表面活性剂对林可霉素发酵生产的影响

在林可霉素发酵过程中,当向培养基中加入表面活性剂十二烷基磺酸钠(SDS)、吐温80(Tween 80)和曲拉通(Triton X-100)时,林可霉素的产量受到较大影响。本研究应用响应面设计法(Response surface design)对表面活性剂的配比进行了优化,得到的优化配比为:十二烷基磺酸钠为31.13 mg/100 mL,吐温80为51.97 mg/100 mL,曲拉通为16.9 mg/100 mL。将该优化配比应用于林可霉素发酵,产量提高了36.67%。     

Evaluation of Small Intestine Grafts Decellularization Methods for Corneal Tissue Engineering

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.     

Virioimmunoassay for studying the interaction of diphtheria toxin with cell membranes]

A model for studying the interaction of diphtheria toxin with cell membranes includes immobilization of purified cell membranes on Sephadex G-25, adsorbtion of toxin on the membranes in the presence of protective colloid, and subsequent detection of adsorbed toxin by means of virioimmunoassay. Diphtheria toxin adsorbed rapidly on membranes of both sensitive (HeLa, macrophages) and resistent to tis action cells, but not on stromaof human erythrocytes. The rate of interaction depends on the concentration of toxin and the temperature of incubation. Adsorbed toxin may be eluted by acidic buffer, 8 M area and 4 M guanidin. HCl, but not by triton X-100, tween 20 and 80, sodium dodecylaulfate and basic buffer.     

Possibilities for Ellman reaction application in the presence of detergents

Triton X-100, triton X-305, twin-80 and sodium deoxycholate in definite concentrations lower the colour intensity of the solution which contains the product of the Ellman reaction. In the case of triton X-100 the colour disappearance is due to resynthesis of the Ellman reagent from 5-thio-2-nitrobenzoate with the presence of detergent in the concentration which is higher than the critical concentration of micelle-formation.     

Effect of the pH of the lysing solution on the sedimentation properties of the nucleoid of intact and irradiated Ehrlich ascites tumor cells

A study was made of the sedimentation properties of Ehrlich ascites tumor cell nucleotide obtained by cell lysis in 1.95 M NaCl, 0.1 M EDTA, 0.02 M tris, and 0.5% triton X-100, at pH 5.0 and 8.0. It was shown that the nucleotide obtained at pH 5.0 has a more compact structure than that obtained at pH 8.0. Irradiation of cells leads to relaxation of the nucleotide decreasing the rate of its sedimentation in a neutral sucrose gradient. The influence of irradiation on the sedimentation properties is more pronounced with pH 5.0 than 8.0.     

Thymocyte membrane changes and modifications of interphase death

A reduction in the rate of interphase death of X-irradiated thymocytes is observed when they are previously treated with triton X-100 or trypsin at low concentrations. The data obtained demonstrate the importance of the structural integrity of the cell surface in the initiation of processes leading to interphase death of lymphoid cells.     

Delayed chloroplast luminescence. Activation and suppression

Effect of diethyl ether, detergent triton X-100, glycerine, sucrose and preliminary heating on delayed luminescence (DL) of chloroplasts has been studied. Ether and triton X-100 in concentrations activating electron transport inhibit DL acting similarly to photophosphorylation uncouplers. Preliminary heating to 42-42C, glycerine and sucrose activate both the electron transport and DL of chloroplasts. Activation of electron transport under these agents is suggested to result not from photophosphorylation uncoupling, but from the changes in the conformation of chloroplast memebranes.     

Effect of triton X-100 on alkaline lipase production by Pseudomonas pseudoalcaligenes F-111

Summary An extracellular alkaline lipase was evaluated from Pseudomonas pseudoalcaligenes F-111, which was isolated from soil using a rhodamine B agar plate with Na₂CO₃. For enzyme production, olive oil, soymeal, triton X-100 and sodium ion were found to be essential. The addition of triton X-100 increased the alkaline lipase by 50- fold. The effect of additives as well as the development of suitable culture conditions for lipase production is discussed.     

Lactate dehydrogenase isoenzymatic spectrum in preimplantation murine embryos

The spectrum of LDH isozymes was studied using different concentration of X-100 triton for enzyme extraction from the eggs and embryos, different systems of electrophoresis and different methods of staining electrophoregrams. Similar results were obtained in all cases. The mouse eggs and embryos were shown to contain only LDH-1; the treatment of oviducal fluid with X-100 triton strengthened the activity of LDH-4 and LDH-5. The detection of the activity of LDH-5 and hybrid isozymes appears to be related to the presence of oviducal fluid in the sample under study.     

Role of hydrophobic elements of the plasma membrane in the reaction of frog urinary bladder cells to antidiuretic hormones

The addition of 10 mg/ml triton X-100 to Ringer solution within the bladder near the serous membrane increases water permeability of the frog bladder wall. At a concentration of 0.1 mg/ml it gives no visible effect on the water and sodium permeability, but inhibits the effects of ADH, forskolin and cAMP. Changes in plasma membrane hydrophobic properties due to the addition of triton X-100 are reversible and ADH effect can be restored after the detergent is removed from the solution. The results obtained show that hydrophobic groups of the plasma membrane contribute to increased water permeability in response to ADH.     

Phosphorylation of membrane proteins from Plasmodium berghei-infected red cells.

Membrane from Plasmodium berghei-infected mouse red cells has a different pattern of phosphorylation by (γ-³²P)ATP from normal membrane. A phosphorylated membrane protein of apparent molecular weight 42,000, absent in membrane from normal cells, can be detected in membrane from infected cells. The new phosphorylated protein can be extracted by 0.1 mM EDTA but not by triton X-100, indicating that it may be red cell actin.     

Relation of the poly(ADP-ribosylation) of proteins to the formation and repair of DNA single-strand breaks in gamma-irradiated permeable Zajdela hepatoma cells

A study was made of the effect of poly(ADP-ribosylation) of proteins on the formation and repair of single-strand DNA breaks in gamma-irradiated (50 Gy) permeable Zajdela ascites hepatoma cells permeabilized by the treatment with 0.05% triton X-100. Incubation of gamma-irradiated permeable cells in conditions promoting DNA synthesis and providing ADP-ribosylation (in the presence of 1 mM NAD) did not cause any substantial changes in the formation of single-strand DNA breaks and did not influence their repair.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle.

The subcellular location of the phase-variable cytadherence-accessory protein HMW3 in Mycoplasma pneumoniae has been examined by biochemical and immunoelectron microscopic techniques. Analysis by Western blot (immunoblot) with HMW3-specific antiserum established the presence of this protein within the M. pneumoniae Triton X-100-insoluble fraction or triton shell. Immunogold labeling of Triton-extracted mycoplasmas with affinity-purified antibodies localized HMW3 to the terminal knob on the rodlike extensions of the triton shell, a location that would correspond to the adherence organelle in whole mycoplasmas. Treatment of triton shells with KI resulted in the selective removal of the adherence-accessory proteins HMW1 to HMW4. Analysis of these triton shells by transmission electron microscopy revealed dramatic ultrastructural changes in the filamentous network and core structure. Immunogold labeling of KI-extracted shells reflected the removal of HMW3 from the disrupted tip structure. An examination of ultrathin sections of wild-type cells by transmission electron microscopy following labeling with HMW3-specific antibodies provided further evidence for the nonrandom distribution of HMW3 and its localization to the terminal portion of filamentous cell extensions. Most colloidal gold molecules were associated with the cell interior, but limited peripheral labeling of the terminal region was also observed. Postfixation antibody labeling of whole cells suggested limited exposure of HMW3 on the mycoplasma surface at the tip structure. However, prefixation antibody labeling failed to indicate surface exposure, raising some uncertainty regarding the relationship of HMW3 with the mycoplasma membrane.     

Association of prolyl hydroxylase activity with membranes

Addition of ionic and nonionic detergents to whole homogenates of liver, kidney and lung prepared by a mild homogenization technique resulted in a two- to three-fold increase of prolyl hydroxylase activity. After subcellular fractionation of whole homogenates of liver, particulate and supernatant fractions were incubated in the presence and absence of triton X-100 and assayed for prolyl hydroxylase activity. All particulate fractions tested were able to release significant amounts of prolyl hydroxylase activity in the presence of triton. The release of enzyme activity by triton was observed with the 1000 × g and 17,000 × g supernatants but not with the 105,000 × g supernatant; thus indicating that detergent does not activate soluble enzyme nor make the substrate more accessible to hydroxylation by the enzyme during incubation. Rigorous homogenization of the 17,000 × g particulate fraction with the Polytron ST system resulted in a substantial loss of the amount of prolyl hydroxylase activity released by treatment with triton. These data suggest that a significant amount of prolyl hydroxylase activity is associated with membranes under physiological conditions.     

Adenylate and guanylate cyclases of cecropia silkmoth fat body.

Adenylate and guanylate cyclases were assayed in silkmoth fat body homogenates by measuring the conversion of [α-³²P]nucleoside triphosphates to cyclic [³²P]nucleotides. Adenylate cyclase was dependent on dithiothreitol, required either Mg²⁺ or Mn²⁺ for activity, was activated by NaF, and inhibited by triton X-100. Guanylate cyclase was not dependent on dithiothreitol, was strictly dependent upon Mn²⁺, unaffected by NaF, and activated by triton X-100. Both cyclases had pH optima near 8.0 and were located chiefly in the particulate fraction of homogenates. Activities of both cyclases were maintained or elevated during the larval-pupal transformation and, in contrast to cyclic nucleotide phosphodiesterases, showed little decline in the early diapausing pupa.     

A study of membrane glycoproteins from the rat brain using D-galactose-specific lectin

Immobilized D-galactose-specific lectin from Zea mais was used to purify rat brain membrane glycoproteins. The membrane glycoproteins preliminarily washed from soluble proteins were solubilized consecutively by 2% triton X-100 and 1% SDS. PAG-electrophoresis with SDS and 2-mercaptoethanol revealed 10 polypeptide bands (Mm 109, 62, 59, 54, 51, 42, 16, 14, 12.5 and 12 kDa) in the membrane fraction of glycoproteins solubilized with triton X-100. Additional solubilization with SDS revealed 3 bands (Mm 109, 62, and 54 kDa). Only 3 polypeptide bands (Mm 62, 59, 42 kDa) were identified when analogous procedure was used for purification of the rat liver glycoproteins. Horse radish peroxidase labelled D-galactose-specific lectin from Zea mais was found to bind to neuron bodies and neurites in the cerebellum. It is suggested that the identified brain-specific membrane glycoproteins may take part in the cell adhesion between neurons.     

Evidence Relating Cessation of Respiration, Cell Envelope Changes, and Death in Ultraviolet-Irradiated Escherichia coli B/r Cells

Ionic and nonionic detergents have little effect on respiring bacteria, but in cultures poisoned with KCN rapid solubilization of the cell membrane, as indicated by turbidity losses, takes place. Ultraviolet radiations cause Escherichia coli cells grown in minimal medium with glycerol as a carbon source to cease respiring and growing about 1 h after irradiation. We tested the effect of the nonionic detergent Triton X-100 on growth and cell membrane dissolution (both measured by turbidity changes), respiration, and viability of unirradiated and irradiated E. coli B/r cells. When the detergent was added to cells immediately after irradiation, a decrease in turbidity occurred only when respiration was about to cease; when it was added after cessation of respiration, the turbidity loss was immediate. In both cases the turbidity loss was about 60%, and disintegration of the cell walls did not take place. 5-Fluorouracil (FU) and thermal (42 C) treatments cause respiration of irradiated cells to be maintained and also cause viability increases. Irradiated cells treated with FU and detergent show no turbidity loss just prior to the time respiration normally ceases, but a loss does occur in irradiated cells incubated with detergent at 42 C. We conclude that FU maintains respiration for all of the cells, but that thermal treatment maintains respiration for only part of the cells. In all cases the detergent had only a negligible effect on the respiration and viability of unirradiated and irradiated cells. We conclude that Triton X-100 causes solubilization of cell membranes of only nonrespiring cells that are not destined to survive.     

Inhibition or activation of Pseudomonas species lipase by 1,2-ethylene-di-N-alkylcarbamates in detergents

1,2-Ethylene-di-N-n-propylcarbamate (1) is characterized as an essential activator of Pseudomonas species lipase while 1,2-ethylene-di-N-n-butyl-, t-butyl-, n-heptyl-, and n-octyl-carbamates (2-5) are characterized as the pseudo substrate inhibitors of the enzyme in the presence of the detergent taurocholate or triton X-100. The inhibition and activation reactions are more sensitive in taurocholate than in triton X-100. From CD studies, the enzyme changes conformations in the presence of the detergent and further alters conformations by addition of the carbamate activator or inhibitor into the enzyme-detergent adduct. Therefore, this study suggests that the conformational change of lipase during interfacial activation is a continuous process to expose the active site of the enzyme to substrate. From 600 MHz (1)H NMR studies, the conformations of the alpha- and beta-methylene moieties of the activator 1,2-ethylene-di-N-n-propylcarbamate in the presence of substrate change after adding taurocholate into the mixture, and the conformations of the beta-methylene moieties of the inhibitor 1,2-ethylene-di-N-n-butylcarbamate in the presence of substrate alter after adding taurocholate into the mixture.     

Concentration of black spots and the effect of detergents on black lipid membranes

Effect of water-soluble detergents, such as triton X-100, saponin and trimethyl octadecylammonium bromide on the concentration of black spots, tension and stability of black lipid membranes was studied. Changes in mechanic stability of the lipid bilayer are discovered, which are in a good correlation with the litic effect of true detergents on cell membranes.