Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 microg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.     

Partial purification and characterization of the organic solvent-tolerant lipase produced by Pseudomonas fluorescens RB02-3 isolated from milk

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50 °C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn2+, Co2+, Cu2+, Ni2+ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd2+, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Quantitative Ultrastructural Investigations of Mitochondrial Development in Leishmania donovani During Transformation*

SYNOPSIS. The ultrastructure of the mitochondrion during transformation of Leishmania donovani from amastigote to promastigote stages was studied by morphometric analysis. There was a slight but significant decrease in relative mitochondrial volume (Vvli) during transformation. This decrease was linear with time for at least 27 hr. No change in relative lipid inclusion volume (Vvli) was observed during transformation. The ratio of inner to outer mitochondrial surface membrane densities (Svmii:Svmio) also remained unchanged. Although the relative number of cristae remains unchanged after transformation, there is an increase in cristae number in the portion of the kinetoplast opposite the flagellum.     

The Ultrastructure of Crithidia fasciculata and Morphological Changes Induced by Growth in Acriflavin*

SYNOPSIS. Crithidia fasciculata is similar to other trypanosomatids in ultrastructure. Of considerable interest is the finding of a lamellar membrane formation attached to the mitochondrion. Some evidence is provided for the tentative hypothesis that this membrane formation is the precursor of mitochondrial membranes. The cellular origin of this structure is unknown. Growth in acriflavin results in a marked alteration of the mitochondrion and kinetoplast. Both structures are deficient in cristae. In addition, the kinetoplast loses its normal fibrillar appearance and becomes a smaller, more electron-dense organelle. These effects are discussed in relation to the proposed involvement of the kinetoplast in the elaboration of functional mitochondria.     

Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P2-Associated,Cytoskeletal Collapse and Late Phase Membrane Blebbing

Pseudomonas aeruginosa is an opportunistic pathogen that is associated with hospital-acquired infections, ventilator-associated pneumonia, and morbidity of immunocompromised individuals. A subpopulation of P. aeruginosa encodes a protein, ExoU, which exhibits acute cytotoxicity. Toxicity is directly related to the phospholipase A₂ activity of the protein after injection into the host cytoplasm via a type III secretion system. ExoU enzymatic activity requires eukaryotic cofactors, ubiquitin or ubiquitin-modified proteins. When administered extracellularly, ExoU is unable to intoxicate epithelial cells in culture, even in the presence of the cofactor. Injection or transfection of ExoU is necessary to observe the acute cytotoxic response. Biochemical approaches indicate that ExoU possesses high affinity to a multifunctional phosphoinositide, phosphatidylinositol 4,5-bisphosphate or PI(4,5)P₂ and that it is capable of utilizing this phospholipid as a substrate. In eukaryotic cells, PI(4,5)P₂ is mainly located in the cytoplasmic side of the plasma membrane and anchors adaptor proteins that are involved in cytoskeletal structures, focal adhesions, and plasma membranes. Time-lapse fluorescent microscopy analyses of infected live cells demonstrate that ExoU intoxication correlates with intracellular damage in the early phases of infection, such as disruption of focal adhesions, cytoskeletal collapse, actin depolymerization, and cell rounding. At later time points, a membrane blebbing phenotype was prominent prior to the loss of the plasma membrane integrity and barrier function. Membrane blebbing appears to accelerate membrane rupture and the release of intracellular markers. Our data suggest that in eukaryotic host cells, intracellular ExoU targets and hydrolyzes PI(4,5)P₂ on the plasma membrane, causing a subsequent disruption of cellular structures and membrane integrity.     

Pseudomonas fluorescens as a potential pathogen: adherence to nerve cells

In order to determine the infectious potential of the psychrotrophic bacterium Pseudomonas fluorescens, a species closely related to the opportunistic pathogen P. aeruginosa, we investigated the binding activity of this bacterium on primary cultures of rat neonate cortical neurons and glial cells, adrenal paraneurons and NG108-15 neuroblastoma cells. Incubated at concentrations of 10(6) and 10(8) CFU/mL, P. fluorescens MF37 exhibited a high binding activity on neurons in the same range as that of P. aeruginosa PAO1. A significant, but lower, adherence of P. fluorescens was also detected on glial cells and adrenal paraneurons. In contrast, when P. fluorescens MF37 or P. aeruginosa PAO1 were incubated with neuroblastoma cells, no binding was observed. In neurons, the association of P. fluorescens with the plasma membrane occurred both on neurites and cell body. Leakage of the cytoplasmic content was frequently noted. Studies performed using the fluorescent probe Hoechst 33258 revealed that in 10% of neurons, P. fluorescens induced the appearance of densely stained clusters of DNA that was typical of an early step of apoptosis. In glial cells exposed to P. fluorescens, marked changes in the morphology of the nucleus, including fragmentation into lobular structures and aggregation of DNA, were also reminiscent of the existence of a possible apoptotic mechanism. Taken together, these results reveal that P. fluorescens can bind to nerve cells and affect their physiology and, in agreement with recent clinical observations, suggest that P. fluorescens could behave as a pathogen.     

Amikacin disrupts the cell envelope of Pseudomonas aeruginosa ATCC 9027

Amikacin, an aminoglycoside known to inhibit protein synthesis, was found to perturb the outer membrane of a sensitive Pseudomonas aeruginosa strain (ATCC 9027). This perturbation was monitored using electron microscopy and biochemical analyses. Following exposure to 20 micrograms amikacin/mL for 15 min, the outer membrane of exponentially growing cells lost 15% of its protein, 18% of its lipopolysaccharide, and 18% of its phosphate. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the whole spectrum of outer membrane protein and lipopolysaccharide was affected. Similarly, atomic absorption spectrophotometry revealed that magnesium and calcium were also lost. When cells were treated with amikacin, electron microscopy of negative stains showed a substantial increase in outer membrane blebbing. Freeze fractures revealed changes in membrane fracture pattern and particle distribution, and thin sections revealed a sequential disruption of the cell envelope beginning at the outer membrane and ending at the plasma membrane. This study supports the proposal that aminoglycoside antibiotics cross the outer membrane of Pseudomonas aeruginosa by displacing metal cations necessary to stabilize the organic constituents of the membrane. Their removal results in loss of the outer membrane and the formation of transient small holes which permit the antibiotic access to the cytoplasmic membrane where it is transported into the cytoplasm.     

Viruses of Entamoeba histolytica II. Morphogenesis of the Polyhedral Particle (ABRM2→HK-9)→HB-301 and the Filamentous Agent (ABRM)2→HK-9

The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses.     

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.     

Cell Blebbing and Membrane Area Homeostasis in Spreading and Retracting Cells

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.     

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Summary In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.Abbreviations b endoplasmic reticulum extension, blebbing - er endoplasmic reticulum - f fibrillar material - g Golgi-like structure - h haustorium - hb haustorial body - hcw haustorial cell wall - hcy haustorial cytoplasm - hf haustorial finger - hocw host cell wall - hocy host cytoplasm - 1 lipid-like droplet - m mitochondrion - mt microtubule - mve multivesicular body - n nucleus - p papilla - ph penetration site of an infection peg - pl plasma membrane - s sheath - sm extrahaustorial membrane - v vacuole - ve vesicle     

Trypanosoma brucei: morphometric changes and loss of infectivity during transformation of bloodstream forms to procyclic culture forms in vitro.

The transformation of Trypanosoma brucei bloodstream forms to procyclic culture forms in the semidefined medium SDM-77 has been studied by light microscopy and quantitative electron microscopy. Stumpy and intermediate forms are able to transform to culture forms whereas slender forms die after approximately 24 hr. The surface coat and infectivity for the mammalian host are lost after 72 hr. Morphometrical analysis of the cells during transformation process revealed: (1) The cytoplasm and the cell surface increased significantly; (2) the volume density of the mitochondrion increased twofold and the surface density of the inner mitochondrial membrane increased threefold; (3) the volume density of the glycosomes remained about constant; (4) the volume density of the lipid inclusions increased up to 72 hr, probably as a result of the complete oxidation of glucose. Transformation as observed by light microscopy was completed in 72 hr. However, quantitative electron microscopy revealed that establishment of the culture form was incomplete even after 11 days.     

Long-term reduction of cold hardiness following ingestion of ice-nucleating bacteria in the Colorado potato beetle,Leptinotarsa decemlineata

We investigated the effect of ingestion of ice-nucleating bacteria on the supercooling capacity and cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a freeze-intolerant species that overwinters as adults in shallow, terrestrial burrows. Ingestion of ice-nucleating bacteria (Enterobacter agglomerans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae), fed on slices of potato tuber, caused an abrupt decrease in supercooling capacity. No change occurred in the supercooling capacity of beetles fed Escherichia coli, as this species lacks ice-nucleating activity. Ingestion rates showed that tubers treated with different species were equally palatable. During diapause induction beetles evacuated food from their guts, but nevertheless retained sufficient ice-nucleating bacteria to diminish supercooling. Beetles fed P. fluorescens and P. putida exhibited reduced supercooling even after an 8-wk exposure to simulated winter conditions. Furthermore, P. fluorescens was isolated 10-wk post-ingestion from diapausing beetles. Our data suggest that ingested bacteria may be retained by insects during entry into diapause and that the cold hardiness of candidate crop pests, such as L. decemlineata, may be reduced by feeding them ice-nucleating bacteria prior to winter diapause.     

The roles of ATP and calcium in morphological changes and cytotoxicity induced by 1,4-benzoquinone in platelets

To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.     

The chondriome of selected trypanosomatids. A three-dimensional study based on serial thick sections and high voltage electron microscopy

The unitary nature of the chondriome of two species of trypanosomatids, Blastocrithidia culicis and Trypanosoma cruzi, has been demonstrated by utilizing serial thick-sectioning techniques combined with high voltage electron microscopy. Profiles of mitochondrial elements seen in thin sections and suspected to be parts of a continuum were confirmed by serial thick sectioning (0.25-0.50 mum thick) and stereopair analysis to be parts of the same mitochondrion. Three-dimensional models obtained from tracings of mitochondrial profiles on cellulose acetate reveal the mitochondrion of B. culicis to consist of a posterior mass with six tubular extensions extending upward and terminating in the anterior apex. The kinetoplast was found suspended between two of the tubular extensions, or less frequently, protuding as a nodule from one of the extensions. A bifurcation of one of the extensions was found in some specimens. The mitochondrion of T. cruzi consists of a triangular- shaped convoluted tubule, the base being the kinetoplast portion while the apex is directed posteriorly. The mitochondrion bifurcates behind the flagellar pocket, lateral to the kinetoplast, sending two entwined extensions into the tenuous anterior apex. Whether the mitochondrion of T. cruzi is unitary in the trypomastigote form was not determined in this study, since only epimastigote forms were used.     

The diversity of lipases from psychrotrophic strains of Pseudomonas : a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene ( lipA ) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens , the gene encodes a lipase of M_r 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M_r approximately 30 kDa comprising sequences from Ps. fragi , Ps. aeruginosa , Ps. fluorescens C9 and Burkholderia , and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

Synaptotagmin II could confer Ca(2+) sensitivity to phagocytosis in human neutrophils

To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.     

Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome

Recently, a novel verocytotoxin named VT2y was described which belongs to the STx family and is produced by Escherichia coli isolated from domestic poultry with swollen head syndrome (SHS). The VT2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines. Morphological evidence (nuclear shrinkage, chromatin condensation and blebbing of the plasma membrane) of apoptosis could be distinguished in 15 min and was maximal at 1 h after treatment with VT2y. This was confirmed by the terminal dUTP nick-end-labeling (TUNEL) method.     

The fine structure of Crithidia fasciculata with special reference to the organelles involved in the ingestion and digestion of protein

Summary As in other trypanosomatids, the cell membrane of Crithidia fasciculata overlies a single layer of microtubules. Each microtubule possesses a large number of periodically arranged drumstick-like appendages and adjacent microtubules are joined by fibrillar connectives. Anteriorly, the microtubules gradually taper to terminate just before or just after entering the reservoir. An attempt is made to correlate microtubule tapering with maintenance of form of the truncated anterior end of the cell. Smooth and coated vesicles are proliferated from the Golgi saccules and the prominent contractile vacuole lies nearby. The single mitochondrion is extensive and expanded at one point to form a capsule for the kinetoplast. The cristae are predominantly plate-like but other configurations do occur. The cytostome, a shallow invagination of the reservoir membrane, is found between two constrictions in the reservoir wall. Supporting the cytostome are several microtubules which penetrate deeply into the cytoplasm. Ingestion of ferritin occurs by pinocytosis from the cytostome and by coated vesicle formation from the reservoir membrane. Digestion probably occurs in multivesicular bodies which contain acid phosphatase activity.