Evidence that the nephrotoxin from the fungus Mortierella wolfii is a protein

1974 ◽  
Vol 20 (11) ◽  
pp. 1513-1516 ◽  
Author(s):  
G. Davey ◽  
J. Kalmakoff
Keyword(s):  
Lethal Dose ◽  
Protein A ◽  
Toxic Substance ◽  
Water Layer ◽  
Iodoacetic Acid ◽  
Exchange Chromatography ◽  
Water Mixture ◽  
Calculated Molecular Weight ◽  
Covalently Linked ◽  
Almost All

A purified toxin from Mortierella wolfii contained about 60% lipid, 39% protein, and less than 0.4% carbohydrate. Labelling of the toxin, with the protein-alkylating agent 3H-iodoacetic acid and subsequent extraction of the lipid with a chloroform–ethanol–water mixture resulted in almost all of the radioactivity (97%) being present in the ethanol–water layer, suggesting that the lipid was not covalently linked to the protein. A threefold reduction in the lipid content of the toxin by ion-exchange chromatography did not reduce toxicity of the toxin, expressed in mean lethal dose (LD50) per unit of protein. The LD50 for mice is 3.3 μg protein. The cumulative findings suggested that the active toxic substance is a protein, with a high proline content (17.2% w/w) and a calculated molecular weight from amino acid analysis of 22 800 daltons.

scholarly journals Rooster comb hyaluronate—protein, a non-covalently linked complex

1986 ◽  
Vol 235 (1) ◽  
pp. 117-123 ◽  
Author(s):  
C P Tsiganos ◽  
D H Vynios ◽  
D L Kalpaxis
Keyword(s):  
Protein Fraction ◽  
Core Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Deae Cellulose ◽  
Solvent Mixture ◽  
Exchange Chromatography ◽  
Cscl Density Gradient ◽  
Covalently Linked

Hyaluronate from rooster comb was isolated by ion-exchange chromatography on DEAE-cellulose from tissue extracts and papain digests. The preparations were labelled with [14C]acetic anhydride and subjected to CsCl-density-gradient centrifugation in 4 M-guanidinium chloride in the presence and absence of 4% ZwittergentTM 3-12. A radioactive protein fraction was separated from the hyaluronate when the zwitterionic detergent was also present. The protein could also be separated from the glycosaminoglycan by chromatography on Sepharose CL-6B eluted with the same solvent mixture. The protein fraction contained three protein bands of Mr 15,000-17,000 as assessed by polyacrylamide-gel electrophoresis in 0.1% SDS, and seemed to lack lysozyme activity. No evidence of other protein or amino acid(s) covalently linked with the hyaluronate was obtained. The hyaluronate-protein complex may be re-formed upon mixing the components, the extent of its formation depending on the conditions used. The results show that, as in chondrosarcoma [Mason, d'Arville, Kimura & Hascall (1982) Biochem. J. 207, 445-457] and teratocarcinoma cells [Prehm (1983) Biochem. J. 211, 191-198] the rooster comb hyaluronate also is not linked covalently to a core protein.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

scholarly journals A SPECIFIC POISON IN THE LIVER EXTRACTS OF RABBITS INOCULATED WITH TYPHOID AND PRODIGIOSUS BACILLI INTRAVENOUSLY

1918 ◽  
Vol 28 (5) ◽  
pp. 571-583
Author(s):  
Julia T. Parker
Keyword(s):  
Anaphylactic Shock ◽  
Liver Extract ◽  
Lethal Dose ◽  
Toxic Substance ◽  
Normal Liver ◽  
Immune Serum ◽  
Minimum Lethal Dose ◽  
Lethal Doses

1. The livers of rabbits inoculated with cultures of Bacillus typhosus or Bacillus prodigiosus under certain conditions contain a toxic substance extractable with salt solution. When the toxic extracts are injected intravenously into normal rabbits the latter animals develop symptoms resembling those of anaphylactic shock and succumb. The lethal doses of the toxic extracts are far smaller than those of normal liver extract. 2. The livers of rabbits injected with typhoid antigen also yield a toxic extract. 3. Boiling as well as filtration through a Berkefeld filter only partially detoxicates the extract. 4. Tolerance to one to two lethal doses of the poisonous extracts can be induced by cautious immunization. 5. Rabbits actively immunized to Bacillus typhosus or Bacillus prodigiosus usually resist one lethal dose of the homologous liver poison; and animals tolerant to the typhoid liver poison resist one minimum lethal dose at least of Bacillus typhosus. 6. Typhoid immune serum is not detoxicating either in vivo or in vitro for the typhoid liver poison. 7. The liver poisons are specific, since rabbits actively immunized to either Bacillus typhosus or Bacillus prodigiosus withstand at least one minimum lethal dose of the homologous but not of the heterologous-liver poisons.

Purification of protein A by ion-exchange chromatography

1989 ◽  
Vol 17 (2) ◽  
pp. 396-397
Author(s):  
GERALDINE M. SCULLY ◽  
PATRICK J. CONSIDINE
Keyword(s):  
Ion Exchange ◽  
Protein A ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography

Film spreading from a miscible drop on a deep liquid layer

2017 ◽  
Vol 829 ◽  
pp. 304-327 ◽  
Author(s):  
Raj Dandekar ◽  
Anurag Pant ◽  
Baburaj A. Puthenveettil
Keyword(s):  
Surface Tension ◽  
Water Layer ◽  
Reynolds Numbers ◽  
Capillary Waves ◽  
Water Mixture ◽  
Inertial Forces ◽  
Viscous Forces ◽  
Dimensionless Velocity ◽  
Deep Water Layer ◽  
Film Spreading

We study the spreading of a film from ethanol–water droplets of radii $0.9~\text{mm}<r_{d}<1.1~\text{mm}$ on the surface of a deep water layer for various concentrations of ethanol in the drop. Since the drop is lighter ($\unicode[STIX]{x1D709}=\unicode[STIX]{x1D70C}_{l}/\unicode[STIX]{x1D70C}_{d}>1.03$), it stays at the surface of the water layer during the spreading of the film from the drop; the film is more viscous than the underlying water layer since $\unicode[STIX]{x1D712}=\unicode[STIX]{x1D707}_{l}/\unicode[STIX]{x1D707}_{d}>0.38$. Inertial forces are not dominant in the spreading since the Reynolds numbers based on the film thickness $h_{f}$ are in the range $0.02<Re_{f}<1.4$. The spreading is surface-tension-driven since the film capillary numbers are in the range $0.0005<Ca_{f}<0.0069$ and the drop Bond numbers are in the range $0.19<Bo_{d}<0.56$. We observe that, when the drop is brought in contact with the water surface, capillary waves propagate from the point of contact, followed by a radially expanding, thin circular film of ethanol–water mixture. The film develops instabilities at some radius to form outward-moving fingers at its periphery while it is still expanding, till the expansion stops at a larger radius. The film then retracts, during which time the remaining major part of the drop, which stays at the centre of the expanding film, thins and develops holes and eventually mixes completely with water. The radius of the expanding front of the film scales as $r_{f}\sim t^{1/4}$ and shows a dependence on the concentration of ethanol in the drop as well as on $r_{d}$, and is independent of the layer height $h_{l}$. Using a balance of surface tension and viscous forces within the film, along with a model for the fraction of the drop that forms the thin film, we obtain an expression for the dimensionless film radius $r_{f}^{\ast }=r_{f}/r_{d}$, in the form $fr_{f}^{\ast }={t_{\unicode[STIX]{x1D707}d}^{\ast }}^{1/4}$, where $t_{\unicode[STIX]{x1D707}d}^{\ast }=t/t_{\unicode[STIX]{x1D707}d}$, with the time scale $t_{\unicode[STIX]{x1D707}d}=\unicode[STIX]{x1D707}_{d}r_{d}/\unicode[STIX]{x0394}\unicode[STIX]{x1D70E}$ and $f$ is a function of $Bo_{d}$. Similarly, we show that the dimensionless velocity of film spreading, $Ca_{d}=u_{f}\unicode[STIX]{x1D707}_{d}/\unicode[STIX]{x0394}\unicode[STIX]{x1D70E}$, scales as $4f^{4}Ca_{d}={r_{f}^{\ast }}^{-3}$.

scholarly journals A novel whey protein synthesized only in late lactation by the mammary gland from the tammar (Macropus eugenii)

1987 ◽  
Vol 241 (3) ◽  
pp. 899-904 ◽  
Author(s):  
K R Nicholas ◽  
M Messer ◽  
C Elliott ◽  
F Maher ◽  
D C Shaw
Keyword(s):  
Mammary Gland ◽  
Amino Acid ◽  
Whey Protein ◽  
Sequence Data ◽  
Protein A ◽  
Gel Filtration ◽  
Tammar Wallaby ◽  
Exchange Chromatography ◽  
Macropus Eugenii ◽  
Apparent Homogeneity

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.

scholarly journals Partial deglycosylation of blood-group-specific glycoproteins

1980 ◽  
Vol 185 (2) ◽  
pp. 327-337 ◽  
Author(s):  
A S R Donald
Keyword(s):  
Acetic Acid ◽  
Amino Acid ◽  
Blood Group ◽  
Time Course ◽  
Gel Filtration ◽  
Lumbricus Terrestris ◽  
Exchange Chromatography ◽  
Acid Reagent ◽  
Almost All ◽  
Cyst Fluids

The time course for the partial deglycosylation of blood-group-specific glycoproteins from human ovarian-cyst fluids with 0.25 M-H2SO4/acetic acid and 6 M-HCl in methanol was studied. Either reagent readily removed about 80% of the carbohydrate from the glycoproteins to leave non-diffusible glycopeptides that contain N-acetylgalactosamine as the predominant sugar. Some changes in amino acid distribution were observed during the deglycosylation, which were attributed to an accelerated break-up of the nonglycosylated regions of the parent glycoprotein. The N-acetylgalactosaminyl-peptides isolated were judged to be polydisperse by gel filtration, and ion-exchange chromatography divided the glycopeptide population into several fractions with differing amino acid compositions. A Lumbricus terrestris hexosaminidase preparation was successful in removing almost all the remaining sugar from the glycopeptides, but caused further rupture of the peptide. When a per O-acetylated glycoprotein was treated with the H2SO4/acetic acid reagent the glycopeptide contained, in addition to N-acetylgalactosamine, about 50% of the sialic acid present in the parent glycoprotein, indicating that most of this sugar is located near the peptide end of the carbohydrate chains.

scholarly journals LuxS-Mediated Signaling in Streptococcus mutans Is Involved in Regulation of Acid and Oxidative Stress Tolerance and Biofilm Formation

2004 ◽  
Vol 186 (9) ◽  
pp. 2682-2691 ◽  
Author(s):  
Zezhang T. Wen ◽  
Robert A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Protein A ◽  
Signal Recognition Particle ◽  
Northern Hybridization ◽  
Wild Type ◽  
Increased Sensitivity ◽  
Mutant Phenotypes ◽  
Almost All

ABSTRACT LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role of luxS of Streptococcus mutans in the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation of luxS resulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes included ffh (a signal recognition particle subunit) and brpA (biofilm regulatory protein A). Interestingly, the luxS mutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). The luxS mutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci, smu486 and smu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of the smu486 smu487 mutant were, in almost all cases, similar to those of the luxS mutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates that luxS-dependent signaling plays critical roles in modulating key virulence properties of S. mutans.

scholarly journals The isolation and partial characterization of ribonuclease A from Bison bison

1973 ◽  
Vol 135 (3) ◽  
pp. 427-441 ◽  
Author(s):  
Gordon R. Stewart ◽  
Kenneth J. Stevenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Content ◽  
Attachment Site ◽  
Iodoacetic Acid ◽  
Exchange Chromatography ◽  
Bison Bison ◽  
Total Amino Acid ◽  
Affinity Column ◽  
Acid Extraction ◽  
Sodium Phosphate Buffer

1. Bison ribonuclease was isolated from pancreas glands of Bison bison by acid extraction, (NH4)2SO4 fractionation, affinity chromatography on Sepharose–5′-(4-aminophenylphosphoryl)uridine 2′,3′-phosphate and ion-exchange chromatography on Bio-Rex-70. 2. The selectivity of the affinity column towards bison ribonuclease in heterogeneous protein solutions was greatly improved by employing piperazine buffers at pH5.3, which decreased non-specific interactions of other proteins. Rapid desorption from the affinity column was obtained with sodium phosphate buffer (pH3). 3. Bison ribonuclease has a total amino acid content very similar to ox ribonuclease. Inactivation of bison ribonuclease with iodoacetic acid leads to the formation of 0.62 residues of π-carboxymethylhistidine and 0.36 residues of τ-carboxymethylhistidine. The amino acid composition of peptides isolated from diagonal peptide ‘maps’ and also of peptides isolated after pH1.6 and 2.4 two-dimensional high-voltage electrophoresis of a digest of bison ribonuclease labelled with pyridoxal 5-phosphate indicates that there is complete homology between ox and bison ribonucleases. 4. The Schiff-base attachment site of pyridoxal 5-phosphate was identified as lysine-41 by NaBH4 reduction followed by peptide isolation.

scholarly journals THE ACID-BASE PROCESSES' CHANGES IN THE BODY OF WHITE RATS UNDER THE INFLUENCE OF NITROGEN-CONTAINING SURFACE-ACTIVE MATERIAL

2020 ◽  
Vol 16 (3) ◽  
pp. 14-19
Author(s):  
V.V. Babienko ◽  
I.V. Sakharova ◽  
L.I. Danilchenko
Keyword(s):  
Peroxidase Activity ◽  
Lethal Dose ◽  
Active Material ◽  
Toxic Substance ◽  
The Body ◽  
Redox Processes ◽  
Biogenic Monoamines ◽  
Sh Groups ◽  
White Rats ◽  
Activity Of Enzymes

Relevance. Everyday contact of the population with surfactants (SAS) in drinking water poses the problem of timely and prompt substantiation of pre-nosological highly sensitive indicators of early manifestations of biological activity of detergents and operational control over the health of the population and the environment. But today the mechanisms of biotransformation, toxicodynamics, toxicokinetics, and metabolic processes that underlie the formation of structural and metabolic disorders when exposed to a surfactant, taking into account possible long-term effects, have not yet been fully elucidated. Objective: to investigate the effect of nitrogen-containing surfactants on redox processes in the body of experimental animals. Materials and methods. The experiments were carried out on 620, and acute experiments on 128 white rats (weight 180-220 g). We used four ionic nitrogen-containing surfactants with specified technical and physicochemical characteristics: FOM 9, FOM 9-4, FOM 9-12, and FOM 9-20. Doses were chosen so as to determine the lethal effect in the lethal dose (LD) range from 0 to 100. The LD50 was calculated. The substances were introduced into the stomach in pure form using a metal probe. The animals were observed for up to 15 days. The time of death of the animals and the total amount of the introduced substance was recorded. The animals were subjected to postmortem examination. Redox processes were qualitatively assessed by the activity of enzymes: cholinesterase, cerulose plasmin, lactate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, peroxidase, catalase, cytochrome oxidase, by the content of SH-groups in the blood, by the concentration of biogenic monoamines. Results. Nitrogen-containing surfactants caused a change in peroxidase activity both upwards and downwards. In all cases, 1/1000 LD50 was inactive. On the 15th day of the experiment, neonol FOM 9-12 reduced the activity of the enzyme, and other substances did not affect it. By the end of the subacute experiment, neonol FOM 9-4 and neonol FOM 9-12 were reduced, and neonol FOM 9-20 increased peroxidase activity. A similar effect was on the activity of catalase: in all groups, except 1/1000 LD50, on day 30 there was a decrease in its activity. Cholinesterase activity increased. For the content of SH-groups in the blood on the 15th day, there was a tendency to decrease, which turned into significant differences on the 30th day in 1/10 LD50. The effect of 1/100 and 1/1000 LD50 did not violate the content of SH-blood groups. A similar effect was on the content of glutathione in the blood. In a subacute experiment, in groups, 1/10 and 1/100 LD50, the content of norepinephrine, tryptophan, serotonin in the liver increased and DOPA and dopamine decreased. The dynamics of adrenaline did not change. The content of dopamine and norepinephrine increased to a lesser extent in the brain; DOPA and adrenaline did not differ from the control; tryptophan increased only under the influence of FOM-9. 1/1000 LD50 did not affect the dynamics of the content of biogenic monoamines. The tested drugs have a similar effect on the body. Conclusions. A more toxic substance in a subacute experiment is FOM-9. The severity of violations in the dynamics of monitoring the activity of enzymes has a close dose-dependence. The effective dose is set at 1/10, the threshold – 1/100, and the inactive – 1/1000 LD50. Common features of the biological action of nitrogen-containing surfactants are the violation of redox processes, bioenergy, oxidative phosphorylation, which under appropriate conditions lead to the pathology of vital organs, functions, and systems of the body.

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