These results led us to investigate morphological and ultrastructural changes inL. amazonensistreated with copaiba oil, using electron microscopy and flow cytometry to assess specific organelles as targets for copaiba oil. In the promastigote and axenic amastigote forms, this copaiba oil caused notable morphological and ultrastructural changes, including extensive mitochondrial damage and denaturation of the plasma membrane. Copaiba oil treatment also induced a decrease in Rh123 fluorescence, suggesting interference with the mitochondrial membrane potential and loss of cell viability with an increase in plasma membrane permeability, as observed by flow cytometry after staining with propidium iodide. In conclusion, copaiba oil could be exploited for the development of new antileishmanial drugs.

Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations

2016 ◽  
Vol 22 (3) ◽  
pp. 630-639 ◽  
Author(s):  
Tanya N. Augustine ◽  
Wendy J. van der Spuy ◽  
Lindsay L. Kaberry ◽  
Millicent Shayi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Diagnostic Tool ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Ultrastructural Changes ◽  
Ultrastructural Alterations ◽  
Scanning Electron

AbstractPlatelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool.

Ultrastructural Changes of Canine Kidneys During 24-Hour Perfusion on a LI-400 Preservation System

1971 ◽  
Vol 29 ◽  
pp. 316-317
Author(s):  
M. O. Magnusson ◽  
D. G. Osborne ◽  
T. Shimoji ◽  
W. S. Kiser ◽  
W. A. Hawk
Keyword(s):  
Electron Microscopy ◽  
Electrolyte Solution ◽  
Ultrastructural Changes ◽  
Midline Incision ◽  
Short Term ◽  
Pentobarbital Anesthesia ◽  
Pulsatile Perfusion

Short term experimental and clinical preservation of kidneys is presently best accomplished by hypothermic continuous pulsatile perfusion with cryoprecipitated and millipore filtered plasma. This study was undertaken to observe ultrastructural changes occurring during 24-hour preservation using the above mentioned method.A kidney was removed through a midline incision from healthy mongrel dogs under pentobarbital anesthesia. The kidneys were flushed immediately after removal with chilled electrolyte solution and placed on a LI-400 preservation system and perfused at 8-10°C. Serial kidney biopsies were obtained at 0-½-1-2-4-8-16 and 24 hours of preservation. All biopsies were prepared for electron microscopy. At the end of the preservation period the kidneys were autografted.

Solid-phase immunoelectron microscopy for rapid diagnosis of enteroviruses

1984 ◽  
Vol 42 ◽  
pp. 226-227 ◽  
Author(s):  
K. Pegg-Feige ◽  
F. W. Doane
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Immunoelectron Microscopy ◽  
Detection Method ◽  
Solid Phase ◽  
Protein A ◽  
Plant Viruses ◽  
Rapid Diagnosis ◽  
Virus Diagnosis ◽  
Antiviral Antibody

Immunoelectron microscopy (IEM) applied to rapid virus diagnosis offers a more sensitive detection method than direct electron microscopy (DEM), and can also be used to serotype viruses. One of several IEM techniques is that introduced by Derrick in 1972, in which antiviral antibody is attached to the support film of an EM specimen grid. Originally developed for plant viruses, it has recently been applied to several animal viruses, especially rotaviruses. We have investigated the use of this solid phase IEM technique (SPIEM) in detecting and identifying enteroviruses (in the form of crude cell culture isolates), and have compared it with a modified “SPIEM-SPA” method in which grids are coated with protein A from Staphylococcus aureus prior to exposure to antiserum.

Effect of Acetone and Kerosene on Skin Ultrastructure

1972 ◽  
Vol 30 ◽  
pp. 92-93
Author(s):  
A. P. Lupulescu ◽  
H. Pinkus ◽  
D. J. Birmingham
Keyword(s):  
Electron Microscopy ◽  
Human Skin ◽  
Osmium Tetroxide ◽  
Human Epidermis ◽  
Ultrastructural Changes ◽  
Chemical Agents ◽  
Skin Ultrastructure ◽  
Volar Surface

Our laboratory is engaged in the study of the effect of different chemical agents on human skin, using electron microscopy. Previous investigations revealed that topical use of a strong alkali (NaOH 1N) or acid (HCl 1N), induces ultrastructural changes in the upper layers of human epidermis. In the current experiments, acetone and kerosene, which are primarily lipid solvents, were topically used on the volar surface of the forearm of Caucasian and Negro volunteers. Skin specimens were bioptically removed after 90 min. exposure and 72. hours later, fixed in 3% buffered glutaraldehyde, postfixed in 1% phosphate osmium tetroxide, then flat embedded in Epon.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

Identification of hepatitis a virus in cell cultures by solid phase immune electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 238-239
Author(s):  
C.D. Humphrey ◽  
T.L. Cromeans ◽  
E.H. Cook ◽  
D.W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Liquid Phase ◽  
Cell Cultures ◽  
Rapid Detection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Immune Electron Microscopy ◽  
Detection And Identification

There is a variety of methods available for the rapid detection and identification of viruses by electron microscopy as described in several reviews. The predominant techniques are classified as direct electron microscopy (DEM), immune electron microscopy (IEM), liquid phase immune electron microscopy (LPIEM) and solid phase immune electron microscopy (SPIEM). Each technique has inherent strengths and weaknesses. However, in recent years, the most progress for identifying viruses has been realized by the utilization of SPIEM.

LC–MS-MS Method for the Determination of Antidepressants and Benzodiazepines in Meconium

2020 ◽  
Vol 44 (6) ◽  
pp. 580-588
Author(s):  
A López-Rabuñal ◽  
E Lendoiro ◽  
M Concheiro ◽  
M López-Rivadulla ◽  
A Cruz ◽  
...  
Keyword(s):  
Extraction Efficiency ◽  
Solid Phase ◽  
Gradient Methods ◽  
Reversed Phase ◽  
Process Efficiency ◽  
Limit Of Quantification ◽  
Mode 2 ◽  
Positive Mode ◽  
Compound Method

Abstract An LC–MS-MS method for the determination of 14 benzodiazepines (BZDs) (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (ADs) (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of ADs and another for BZDs. Analytes were monitored by tandem mass spectrometry employing electrospray positive mode in MRM mode (2 transitions per compound). Method validation included: linearity [n = 5, limit of quantification (LOQ) to 400 ng/g], limits of detection (n = 6, 1–20 ng/g), LOQ (n = 9, 5–20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6–111.5%), imprecision (n = 15, 0–14.6%), matrix effect (n = 10, −73 to 194.9%), extraction efficiency (n = 6, 35.9–91.2%), process efficiency (n = 6, 20.1–188.2%), stability 72 h in the autosampler (n = 3, −8.5 to 9%) and freeze/thaw stability (n = 3, −1.2 to −47%). The method was applied to four meconium specimens, which were analyzed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC–MS-MS method allows a high throughput determination of the most common BZDs and ADs in meconium, which could be useful in clinical and forensic settings.

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