scholarly journals Optimized Method for Pseudomonas aeruginosa Integrative Filamentous Bacteriophage Propagation

2022 ◽  
Vol 12 ◽  
Author(s):  
Damir Gavric ◽  
Petar Knezevic
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gradient Centrifugation ◽  
Virus Titer ◽  
Single Layer ◽  
Filamentous Phage ◽  
Equilibrium Density ◽  
Phenotypic Traits ◽  
Filamentous Bacteriophages ◽  
High Virus ◽  
First Time

Filamentous bacteriophages frequently infect Pseudomonas aeruginosa and alter its phenotypic traits, including virulence factors. The first step in examination of these phages is to obtain suspensions with high virus titer, but as there are no methods for integrative filamentous phage multiplication, the aim was to design, describe, and compare two methods for this purpose. As models, three strains of Pseudomonas aeruginosa, containing (pro)phages Pf4, Pf5, and PfLES were used (PAO1, UCBPP-PA14, and LESB58, respectively). Method 1 comprised propagation of phages in 6 L of bacterial culture for 48 h, and method 2 applied 600 mL culture and incubation for 6 days with centrifugation and addition of new medium and inoculum at 2-day intervals. In method 1, phages were propagated by culture agitation, followed by centrifugation and filtration (0.45 and 0.22 μm), and in method 2, cultures were agitated and centrifuged several times to remove bacteria without filtration. Regardless of the propagation method, supernatants were subjected to concentration by PEG8000 and CsCl equilibrium density gradient centrifugation, and phage bands were removed after ultracentrifugation and dialyzed. In the obtained suspensions, phage titer was determined, and concentration of isolated ssDNA from virions was measured. When propagation method 2 was compared with method 1, the phage bands in CsCl were much thicker, phage number was 3.5–7.4 logs greater, and concentration of ssDNA was 7.6–22.4 times higher. When phage count was monitored from days 2 to 6, virion numbers increased for 1.8–5.6 logs, depending on phage. We also observed that filamentous phage plaques faded after 8 h of incubation when the double layer agar spot method was applied, whereas the plaques were visible for 24 h on single-layer agar. Finally, for the first time, we confirmed existence of replicative form and virions of PfLES (pro)phage as well as its ability to produce plaques. Similarly, for the first time, we confirmed plaque production of Pf5 (pro)phage present in P. aeruginosa strain UCBPP-PA14. The described method 2 has many advantages and can be further improved and adopted for filamentous phages of other hosts.

scholarly journals Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitions of platelet aggregation; enrichment achieved with a two-step separation technique.

1976 ◽  
Vol 69 (1) ◽  
pp. 159-172 ◽  
Author(s):  
R F Levine ◽  
M E Fedorko
Keyword(s):  
Guinea Pig ◽  
Gradient Centrifugation ◽  
Velocity Sedimentation ◽  
High Yield ◽  
Equilibrium Density ◽  
Large Size ◽  
Membrane Topography ◽  
First Time ◽  
Better Than

Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro.

Perforation mechanics of UHMWPE soft ballistic sub-laminate and soft ballistic armor pack: A finite element study

2021 ◽  
pp. 089270572110420
Author(s):  
Bazle Z (Gama) Haque ◽  
John W Gillespie
Keyword(s):  
Finite Element ◽  
Single Layer ◽  
Areal Density ◽  
Element Model ◽  
Transverse Impact ◽  
Free Standing ◽  
Uhmwpe Fibers ◽  
Ballistic Limit Velocity ◽  
Definition Of ◽  
First Time

Soft-ballistic sub-laminate (SBSL) made from ultra-high molecular weight polyethylene (UHMWPE) fibers in [0/90] stacking sequence are the building block of a multi-layer soft-ballistic armor pack (SBAP, aka Soft Armor). A systematic study of the perforation dynamics of a single layer SBSL and several multi-layer SBAPs (2, 3, 4, 8, 16, 24, 32 layers) is presented for the first time in the literature. A previously validated finite element model of transverse impact on a single layer is used to study the perforation mechanics of multi-layer SBAPs with friction between individual layers. Following the classical definition of ballistic limit velocity, a minimum perforation velocity has been determined for free-standing single layer SBSL and multi-layer SBAPs. For the multi-layer SBAPs, complete perforations have been identified as progressive perforation of individual layers through the thickness. The minimum perforation velocities of multi-layer SBAPS is linear with the areal density for the eight (8) layer target and thicker. Large deformation behavior and perforation mechanics of the SBAPs is discussed in detail.

Mapping subcellular distribution of Na+-K+-ATPase in rat parotid gland

1986 ◽  
Vol 250 (3) ◽  
pp. C430-C441 ◽  
Author(s):  
C. N. Conteas ◽  
A. A. McDonough ◽  
T. R. Kozlowski ◽  
C. B. Hensley ◽  
R. L. Wood ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Sedimentation Equilibrium ◽  
Equilibrium Density ◽  
Phase System ◽  
Gamma Glutamyl Transpeptidase ◽  
Gamma Glutamyl ◽  
Rat Parotid ◽  
Glutamyl Transpeptidase

Recent subcellular fractionation studies have raised the possibility that Na+-K+-ATPase might be present in both the apical and the basal-lateral membranes of exocrine gland acinar cells. Analytical fractionation and immunofluorescence microscopy studies of rat parotid glands were performed to confirm this interpretation. The distributions of biochemical markers after analyses based on differential sedimentation, equilibrium density-gradient centrifugation, and partitioning in an aqueous polymer two-phase system defined a total of 15 physically and biochemically distinct membrane populations. Among these populations, it was possible to select one (designated population i) with the characteristics expected of acinar cell basal-lateral plasma membranes. It contained Na+-K+-ATPase enriched 33-fold, and gamma-glutamyl transpeptidase enriched 23-fold with respect to the initial homogenate. A second population (designated population c) had the characteristics expected of acinar cell apical plasma membranes; it contained Na+-K+-ATPase enriched 28-fold, and gamma-glutamyl transpeptidase enriched 53-fold with respect to the initial homogenate. Although the identification of population c remains provisional, immunofluorescence studies verified that Na+-K+-ATPase is present in both the apical and the basal-lateral acinar cell plasma membranes. In view of these results, it is likely that the apical Na+-K+-ATPase would participate in series with basal-lateral sodium- and chloride-entry pathways in driving the secretory electrolyte fluxes.

scholarly journals Pf Filamentous Phage Requires UvrD for Replication in Pseudomonas aeruginosa

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Eriel Martínez ◽  
Javier Campos-Gómez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Repair ◽  
Dna Helicase ◽  
Filamentous Phage ◽  
Rolling Circle Replication ◽  
Content Type ◽  
Rolling Circle ◽  
Initiator Protein ◽  
Biofilm Development ◽  
Filamentous Phages

ABSTRACT Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa, plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous phages among diverse bacteria genera, adding a new, previously unrecognized function of this accessory helicase. Pf is a lysogenic filamentous phage that promotes biofilm development in Pseudomonas aeruginosa. Pf replicates by a rolling circle replication system which depends on a phage-encoded initiator protein and host factors usually involved in chromosome replication. Rep, an accessory replicative DNA helicase, is crucial for replication of filamentous phages in Escherichia coli. In contrast, here we show that, instead of depending on Rep, Pf replication depends on UvrD, an accessory helicase implicated in DNA repair. In this study, we also identified the initiator protein of Pf and found that it shares similarities with that of Vibrio phages CTXφ and VGJφ, which also depend on UvrD for replication. A structural comparative analysis of the initiator proteins of most known filamentous phages described thus far suggested that UvrD, known as a nonreplicative helicase, is involved in rolling circle replication of filamentous phages in diverse bacteria genera. This report consolidates knowledge on the new role of UvrD in filamentous phage replication, a function previously thought to be exclusive of Rep helicase. IMPORTANCE Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa, plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous phages among diverse bacteria genera, adding a new, previously unrecognized function of this accessory helicase.

scholarly journals Analysis of the role of p200-containing vesicles in post-Golgi traffic.

1996 ◽  
Vol 7 (6) ◽  
pp. 961-974 ◽  
Author(s):  
E Ikonen ◽  
R G Parton ◽  
F Lafont ◽  
K Simons
Keyword(s):  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Apical Surface ◽  
Transport Pathways ◽  
Permeabilized Cells ◽  
Influenza Hemagglutinin ◽  
Streptolysin O ◽  
Marker Proteins ◽  
Viral Marker ◽  
Golgi Network

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.

Dna Synthesis in Purified Populations of Avian Erythroid Cells

1972 ◽  
Vol 10 (1) ◽  
pp. 27-46
Author(s):  
A. F. WILLIAMS
Keyword(s):  
Biochemical Characterization ◽  
Bone Marrow Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Experimental System ◽  
Equilibrium Density ◽  
Polychromatic Erythrocytes ◽  
Type Size ◽  
Haemoglobin Content

By the use of equilibrium density-gradient centrifugation erythroblasts and early polychromatic erythrocytes have been isolated from avian anaemic bone marrow. Cells from both the unfractionated and purified preparations have been characterized in terms of their histological type, size, haemoglobin content and ability to synthesize DNA. Erythroblasts were the only cells to synthesize DNA and it appeared that their progeny, the polychromatic erythrocyte, failed to enter a new S phase. The experimental system described allows biochemical characterization of earlier stages of avian erythropoiesis than has previously been possible.

scholarly journals Pseudomonas aeruginosa Requires the DNA-Specific Endonuclease EndA To Degrade Extracellular Genomic DNA To Disperse from the Biofilm

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Kathryn E. Cherny ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Genomic Dna ◽  
Early Stage ◽  
Content Type ◽  
Biofilm Dispersion ◽  
Biofilm Structure ◽  
First Time ◽  
Biofilm Matrix ◽  
Dispersed Cells

ABSTRACT The dispersion of biofilms is an active process resulting in the release of planktonic cells from the biofilm structure. While much is known about the process of dispersion cue perception and the subsequent modulation of the c-di-GMP pool, little is known about subsequent events resulting in the release of cells from the biofilm. Given that dispersion coincides with void formation and an overall erosion of the biofilm structure, we asked whether dispersion involves degradation of the biofilm matrix. Here, we focused on extracellular genomic DNA (eDNA) due to its almost universal presence in the matrix of biofilm-forming species. We identified two probable nucleases, endA and eddB, and eddA encoding a phosphatase that were significantly increased in transcript abundance in dispersed cells. However, only inactivation of endA but not eddA or eddB impaired dispersion by Pseudomonas aeruginosa biofilms in response to glutamate and nitric oxide (NO). Heterologously produced EndA was found to be secreted and active in degrading genomic DNA. While endA inactivation had little effect on biofilm formation and the presence of eDNA in biofilms, eDNA degradation upon induction of dispersion was impaired. In contrast, induction of endA expression coincided with eDNA degradation and resulted in biofilm dispersion. Thus, released cells demonstrated a hyperattaching phenotype but remained as resistant to tobramycin as biofilm cells from which they egress, indicating EndA-dispersed cells adopted some but not all of the phenotypes associated with dispersed cells. Our findings indicate for the first time a role of DNase EndA in dispersion and suggest weakening of the biofilm matrix is a requisite for biofilm dispersion. IMPORTANCE The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

Triton WR-1339 injected into rats enters renal renin granules

1988 ◽  
Vol 255 (3) ◽  
pp. E353-E356
Author(s):  
B. J. Morris
Keyword(s):  
Plasma Proteins ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Extracellular Fluid ◽  
Sucrose Density Gradient ◽  
Equilibrium Density ◽  
Juxtaglomerular Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton Wr 1339 ◽  
Extracellular Substances

To test directly the possibility that substances in extracellular fluid can gain access to renin storage granules in renal juxtaglomerular cells, rats were injected with Triton WR-1339, which binds to plasma proteins. A heavy granule fraction was prepared, and isopycnic sucrose density gradient centrifugation was performed. The renin granule peak was found to be altered from a mean equilibrium density of 1.202 g/ml in control rats to 1.196 g/ml for rats injected with Triton WR-1339 (P less than 0.005). The distribution of angiotensinogen, which is bound in kidney granules having a different buoyant density, was also examined and found to be unaltered. After injection, Triton WR-1339 binds to circulating plasma proteins. The results for renin support the possibility of pinocytotic uptake of protein-Triton WR-1339 complexes by the juxtaglomerular cells with subsequent fusion of the endocytotic lysosomal vacuoles with renin granules accounting for the translocation of ingested substances into the granule matrix. If so, the potential would therefore exist for interaction(s) of ingested extracellular substances with renin or other components in the granules. The present study has therefore demonstrated directly that endogenous extracellular substances may enter renin granules.

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