Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.     

Hydrolysis and rearrangement reactions of riboflavin phosphates. An explicit kinetic study

Four isomeric monophosphates and five isomeric bisphosphates of riboflavin were isolated from commercial FMN or were prepared by acid-catalyzed isomerization. The reaction of riboflavin monophosphates in aqueous solution was studied in the pH range between 0 and 9 under various conditions. The predominant reaction at pH values below 2 is the acid-catalyzed migration of the phosphate group. A detailed kinetic study of this reaction was performed by high pressure liquid chromatography. Experimental data were fitted with computer-generated curves based on an algorithm for a network of first-order reactions. Rate constants and activation parameters were obtained for the temperature range of 50-80 degrees C at pH 1. At thermodynamic equilibrium, the reaction mixture contains about 66% 5'-phosphate, 11% 4'-phosphate, 8% 3'-phosphate, and 15% 2'-phosphate (pH 1.0, 50 degrees C). In the pH range between 3 and 7, the hydrolysis of FMN is the prevailing reaction with a rate maximum at about pH 4. The same experimental approach was used in a subsequent kinetic study on the isomerization of riboflavin bisphosphates. The formation of five out of six possible isomers was studied quantitatively and rate constants for each partial reaction were obtained.     

Influence of cooling rates and plunging temperatures in an interrupted slow-freezing procedure for semen of the African catfish, Clarias gariepinus

The objective of this study was to optimize interrupted slow-freezing protocols for African catfish semen. Semen diluted with methanol and extender was frozen in 1-ml vials in a programmable freezer. The temperatures of the freezer (T(chamber)) and of the semen (T(semen)) were measured simultaneously. We first tested two-step freezing protocols with different cooling rates (-2, -5, and -10 degrees C/min) and different temperatures at plunging into liquid N2. The difference between T(semen) and T(chamber) increased with faster cooling rates. In all programs, survival of spermatozoa, expressed as hatching rates, increased from near zero when T(semen) at plunging was higher than -30 degrees C to values equal to those of control when T(semen) at plunging was equal to or lower than -38 degrees C. The inclusion of an isothermal holding period before plunging into liquid N2 (three-step freezing protocols) resulted in an equilibration between T(semen) and T(chamber) and improved semen survival. Semen could be plunged at temperatures as high as -36 degrees C when cooled at -5 or -10 degrees C/min, without compromising postthaw semen survival. Cooling at -2 degrees C/min in combination with a 5-min holding period reduced postthaw survival. We conclude that with slow cooling rates of -2 to -5 degrees C/min, hatching rates can be maximized by plunging as soon as T(semen) reaches -38 degrees C. The isothermal holding period is beneficial when faster rates are used. A simple and efficient protocol for freezing African catfish semen can be obtained by cooling at a rate of -5 to -10 degrees C/min combined with a 5-min holding period in the freezer, at -40 degrees C.     

Artifact formation during transmethylation of lipid peroxides.

By-product formation during base-catalyzed transesterification of lipid peroxides was observed in model experiments. A partially oxidized linoleic acid methyl ester as well as purified hydroxylinoleates served as test compounds. By-products formed during a simulated transmethylation of purified hydroxylinoleates were separated from the main components by means of thin-layer chromatography. The loss attributable to these by-products amounted to 10%. Oxygenated fatty acid derivatives were completely destroyed by acid-catalyzed transmethylation. Catalytic hydrogenation prior to base-catalyzed transmethylation proved to be a simple means to minimize side-product formation. By using this technique the yield of hydroxystearates from a partially autoxidized linoleic acid methyl ester preparation was improved significantly.     

Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.     

Acid-catalyzed esterification of Zanthoxylum bungeanum seed oil with high free fatty acids for biodiesel production

A technique to produce biodiesel from crude Zanthoxylum bungeanum seed oil (ZSO) with high free fatty acids (FFA) was developed. The acid value of ZSO was reduced to 1.16mg KOH/g from 45.51mg KOH/g by only one-step acid-catalyzed esterification with methanol-to-oil molar ratio 24:1, H(2)SO(4) 2%, temperature 60 degrees C and reaction time 80min, which was selected as optimum for the acid-catalyzed esterification. During the acid-catalyzed esterification, FFA was converted into fatty acid methyl esters, which was confirmed by (1)H NMR spectrum. Compared with the other two-step pretreatment procedure, this one-step pretreatment can reduce the production cost of ZSO biodiesel. Alkaline-catalyzed transesterification converted the pretreated ZSO into ZSO biodiesel. The yield of ZSO biodiesel was above 98% determined by (1)H NMR spectrum. This study supports the use of crude ZSO as a viable and valuable raw feedstock for biodiesel production.     

Separation by high-performance liquid chromatography of penicillins with C4 to C10 aliphatic side chains

A suitable and rapid method for the simultaneous determination of different aliphatic penicillins is described. Butyryl-, pentanoyl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl- and decanoylpenicillin can be completely separated by high-performance liquid chromatography using an isocratic elution mode (50 mM H2KPO4, pH 5.0:methanol, 45:55 v/v). Under these conditions, retention times for C4 to C10 aliphatic side-chain penicillins were 2.5, 2.8, 4.1, 5.8, 8.9, 15.3, and 28.2 min. The benzylpenicillin retention time was 3.3 min.     

Quantitative analysis of valienamine in the microbial degradation of validamycin A after derivatization with p-nitrofluorobenzene by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 degrees C. With the mobile phases consisting of acetonitrile-water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 microl, the column temperature 40 degrees C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5-150.0 microg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from -0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.     

Cryopreservation of chum salmon blastomeres by the straw method

In order to preserve genetic resources of chum salmon, Oncorhynchus keta, optimum conditions for cryopreservation of isolated blastomeres were investigated. Survival rates under various conditions were compared: the nature and the concentration of cryoprotectants before and after freezing, the seeding temperature, and the developmental stages of donor embryos. Isolated blastomeres immersed for 30 min in Eagle's MEM containing both a cryoprotectant and 10% fetal bovine serum (FBS) at 10 degrees C were transferred into a straw and frozen at 1 degrees C/min to -30 degrees C by a programmable freezer before being plunged into liquid nitrogen. Ice seeding was carried out at -5 to -15 degrees C. Frozen blastomeres were thawed in water at 15 degrees C. Blastomeres cryopreserved with MEM containing 10% dimethyl sulfoxide (Me(2)SO) and 10% FBS (10% Me(2)SO/MEM10) showed higher survival rates than those cryopreserved with MEM containing 10% FBS and 10% glycerol, ethyleneglycol, 1, 2-propanediol, or sucrose. Blastomeres treated with 10% Me(2)SO/MEM10 showed higher survival rates than those treated with MEM containing only 10% Me(2)SO. Blastomeres seeded above -10 degrees C showed higher survival rates than non-seeded ones. Frozen blastomeres at advanced stages demonstrated high survival rates. Blastomeres cryopreserved under optimum conditions showed survival rates of 59.3+/-2.8%. These results indicate that 10% Me(2)SO/MEM10 is a suitable cryoprotectant medium to cryopreserve chum salmon blastomeres, that seeding should be carried out above -10 degrees C on pre-freezing, and that blastomeres at the blastula stage should be used as material.     

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole-imidazole polyamides in rat plasma

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)-imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py-Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mmx15.0 cm TOSOH Co., Japan) column maintained at 40 degrees C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0-10 min, A: 100-20%, B: 0-80% linear gradient; 10-15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py-Im polyamides after intravenous injection.     

Pretreatment efficiency and structural characterization of rice straw by an integrated process of dilute-acid and steam explosion for bioethanol production

The combined pretreatment of rice straw using dilute-acid and steam explosion followed by enzymatic hydrolysis was investigated and compared with acid-catalyzed steam explosion pretreatment. In addition to measuring the chemical composition, including glucan, xylan and lignin content, changes in rice straw features after pretreatment were investigated in terms of the straw's physical properties. These properties included crystallinity, surface area, mean particle size and scanning electron microscopy imagery. The effect of acid concentration on the acid-catalyzed steam explosion was studied in a range between 1% and 15% acid at 180°C for 2 min. We also investigated the influence of the residence time of the steam explosion in the combined pretreatment and the optimum conditions for the dilute-acid hydrolysis step in order to develop an integrated process for the dilute-acid and steam explosion. The optimum operational conditions for the first dilute-acid hydrolysis step were determined to be 165°C for 2 min with 2% H(2)SO(4) and for the second steam explosion step was to be carried out at 180°C for 20 min; this gave the most favorable combination in terms of an integrated process. We found that rice straw pretreated by the dilute-acid/steam explosions had a higher xylose yield, a lower level of inhibitor in the hydrolysate and a greater degree of enzymatic hydrolysis; this resulted in a 1.5-fold increase in the overall sugar yield when compared to the acid-catalyzed steam explosion.     

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.     

A homotetrameric agglutinin with antiproliferative and mitogenic activities from haricot beans

A homotetrameric agglutinin with a molecular mass of 130 kDa was isolated from seeds of the haricot bean. The agglutinin was isolated using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 200. Haricot bean agglutinin was adsorbed on DEAE-cellulose and Affi-gel blue gel. The hemagglutinating activity of the agglutinin was stable up to 40 degrees C. It underwent a 40% decline when the temperature was raised to 50 degrees C and a complete loss when the temperature was further increased to 80 degrees C. The hemagglutinating activity exhibited a time-dependent loss in activity when the agglutinin was incubated at 100 degrees C for different durations. No activity was discernible when the agglutinin was left at 100 degrees C for 1 min. The activity also underwent a decline in the presence of 500 mM FeCl(3) and CaCl(2). Haricot bean agglutinin manifested a weaker mitogenic activity than concanavalin A toward mouse splenocytes. It exhibited antiproliferative activity toward the tumor cell lines M1 [leukemia], HepG2 [hepatoma] and L1210 [leukemia] cells.     

Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC

Arachidonic acid (AA) was separated and purified from microbial lipids by the combined method of urea inclusion reaction and reversed-phase high performance liquid chromatography. At first, AA was concentrated from free fatty acids made from microbial lipids by a urea inclusion reaction. The optimum conditions were as follows: methanol was the suitable solvent, the ratio of free fatty acids to urea to methanol was 1:2:8 (wt/wt), and the temperature of the urea inclusion reaction was -10 degrees C. The AA content was increased from 38% to 79%, and then AA was purified on a C(18) preparative column (300 mm x 30 mm I.D., d(p)=15 microm), using methanol-water (95:5, v/v) as the mobile phase, at a flow rate of 5 mL/min. The purity of AA after two steps purification reached 99%. This result indicates that the combined method of the urea inclusion reaction and reversed-phase high performance liquid chromatography is a promising technique for purification of AA.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of superoxide dismutase from chicken liver

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme has been isolated, purified and partially characterized from chicken liver. The following steps were carried out in order to purify chicken liver SOD. Initially, the liver was homogenized and hemoglobin was removed. Subsequently protein precipitation was effected with (NH(4))(2)SO(4), methanol, (NH(4))(2)SO(4)-methanol and polyethylene glycol methods. The product from polyethylene glycol-3350 precipitation was found to have the highest SOD activity. Polyethylene glycol was removed by chromatography using a PD-10 column. After passing through an ultrafilter, the superoxide dismutase was fractionated by DEAE-ion chromatography and then Sephadex G-75 gel filtration chromatography. During this purification procedure, a specific activity of 4818.2 IU/mg was reached, corresponding to 285.8-fold purification. The purified enzyme, which was characterized as cyanide-sensitive SOD, contained two subunits having Cu and Zn elements with a molecular weight of 16000+/-500 for each. The optimum pH of purified CuZnSOD was determined to be 8.9. The enzyme was found to have good pH stability in the pH range 6.0-7.5 at 25 degrees C over a 2-h incubation period and displayed good thermal stability up to 45 degrees C at pH 7.4 over a 1-h incubation period. The SOD enzyme was not inhibited by DTT and beta-mercaptoethanol, but inhibited by CN(-) and H(2)O(2). In the presence of 2 mM iodoacetamide, the enzyme showed an approximately 40% activity loss. Finally, the inhibitory effect of ionic strength on SOD was also investigated.     

A new procedure for the isomerization of vitamin D and its metabolites

A new procedure for the isomerization of vitamin D and its metabolites is described. Vitamin D or its metabolites are dissolved in 100 microliters methanol and 10 M HC1 in 2-butanol is used as the reagent for isomerization. The isomerization reaction is carried out at 5 degrees C for 2-3 min which gives quantitative yields of isotachysterols down to 10 ng level without use of any carrier.     

Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris

A full-length xylanase gene, encoding 326 amino acids belonging to the fungal glycosyl hydrolase family 10, from Aspergillus terreus BCC129 was cloned and sequenced. Sequence analysis suggested that the first 25 amino acids of this enzyme is the signal peptide. Therefore, only the mature xylanase gene of 906 bp was cloned into a yeast expression vector, pPICZalphaA, for heterologous expression in Pichia pastoris. A band of approximately, 33 kDa was observed on the SDS-PAGE gel after one day of methanol induction. The expressed enzyme was purified by gel filtration chromatography. The purified recombinant xylanase demonstrated optimal activity at 60 degrees C, pH 5.0 and a Km of 4.8 +/- 0.07 mg/ml and a Vmax of 757 +/- 14.54 micromol/min mg, using birchwood xylan as a substrate. Additionally, the purified enzyme demonstrated broad pH stability from 4 to 10 when incubated at 40 degrees C for 4 h. It also showed a moderate thermal stability since it retained 90% of its activity when incubated at 50 degrees C, 30 min, making this enzyme a potential use in the animal feed and paper and pulp industries.     

蜜环菌胞外漆酶的合成、纯化及性质研究

研究了蜜环菌胞外漆酶合成条件和酶学性质。实验表明,培养基初始pH5.5、培养温度25℃有利于菌株产酶;与麦芽糖、山梨糖和半乳糖相比,纤维二糖和棉子糖作为碳源时漆酶产量更高;有机氮源比无机氮源有利于漆酶合成。泥炭提取液可显著诱导漆酶生成,当其含量为50%时,菌株漆酶最高产量是对照组的7倍。在蜜环菌发酵上清液中检测到3个漆酶同功酶组分,其主要活性（约占75%）组份漆酶A经 (NH₄)₂SO₄沉淀、制备级PAGE电泳和阴离子交换柱层析被分离纯化至电泳均一,SDSPAGE法测得酶亚基分子量59kD,凝胶过滤色谱法测定活性酶分子量58kD。纯化的漆酶A等电点pI为4.0,氧化愈创木酚的最适反应pH为5.6,最适温度为60℃,在60℃和65℃时半衰期分别为45min和36.8min,在pH5.2～7.2范围内稳定性较好。100mmol/L Cl^-对该酶有显著抑制作用,1mmol/L SO^2-₄ 对漆酶有激活作用,1mmol/L NaN₃可完全抑制酶活性,10 mmol/L EDTA对漆酶活没有明显影响,1mmol/L Cu²⁺对漆酶有激活作用。以愈创木酚为底物时,测得酶的K_m=1.026mmol/L,V_max=5μmol/(min·mg);以ABTS为底物时,测得其K_m=0.22mmol/L,V_max=69μmol/(min·mg)。