scholarly journals Preparation and purification of polymerized actin from sea urchin egg extracts.

1975 ◽  
Vol 66 (2) ◽  
pp. 305-315 ◽  
Author(s):  
R E Kane
Keyword(s):  
Sea Urchin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mammalian Brain ◽  
Simple Method ◽  
Cytoplasmic Proteins ◽  
Sea Urchin Egg ◽  
Egg Extracts ◽  
Low Levels ◽  
Birefringent Fibrils

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

scholarly journals Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts.

1976 ◽  
Vol 71 (3) ◽  
pp. 704-714 ◽  
Author(s):  
R E Kane
Keyword(s):  
Sea Urchin ◽  
Low Temperatures ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Complex Structure ◽  
The Other ◽  
Cytoplasmic Proteins ◽  
Sea Urchin Egg ◽  
Low Concentrations ◽  
Egg Extracts

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.

Influence of vitamins and osmolites on growth and bacteriocin production byLactobacillus salivariusCRL 1328 in a chemically defined medium

2009 ◽  
Vol 55 (3) ◽  
pp. 304-310 ◽  
Author(s):  
Esteban Vera Pingitore ◽  
Elvira María Hebert ◽  
Fernando Sesma ◽  
María Elena Nader-Macías
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Defined Medium ◽  
Bacteriocin Production ◽  
Biologically Active ◽  
Chemically Defined Medium ◽  
Simple Method ◽  
Protein Gel ◽  
Rate Maximum ◽  
Human Vagina

The aim of this study was to analyze the influence of vitamins, glycerol, and salts on the growth and bacteriocin production by Lactobacillus salivarius CRL 1328, a human vagina isolate, by using a chemically defined medium to determine the optimal conditions for salivaricin production. The single omission of d-biotin, thiamine, p-aminobenzoic acid, folic acid, or cyanocobalamin did not affect the bacterial growth, whereas the removal of nicotinic acid, riboflavin, and pyridoxal produced a decrease of about 30% in the growth rate. Maximum salivaricin activity was observed after the addition of 5 or 10 g/L of NaCl. On the basis of the nutritional requirements and the levels of salivaricin production, a new optimized and simplified defined medium (SDM–NaCl) for L. salivarius CRL 1328 bacteriocin production was formulated. The kinetics of salivaricin production in SDM–NaCl and in the complex media LAPTg revealed that bacteriocin production was growth linked. A combination of tricine – sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine–SDS–PAGE), Lumitein protein gel staining, and a bioassay for antibacterial activity indicated that the molecular mass of salivaricin CRL 1328 is about 4.5 kDa. The partially purified bacteriocin, obtained from SDM–NaCl after concentration, allowed for the design of a relatively simple method for the recovery of a biologically active protein.

scholarly journals DIRECT ISOLATION OF THE HYALINE LAYER PROTEIN RELEASED FROM THE CORTICAL GRANULES OF THE SEA URCHIN EGG AT FERTILIZATION

1970 ◽  
Vol 45 (3) ◽  
pp. 615-622 ◽  
Author(s):  
R. E. Kane
Keyword(s):  
Protein Content ◽  
Sea Urchin ◽  
Insoluble Fraction ◽  
Glycerol Solution ◽  
Cortical Granules ◽  
Simple Method ◽  
Quantitative Measurements ◽  
Hawaiian Species ◽  
Sea Urchin Egg

Treatment of the eggs of the sea urchin with a 1 M solution of glycerol at fertilization allows the recovery from this solution of the protein released from the cortical granules, including that which would normally give rise to the hyaline layer. The calcium-gelable protein previously extracted from whole eggs and from isolated cortical material was found to be present in the glycerol solution, confirming its localization in the cortical granules and its role in the hyaline layer. Quantitative measurements on the eggs of two Hawaiian species, Colobocentrotus atratus and Pseudoboletia indiana, which have the widest variation in the gel protein content, demonstrated that a proportionate amount of this material was released at fertilization in these species, which correlates with the thickness of the hyaline layer in the two cases. In addition, the calcium-insoluble fraction of Sakai can be extracted from these eggs after removal of the hyaline protein by glycerol, showing that this is a different material. A simple method for the separation of the hyaline protein from the calcium-insoluble fraction in solution is provided.

scholarly journals Changes in α- and ß-amylase during Storage of Sweetpotato Lines with Varying Starch Hydrolysis Potential

1993 ◽  
Vol 118 (2) ◽  
pp. 236-242 ◽  
Author(s):  
Teresa A. Morrison ◽  
Russell Pressey ◽  
Stanley J. Kays
Keyword(s):  
Reducing Sugars ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Storage Period ◽  
Total Sugar ◽  
Starch Hydrolysis ◽  
Storage Root ◽  
High Starch ◽  
Low Levels

Staple-type lines of sweetpotato [Ipomoea batatus (L.) Lam.] do not sweeten significantly upon cooking as compared to the traditional-type lines. Four lines exhibiting distinct differences in sweetness after cooking were evaluated for changes in α- and ß-amylase activity and reducing sugars (by HPLC) at harvest, after curing, and at intervals during 180 days of storage. The traditional cultivar `Jewel' and staple-type line `Sumor' displayed high a- and ß-amylase activities, which rose from low levels at harvest to peak levels ≈ 90 days into the storage period. Staple-type lines `99' and `86' displayed significantly lower a- and ß-amylase activities. By using polyclonal sweetpotato ß-amylase antibody and western blot following native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was confirmed that a lower level of ß-amylase synthesis existed in `99' and `86'. Quantitatively, `Jewel', `Sumor', and an additional staple-type line, `HiDry', had 361,374, and 365 μg ß-amylase protein per gram of fresh storage root tissue, respectively, while `99' and `86' possessed <60 and 12 μg·g-1, respectively. In raw roots, individual (glucose, fructose, and sucrose) and total sugar concentrations were significantly higher in `Jewel' than in `Sumor', `99', or `86'. Only trace amounts of maltose were found in raw roots of any line. Sucrose, glucose, and fructose concentrations decreased with baking in all lines except `86', in which they increased. There was substantial maltose produced by baking `Jewel' and `Sumor', but only trace amounts found in baked `99' and `86'. Sweetpotato germplasm can be separated into four general classes based on initial sugar concentration and changes during cooking: 1) low sugars/low starch hydrolysis, 2) low sugars/high starch hydrolysis, 3) high sugars/low starch hydrolysis, and 4) high sugars/high starch hydrolysis. At least two mechanisms may confer the lack of starch hydrolysis and subsequent sweetening in staple-type sweetpotato: 1) inhibition of ß-amylase synthesis, and 2) a nonenzyme mediated mechanism.

scholarly journals Specificity of Rabbit Antisera against Lipopolysaccharide of Acinetobacter

1998 ◽  
Vol 36 (5) ◽  
pp. 1245-1250 ◽  
Author(s):  
Ralph Pantophlet ◽  
Lore Brade ◽  
Lenie Dijkshoorn ◽  
Helmut Brade
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Accurate Identification ◽  
Simple Method ◽  
Whole Cell ◽  
Cell Lysates ◽  
O Antigen ◽  
Homologous Antigen ◽  
Routine Identification

Acinetobacter has been reported to be involved in hospital-acquired infections with increasing frequency. However, clinical laboratories still lack simple methods that allow the accurate identification of Acinetobacter strains at the species level. For this study, proteinase K-digested whole-cell lysates from 44 clinical and environmental isolates were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with hyperimmune rabbit sera to examine the possibility of developing a serotyping scheme based on the O antigen of Acinetobacterlipopolysaccharide (LPS). The antisera, obtained by immunization of rabbits with 13 of the heat-killed isolates investigated, were characterized by Western blotting and enzyme immunoassay by using proteinase K-digested whole-cell lysates and phenol-water-extracted LPS as antigens. In both assays, the antisera were shown to be highly specific for the homologous antigen. In addition, assignment ofAcinetobacter LPS to the smooth or the rough phenotype was shown not to be reliable when it was based only on the results obtained with silver-stained gels. O-antigen reactivity, determined by Western blot analysis, was observed with 11 of the 31 isolates, most of which belonged to the species Acinetobacter baumannii (DNA group 2) and the unnamed DNA group 3. Interestingly, some O antigens were found in a DNA group different from that of the strain used for immunization. The results indicate that O serotyping ofAcinetobacter strains is feasible and thus may provide a simple method for the routine identification of these opportunistic pathogens.

scholarly journals Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

2003 ◽  
Vol 12 (3) ◽  
pp. 179-183 ◽  
Author(s):  
E. Jablonska ◽  
M. Marcinczyk
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytoplasmic Proteins ◽  
Wide Range ◽  
Interleukin 15 ◽  
Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

scholarly journals Interconversion of structural and contractile actin gels by insertion of myosin during assembly.

1983 ◽  
Vol 97 (6) ◽  
pp. 1745-1752 ◽  
Author(s):  
R E Kane
Keyword(s):  
Sea Urchin ◽  
Actin Polymerization ◽  
Small Volume ◽  
Final Concentration ◽  
Cytoplasmic Structure ◽  
Cytoplasmic Proteins ◽  
Sea Urchin Egg ◽  
Low Salt ◽  
Sequential Combination ◽  
Characteristic Banding

Extracts of the soluble cytoplasmic proteins of the sea urchin egg form gels of different composition and properties depending on the temperature used to induce actin polymerization. At temperatures that inactivate myosin, a gel composed of actin, fascin, and a 220,000-mol-wt protein is formed. Fascin binds actin into highly organized units with a characteristic banding pattern, and these actin-fascin units are the structural core of the sea urchin microvilli formed after fertilization and of the urchin coelomocyte filopods. Under milder conditions a more complex myosin-containing gel is formed, which contracts to a small fraction of its original volume within an hour after formation. What has been called "structural" gel can be assembled by combining actin, fascin, and the 220,000-mol-wt protein in 50-100 mM KCl; the aim of the experiments reported here was to determine whether myosin could be included during assembly, thereby interconverting structural and contractile gel. This approach is limited by the aggregation of sea urchin myosin at the low salt concentrations utilized in gel assembly. A method has been devised for the sequential combination of these components under controlled KCl and ATP concentrations that allows the formation of a gel containing dispersed myosin at a final concentration of 60-100 mM KCl. These gels are stable at low (approximately 10 micron) ATP concentrations, but contract to a small volume in the presence of higher (approximately 100 micron) ATP. Contraction can be controlled by forming a stable gel at low ATP and then overlaying it with a solution containing sufficient ATP to induce contraction. This system may provide a useful model for the study of the interrelations between cytoplasmic structure and motility.

scholarly journals Simple Method for Repurification of Endotoxins for Biological Use

2007 ◽  
Vol 73 (6) ◽  
pp. 1803-1808 ◽  
Author(s):  
Alina Tirsoaga ◽  
Alexey Novikov ◽  
Minou Adib-Conquy ◽  
Catherine Werts ◽  
Catherine Fitting ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
Ionization Mass ◽  
Toll Like Receptor ◽  
Simple Method ◽  
Spectrometry Analysis ◽  
Toll Like Receptor 2 ◽  
Oligomerization Domain

ABSTRACT A method for obtaining highly purified endotoxin (lipopolysaccharide [LPS]) in a few hours by repurification of commercial or laboratory preparations was devised. It avoids the use of phenol, which is not suitable for phenol-soluble lipopolysaccharides nor for some industrial purposes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry analysis confirmed the integrity of the purified LPSs. The purified products did not activate Toll-like receptor 2 (TLR2), nuclear oligomerization domain 1 (NOD1), or NOD2 but did activate TLR4. Applied to different lipopolysaccharides, the method also improved their mass spectra, thus facilitating their structural analysis.

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