scholarly journals Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

2021 ◽  
Author(s):  
RB Gorodnichev ◽  
MA Kornienko ◽  
NS Kuptsov ◽  
AD Efimov ◽  
VI Bogdan ◽  
...  
Keyword(s):  
Density Gradient ◽  
Sucrose Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Phage Lysate ◽  
Purification Method ◽  
Cscl Density Gradient ◽  
Cell Components ◽  
Purification Methods ◽  
Sucrose Gradient Ultracentrifugation

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

scholarly journals THE SEDIMENTATION PROPERTIES OF THE SKIN-SENSITIZING ANTIBODIES OF RAGWEED-SENSITIVE PATIENTS

1964 ◽  
Vol 120 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Burton R. Andersen ◽  
Wilton E. Vannier
Keyword(s):  
Density Gradient ◽  
Sedimentation Rate ◽  
Gradient Method ◽  
Sucrose Density Gradient ◽  
Major Portion ◽  
Density Gradient Ultracentrifugation ◽  
Cell Method ◽  
Average Value ◽  
Γ Globulins

The sedimentation coefficients of the skin-sensitizing antibodies to ragweed were evaluated by the moving partition cell method and the sucrose density gradient method. The most reliable results were obtained by sucrose density gradient ultracentrifugation which showed that the major portion of skin-sensitizing antibodies to ragweed sediment with an average value of 7.7S (7.4 to 7.9S). This is about one S unit faster than γ-globulins (6.8S). The data from the moving partition cell method are in agreement with these results. Our studies failed to demonstrate heterogeneity of the skin-sensitizing antibodies with regard to sedimentation rate.

scholarly journals Subcellular localization of ferritin and iron taken up by rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 685-688 ◽  
Author(s):  
J C Sibille ◽  
M Ciriolo ◽  
H Kondo ◽  
R R Crichton ◽  
P Aisen
Keyword(s):  
Acid Phosphatase ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Density Gradient Ultracentrifugation

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

scholarly journals Hypoprothrombinemia: case report

Blood ◽  
1978 ◽  
Vol 51 (2) ◽  
pp. 299-306
Author(s):  
RR Montgomery ◽  
A Otsuka ◽  
WE Hathaway
Keyword(s):  
Case Report ◽  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Factor Ii ◽  
History Of

A patient with a significant history of spontaneous and posttraumatic bleeding was found to have hypoprothrombinemia. His prothrombin (factor II) activity by clotting assay was 9.5% and his factor II antigen was 5%. Crossed immunoelectrophoresis and sucrose density gradient ultracentrifugation of the patient's plasma showed his prothrombin to be qualitatively indistinguishable from normal prothrombin by these techniques.

scholarly journals Oral toxicities of Clostridium botulinum type C and D toxins of different molecular sizes

1980 ◽  
Vol 28 (2) ◽  
pp. 303-309
Author(s):  
I Ohishi ◽  
G Sakaguchi
Keyword(s):  
Density Gradient ◽  
Clostridium Botulinum ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Botulinum Toxins ◽  
Molecular Dissociation ◽  
Botulinum Type ◽  
Molecular Sizes ◽  
Type C ◽  
Progenitor Toxin

Clostridium botulinum type C progenitor toxins of different molecule sizes, C-L (16S) and C-M (12S), were purified from cultures of strains 573, Stockholm, and CB-19. C-L toxin showed some hemaggglutinin activity, whereas C-M toxin did not. Neither C-L nor C-M toxin was activated upon trypsinization. Molecular dissociation of purified type C-L and C-M toxins into toxic and nontoxic components was demonstrated by sucrose density gradient ultracentrifugation and diethylaminoethyl-Sephadex chromatography at pH 8.0. The molecular construction of type C progenitor toxin appears to be analogous to that reported for botulinum toxins of other types. C-L and D-L toxins showed higher oral toxicities to mice than did C-M or D-M toxin. Such higher oral toxicities were ascribed to the higher stabilities of these toxins in gastric and intestinal juices.

scholarly journals CHARACTERIZATION OF IMMUNOGLOBULIN STRUCTURES FROM THE SURFACE OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

1971 ◽  
Vol 134 (1) ◽  
pp. 265-280 ◽  
Author(s):  
Trond Eskeland ◽  
Eva Klein ◽  
Masaharu Inoue ◽  
Bo Johansson
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Density Gradient Ultracentrifugation ◽  
Freezing And Thawing ◽  
Passive Hemagglutination

Chronic lymphocytic leukemia cells with relatively large amounts of mu and kappa immunoglobulin structures on the surface, and apparently very small amounts intracellularly, were subjected to homogenization or washing after freezing and thawing. After a light centrifugation, which sedimented the nuclei and unbroken cells, most of the immunoglobulin structures were found in the supernatant. Ultracentrifugation, which was performed to remove the membranes from the supernatant, sedimented only half the amount of the immunoglobulin structures. By sucrose density gradient ultracentrifugation and Sephadex G-200 filtration, the unsedimented immunoglobulin structures were shown to consist of 7S IgM and free kappa chains. About 80,000 7S IgM molecules were calculated to be present on each cell. The amount of kappa chains not associated with IgM was estimated to be equal to the amount of kappa chains in IgM. Inhibition of passive hemagglutination was used to detect and quantitate the immunoglobulin structures.

scholarly journals Soluble C3 proconvertase and convertase of the classical pathway of human complement. Conditions of stabilization in vitro

1985 ◽  
Vol 226 (2) ◽  
pp. 429-436 ◽  
Author(s):  
M B Villiers ◽  
N M Thielens ◽  
M G Colomb
Keyword(s):  
Density Gradient ◽  
Binding Protein ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Classical Pathway ◽  
Human Complement ◽  
Proteolytic Fragment ◽  
The Individual

Soluble classical-pathway C3 convertase and proconvertase were prepared from purified C4b-C2ox complex in the presence of Ni2+; the two complexes, stable for at least 15 h at 4 degrees C, were isolated by sucrose-density-gradient ultracentrifugation. The C3 convertase alone was able to cleave C3, and its decay was accelerated in the presence of C4-binding protein. The individual roles of Ni2+ and I2 treatment of C2 in the stabilization of the complexes seemed to be different and additive. 63Ni2+ binding coupled to h.p.l.c. analysis showed that 63Ni2+ bound only to the C2ox proteolytic fragment a (1 mol/mol) with a Kd of 26 microM. Competition studies between Ni2+ and Mg2+ indicated that only half of the Ni2+ bound to the C3 convertase was removed by Mg2+, whereas, in the same conditions, Ni2+ bound to C2ox proteolytic fragment a was not displaced, suggesting the presence of two sets of sites on the convertase. EDTA prevented the formation of both C3 convertase and proconvertase; EDTA had no effect on the preformed C3 convertase, whereas it dissociated the preformed proconvertase.

scholarly journals Reconstitution of an Allophycocyanin Trimer Complex Containing the C-Terminal 21-23 kDa Domain of the Core-Membrane Linker Polypeptide Lcm

1994 ◽  
Vol 49 (5-6) ◽  
pp. 331-336 ◽  
Author(s):  
Lothar Gottschalk ◽  
Friedrich Lottspeich ◽  
Hugo Scheer
Keyword(s):  
Density Gradient ◽  
Fluorescence Emission ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Preparative Scale ◽  
Small Core ◽  
The Core ◽  
Spectral Changes ◽  
Sds Page ◽  
Molecular Masses

Abstract Allophycocyanin (AP) was isolated from extracts of the cyanobacterium Mastigocladus laminosus. A fraction enriched in AP-associated polypeptides with apparent molecular masses of 21 -23 kD a in SDS-PAGE, was isolated on a preparative scale and identified as a homolo­gous mixture of C-terminal fragments of the core-membrane linker polypeptide Lcm. The complex (αAPβAP)3 ·21 -23 kD a was reconstituted and characterized by sucrose density gradient ultracentrifugation, absorption, fluorescence emission and circular dichroism spectroscopy. The 21 -23 kD a polypeptides were found to induce spectral changes in AP similar to those induced by the small core linker polypeptide Lc8.9. Possible functions of the complex in phycobilisomes are discussed.

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