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1.
Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color. 相似文献
2.
The diaminobenzoic acid dihydrochloride fluorescence method has been examined to determine optimal conditions for the assay of DNA in perchloric acid extracts and in pretreated Chlamydomonas cells on glass-fiber filters. Maximal fluorescence yield was obtained by allowing: (a) the perchloric acid extract to react with 4–6% diaminobenzoic acid dihydrochloride for 20–40 min at 60–80°C at about pH 3.0; (b) the pretreated cells on glass-fiber filters to react with a 20% solution of diaminobenzoic acid dihydrochloride for 40 min at 60°C. 相似文献
3.
JONATHAN I. RAVDIN CHERYL F. MURPHY PAUL H. SCHLESINGER 《The Journal of eukaryotic microbiology》1988,35(1):159-163
ABSTRACT. We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed 125iodine (125I) labeled tyrosine conjugated dextran; 125I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of 125I-dextran released after 120 min at 37°C, 31°C, 25°C, and 4°C, respectively (P < 0.01 for each). Exocytosis at 37°C was inhibited by cytochalasin D (10 μg/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 μM) (P < 0.01 for each at ≥ 60 min). Calcium ionophore A23187 (1 μM) enhanced exocytosis at 5 and 15 min (P < 0.01). Elevation of vesicle pH with NH4Cl (10 mM) had no effect on release of 125I-dextran; phorbol myristate acetate (10?6 M) increased exocytosis by 46% at 30 min (P < 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased 125I-dextran release into the cell supernatant after 30 and 60 min at 37°C (by 40% and 42%, respectively, P < 0.01); release of 125I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters. 相似文献
4.
Gen-ichi Danno 《Bioscience, biotechnology, and biochemistry》2013,77(2):264-273
An alkaline proteinase of Aspergillus sulphureus (Fresenius) Thorn et Church has been purified in good yields from wheat bran culture by fractionation with ammonium sulfate, treatment with acrynol, and DEAE-Sephadex A-50 column chromatography. The crystalline preparation was homogeneous on sedimentation analysis and polyacrylamide gel zone electrophoresis. The molecular weight was calculated to be 23,000 by gel filtration. The amino acid composition of the enzyme was determined. The enzyme did not precipitate with acrynol. Optimum pH for the hydrolysis of casein was 7 to 10 at 35°G for 15 min. Optimum temperature was 50°C at pH 7 for 10 min. The enzyme was highly stable at the range of pH 6 to 11 at 5°C, whereas relatively stable at pH 6 to 7 at 35°C. Metalic salts tested did not affect activity. Chelating agents, sulfhydryl reagents, TPCK, and oxidizing or reducing reagents tested, except iodine, had no effect on the activity. Diisopro-pylfluorophosphate and N-bromosuccinimide almost completely inactivated the proteinase. 相似文献
5.
L J Menz 《Cryobiology》1975,12(4):405-416
Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work. 相似文献
6.
Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH4)2SO4 precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking
agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were
found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C.
100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing
412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting
in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5%
inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity. 相似文献
7.
《Biotechnic & histochemistry》2013,88(6):331-334
Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl2, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na2S2O3 (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation. 相似文献
8.
《Prostaglandins and medicine》1980,4(4):263-273
Prostacyclin (PGI2) stimulates platelet adenylate cyclase, elevates intracellular levels of cyclic adenosine monophosphate and blocks the response to aggregating agents. It is rapidly hydrolyzed (T 1–5 min) to 6-keto prostaglandin F1α at acid or neutral pH.As a result, platelets incubated with PGI2 will recover spontaneously and respond to aggregating agents within 15–60 min, depending on the initial PGI2 concentration. In the present study we have evaluated the influence of temperature and pH on the stability of PGI2 and its effects on platelet function. PGI2 in Tris buffer was stabilized at several pH levels and stored at 37°C, 23°C, and 4°C. Inhibitory influence on platelet function was lost rapidly at pH 7.2–7.4, lasted several hours at pH 7.8 and was retained indefinitely at pH 8 or above. PGI2 (2.8 hM) completely inhibited the response to arachidonic acid for 15 min. at pH 7.4, for at least 1 hour at pH 7.8 and showed no reversal of inhibition after 48 hours at pH 8. However, PGI2 inhibited samples at pH 8 completely recovered their sensitivity to arachidonic acid when the pH was reduced to 7.4. These findings indicate that the biological activity of PGI2, though labile at neutral pH, is stable at pH 8 and can inhibit cAMP mediated platelet functions for at least 48 hours. Because of its pH dependence, PGI2 may be a useful agent for prolonging the sensitivity of stored platelets. 相似文献
9.
One clone (ACPGA001) exhibiting penicillin G acylase (PGA) activity was screened from a metagenomic library by using a medium containing penicillin G. A novel PGA gene from the inserted fragment of ACPGA001 was obtained by sequencing. The amino acid sequence of ACPGA001 PGA exhibited <33 % similarity to PGAs retrieved from GenBank. This gene was expressed in Escherichia coli M15 and the recombinant protein was purified and characterized. The ACPGA001 PGA exhibited a maximum activity at 60 °C and showed high activity at pH 4–10 with an optimum pH of 8.0. This enzyme was stable at 40 °C for 70 min with a half-life of 60 min at 55 °C. These beneficial characteristics of ACPGA001 PGA provide some advantages for the potential application of ACPGA001 PGA in industry. 相似文献
10.
Ancelmo Rabelo de Souza Ana Luísa Silva Sampaio Moreira da Costa Demonte Karina de Araujo Costa Mariana Alcantara Cardoso Faria Ricardo Durães-Carvalho Marcelo Lancellotti Carlos Francisco Sampaio Bonafe 《Applied microbiology and biotechnology》2013,97(16):7417-7425
Mycobacterium abscessus is an important hospital-acquired pathogen involved in infections associated with medical, surgical, and biopharmaceutical materials. In this work, we investigated the pressure-induced inactivation of two strains [2544 and American Type Culture Collection (ATCC) 19977] of M. abscessus in combination with different temperatures and pH conditions. For strain 2544, exposure to 250 MPa for 90 min did not significantly inactivate the bacteria at 20 °C, whereas at ?15 °C, there was complete inactivation. Exposure to 250 MPa at ≥60 °C caused rapid inactivation, with no viable bacteria after 45 min. With 45 min of exposure, there were no viable bacteria at any temperature when a higher pressure (350 MPa) was used. Extremes of pH (4 or 9) also markedly enhanced the pressure-induced inactivation of bacteria at 250 MPa, with complete inactivation after 45 min. In comparison, exposure of this strain to the disinfecting agent glutaraldehyde (0.5 %) resulted in total inactivation within 5 min. Strain 19977 was more sensitive to high pressure but less sensitive to glutaraldehyde than strain 2544. These results indicate that high hydrostatic pressure in combination with other physical parameters may be useful in reducing the mycobacterial contamination of medical materials and pharmaceuticals that are sensitive to autoclaving. 相似文献
11.
《Process Biochemistry》2004,39(5):535-539
Bovine pancreatic α-chymotrypsin was covalently modified with the O-carboxymethyl poly-β-cyclodextrin (M=1.3×104, 40% COOH groups) using a water-soluble carbodiimide as a coupling agent. The conjugate prepared by this procedure retained high proteolytic and esterolytic activity and contained about 74% carbohydrate by weight of transformed protein. The optimum temperature for α-chymotrypsin was increased by 5 °C after this transformation. The thermostability of the polymer–enzyme adduct was also increased by 5 °C. The conjugate prepared was also more resistant to thermal inactivation at different temperatures, ranging from 45 to 55 °C. Additionally, the modified enzyme was 11-fold more stable at pH 9.0. The direct influence of supramolecular interactions between the hydrophobic amino acid residues located at the surface of the protease and the attached polycyclodextrin moieties on α-chymotrypsin stabilization was demonstrated. 相似文献
12.
Yoshio Tsujisaka Mieko Iwai Yoshio Tominaga 《Bioscience, biotechnology, and biochemistry》2013,77(6):1457-1464
Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.It was estimated with the crystalline enzyme that the sedimentation constant (s20, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min. 相似文献
13.
Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured
in liquid Murashige and Skoog (MS) medium containing 0.2 mg l−1 α-naphthalene acetic acid and 0.5 mg l−1 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m−2 s−1) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped
in a 0.5 M CaCl2 solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then,
these were dehydrated with a plant vitrification solution 2 (PVS2) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8,
for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs
were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for
three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots,
and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was
above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum. 相似文献
14.
An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6–10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate. 相似文献
15.
Spore suspensions of 15 strains in 15 species of Micromonospora prepared with ultrasonication-technique were tested for resistance to moist heat, acid, alkali, and organic solvents (5 alcohols, 4 ketones and ether). More than 50% spore-survival was found in most organisms heated at 60°C for 20min, but less than 0.5% survived at 80°C. The spore-viability did not change at pH 6 to 8, but decreased beyond this range, and remarkably at acidic pH. A maximum reduction in viability was found with most organic solvents at a concentration of around 80%, and the spores were more resistant to ketone than alcohols and dioxane. Several Streptomyces species were also studied, and their spores were less resistant to heat and organic solvents than those of Micromonospora. 相似文献
16.
《Biotechnic & histochemistry》2013,88(5-6):253-255
Here we describe a method for gross staining of gray matter in slices of formaldehyde-fixed human brain. After protection of white matter with 4% phenol at 60°C for 5 min followed by a cold water wash, the gray matter was stained for 10-15 min at 20-25°C with 1% aqueous copper(II) phthalocyanine tetrasulfonic acid tetrasodium salt (CPTS). The staining resisted all attempts to be washed from the gray matter. Stained slices can be stored indefinitely in slightly acidified water, or plastinated as permanent dry specimens. 相似文献
17.
STEVE J. UPTON WILLIAM L. CURRENT SUSAN M. BARNARD JOHN V. ERNST 《The Journal of eukaryotic microbiology》1984,31(2):293-297
In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO2 atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation. 相似文献
18.
The bone morphogenetic property of bone matrix is degraded at 25 ° to 37 °C within 24 hours after a bone is removed from the body. The degradation occurs in the intact undemineralized bone from the action of endogenous enzymes, presumably neutral proteinases at pH optima of 7 · 0 to 7 · 4. Degradation is: more rapid at physiologic than at acid pH; heat inactivated in the range between 40 ° and 60 °C; slow at 2 °C over a period of 7 days in EDTA at pH 7 · 4. Degradation is inhibited by iodoacetic acid at concentrations as low as 3 · 0 mmoles per liter either in phosphate buffer or EDTA. Degradative activity of endogenous enzymes, as measured by the yield of bone from implants of matrix, is comparable to those obtained from matrix treated with trypsin at 15 °C, pH 7 · 6 over a period of 12 hours. These enzymes include a neutral proteinase (BMP-ase) which degrades bone morphogenetic protein (BMP) without mobilizing bone collagen hydroxy-proline as rapidly and as selectively as a specific functional entity. Observations on carboxypeptidase A and thermolysin cleavage of phenylalanine groups and data on acetylation of tyrosyl groups reducing bone yield suggest aromatic amino acids may be necessary for the biologically active conformation of BMP. 相似文献
19.
Li Tan Yue-Qin Tang Hiroto Nishimura Shouta Takei Shigeru Morimura 《Preparative biochemistry & biotechnology》2013,43(7):682-695
Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid–alkali combination (1% H2SO4, 150°C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35°C for 36 hr after presaccharification at 50°C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover. 相似文献
20.
Cynthia Sequeiros Marisol Vallejo Emilio Rogelio Marguet Nelda Lila Olivera 《Archives of microbiology》2010,192(4):237-245
After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity
against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria,
but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the
antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source
of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed
that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between
pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that
the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments
with fish. 相似文献