scholarly journals Characterization of polymer release from the flagellar pocket of Leishmania mexicana promastigotes.

1994 ◽  
Vol 125 (2) ◽  
pp. 321-331 ◽  
Author(s):  
Y D Stierhof ◽  
T Ilg ◽  
D G Russell ◽  
H Hohenberg ◽  
P Overath
Keyword(s):  
Culture Medium ◽  
Protozoan Parasite ◽  
Flagellar Pocket ◽  
Leishmania Mexicana ◽  
Electron Microscopic ◽  
Fibrous Network ◽  
Intracellular Compartments ◽  
Polymeric Structures ◽  
Microscopic Techniques

Trypanosomatids contain a unique compartment, the flagellar pocket, formed by an invagination of the plasma membrane at the base of the flagellum, which is considered to be the sole cellular site for endocytosis and exocytosis of macromolecules. The culture supernatant of Leishmania mexicana promastigotes, the insect stage of this protozoan parasite, contains two types of polymers: a filamentous acid phosphatase (sAP) composed of a 100-kD phosphoglycoprotein with non-covalently associated proteo high molecular weight phosphoglycan (proteo-HMWPG) and fibrous material termed network consisting of complex phosphoglycans. Secretion of both polymers is investigated using mAbs and a combination of light and electron microscopic techniques. Long filaments of sAP are detectable in the lumen of the flagellar pocket. Both sAP filaments and network material emerge from the ostium of the flagellar pocket. While sAP filaments detach from the cells, the fibrous network frequently remains associated with the anterior end of the parasites and can be found in the center of cell aggregates. The related species L. major forms similar networks. Since polymeric structures cannot be detected in intracellular compartments, it is proposed that monomeric or, possibly, oligomeric subunits synthesized in the cells are secreted into the flagellar pocket. Polymer formation from subunits is suggested to occur in the lumen of the pocket before release into the culture medium or, naturally, into the gut of infected sandflies.

Processing and trafficking of cysteine proteases in Leishmania mexicana

2000 ◽  
Vol 113 (22) ◽  
pp. 4035-4041 ◽  
Author(s):  
D.R. Brooks ◽  
L. Tetley ◽  
G.H. Coombs ◽  
J.C. Mottram
Keyword(s):  
Cysteine Protease ◽  
Mammalian Cells ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Flagellar Pocket ◽  
Leishmania Mexicana

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.

scholarly journals Purification and molecular characterization of the NAD+-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica

1994 ◽  
Vol 303 (3) ◽  
pp. 743-748 ◽  
Author(s):  
I Bruchhaus ◽  
E Tannich
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Dna Sequences ◽  
Microscopic Analysis ◽  
Kinetic Studies ◽  
Protozoan Parasite ◽  
Striking Similarity ◽  
Electron Microscopic ◽  
Catalytic Functions

A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

Characterization of BCG Vaccine by coulter counter and scanning electron microscopic techniques

1991 ◽  
Vol 8 (1-4) ◽  
pp. 194-199 ◽  
Author(s):  
Michael J. Groves ◽  
Melvin E. Klegerman ◽  
Priscilla O. Devadoss ◽  
Onkar N. Singh ◽  
Yueying Zong
Keyword(s):  
Coulter Counter ◽  
Bcg Vaccine ◽  
Electron Microscopic ◽  
Scanning Electron Microscopic ◽  
Microscopic Techniques ◽  
Scanning Electron

scholarly journals Protein Serine/Threonine Phosphatase Type 2C of Leishmania mexicana

2021 ◽  
Vol 11 ◽  
Author(s):  
Alma Reyna Escalona-Montaño ◽  
Mariana Zuñiga-Fabián ◽  
Nallely Cabrera ◽  
Ricardo Mondragón-Flores ◽  
Jenny Nancy Gómez-Sandoval ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Specific Inhibitor ◽  
Protozoan Parasite ◽  
Structural Diversity ◽  
Flagellar Pocket ◽  
Leishmania Mexicana ◽  
Phosphatase Inhibitors ◽  
Cell Functions ◽  
Transmission Electron ◽  
The Family

Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in Leishmania, a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in Leishmania mexicana (LmxPP2C). Recombinant LmxPP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn+2). LmxPP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-LmxPP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized LmxPP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, LmxPP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.

Ultrastructural and Histochemical Characterization of Secretory Granules in Human Lung Adenocarcinoma Cells

2000 ◽  
Vol 6 (S2) ◽  
pp. 480-481
Author(s):  
R. G. Aktas ◽  
E. Demiralay ◽  
S. Altaner ◽  
L. Candan ◽  
A. K. Kutlu
Keyword(s):  
Secretory Granules ◽  
Secretory Cells ◽  
Electron Microscopic ◽  
Epitheloid Cells ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Excellent Research ◽  
Tumoral Cells ◽  
Microscopic Techniques

Histochemical methods offer an excellent research tool for the characterization of glycoproteins in the secretory cells, thus contributing to the elucidation of the pathophysiology of different diseases. The different staining characteristics of mucins can be useful in diagnostic histopathology. It has been proposed that the reduction in sulphated glycoproteins and an increase in sialomucins in intestinal mucosa was an indicator of premalignant changes in carcinoma of the bowel. It has subsequently argued that this change may be consequence rather than a precursor of neoplasia. It may still be of some value as a marker of a premalignant change although it is somewhat variable. Previous studies have demonstrated the characterization of glycoproteins in different type of epitheloid cells in normal and pathologic conditions by using histochemical and electron microscopic techniques. However; little information has been available concerning the exact features of secretory granules in both normal and tumoral cells in lung.

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