scholarly journals Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

2004 ◽  
Vol 164 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Michael Klemba ◽  
Wandy Beatty ◽  
Ilya Gluzman ◽  
Daniel E. Goldberg
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Food Vacuole ◽  
Protein Fluorescence ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Plasmepsin Ii ◽  
Green Fluorescent ◽  
Secretory System

fA amily of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II–GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.

scholarly journals Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum

2007 ◽  
Vol 407 (3) ◽  
pp. 343-354 ◽  
Author(s):  
Nectarios Klonis ◽  
Olivia Tan ◽  
Katherine Jackson ◽  
Daniel Goldberg ◽  
Michael Klemba ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescent Protein ◽  
Ph Value ◽  
Ph Dependence ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Intracellular Compartments ◽  
Green Fluorescent ◽  
Haem Detoxification ◽  
Endocytic Vesicles

The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4–5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems.

Food vacuole-associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum

2004 ◽  
Vol 54 (1) ◽  
pp. 109-122 ◽  
Author(s):  
Katherine E. Jackson ◽  
Nectarios Klonis ◽  
David J. P. Ferguson ◽  
Akinola Adisa ◽  
Con Dogovski ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Food Vacuole ◽  
Lipid Bodies ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

scholarly journals Effect of sequential C‐terminal tryptophans on green fluorescent protein fluorescence

FEBS Open Bio ◽  
2018 ◽  
Vol 8 (7) ◽  
pp. 1176-1183
Author(s):  
Shirou Tsuchida ◽  
Takumi Kanashiki ◽  
Shuhei Izumiya ◽  
Takuya Ichikawa ◽  
Ryusuke Kurosawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Protein Fluorescence ◽  
Green Fluorescent

scholarly journals Fate of haem iron in the malaria parasite Plasmodium falciparum

2002 ◽  
Vol 365 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Timothy J. EGAN ◽  
Jill M. COMBRINCK ◽  
Joanne EGAN ◽  
Giovanni R. HEARNE ◽  
Helder M. MARQUES ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Chemical Analysis ◽  
Malaria Parasite ◽  
Spectroscopic Imaging ◽  
Food Vacuole ◽  
Electron Spectroscopic Imaging ◽  
Iron Species ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Haem Iron

Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61±2% of the iron within parasitized erythrocytes, of which 92±6% is located within the food vacuole. Of this, 88±9% is in the form of haemozoin. 57Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion.

scholarly journals Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence

2003 ◽  
Vol 69 (7) ◽  
pp. 4214-4218 ◽  
Author(s):  
J. Reunanen ◽  
P. E. J. Saris
Keyword(s):  
Green Fluorescent Protein ◽  
Lactococcus Lactis ◽  
Culture Supernatant ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Regulatory Proteins ◽  
Protein Fluorescence ◽  
Gene Encoding ◽  
Two Component ◽  
Green Fluorescent

ABSTRACT A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 μg of nisin in cheese, and 1 μg of nisin per ml in salad dressings.

scholarly journals Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere

1999 ◽  
Vol 65 (10) ◽  
pp. 4646-4651 ◽  
Author(s):  
Bo Normander ◽  
Niels B. Hendriksen ◽  
Ole Nybroe
Keyword(s):  
Green Fluorescent Protein ◽  
Pseudomonas Fluorescens ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protein Fluorescence ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The gfp-tagged Pseudomonas fluorescensbiocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.

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