Cell swelling induced by the permeant molecules urea or glycerol induces immediate high amplitude thyrotropin and prolactin secretion by perifused adenohypophyseal cells

The permeant molecules, urea and glycerol, evoked a prompt secretory burst of TSH and PRL when added to the extracellular medium of acutely dispersed anterior pituitary cells. Secretion of both hormones was proportional to the concentration of urea or glycerol between 26 and 104 mM (r greater than 0.89, P less than 0.001). Equivalent concentrations of the impermeant molecule, mannitol, did not induce secretion. The acute TSH and PRL secretory responses to TRH, hyposmolarity, and permeant molecules were qualitatively indistinguishable. These data support our hypothesis that cell swelling and resultant plasmalemma expansion is a potent inducer of hormone secretion. Since the secretory response to permeant molecules was not reduced in a Ca2+-free medium containing 0.1 mM EGTA, an increase in Ca2+ transport across the plasmalemma to raise cytosol Ca2+ concentration does not appear involved.     

Stopped-flow spectrophotometric assay of glycerol permeation in Escherichia coli: applicability and limitations.

The passive permeation and facilitated diffusion of glycerol in various strains of Escherichia coli have been studied by stopped-flow spectrophotometry. Contrary to the prediction for glycerol entry by simple diffusion, the reciprocal relaxation time (1/tau, s-1) for the passive permeation of glycerol in cells grown in the presence of glucose was not constant but decreased as the glycerol concentration increased above 100 mM. This anomaly was not due to refractive index differences or to the presence of residual levels of the glycerol facilitator protein in non-induced cells. Although reciprocal relaxation times for glycerol-induced E. coli exhibited the expected elevation relative to non-induced cells, a similar anomalous decrease 1/tar (s-1) with increasing glycerol concentration was observed. In addition, at early times after suspension in dilute buffer, the 1/tau (s-1) values obtained for induced or non-induced E. coli swelling in glycerol were considerably greater than for organisms incubated in dilute buffer for longer times. We concluded that either this spectrophotometric technique was not monitoring solely the permeation of glycerol into E. coli, or concentrations of glycerol above 100 mM significantly perturbed the structure of the E. coli cell envelope.     

葡萄生轴霜霉菌孢子囊的长期保存

为了解决葡萄生轴霜霉菌的活体保存问题,本文开展了多种保藏方法的研究。采用二甲基亚砜和甘油分别于不同低温条件下对孢子囊进行了保存,取出后进行接种试验。结果表明,孢子囊用不同保护剂处理后保存于-76℃中5d,10%二甲基亚砜处理的孢子囊经接种后发病面积百分率为68.3%,其致病性明显强于25%甘油处理;孢子囊用不同浓度的二甲基亚砜保存于-76℃中30d,5%和10%二甲基亚砜处理后致病性差异不显著,但明显高于15%、20%、25%二甲基亚砜处理的孢子囊;5%和10%二甲基亚砜处理的孢子囊,在-76℃中保存5d和15d后,致病性明显高于-20℃的处理;孢子囊经-76℃保存240d后,10%二甲基亚砜处理的孢子囊无致病性,而5%二甲基亚砜处理的发病面积百分率为56.3%,与对照无显著差异,说明孢子囊用含5%的二甲基亚砜处理能在-76℃中长期保存。     

Developmental capacity of bovine oocytes frozen in different cryoprotectants

Iso-osmolar concentrations of 1.6 M of 1,2-propanediol, glycerol and dimethylsulfoxide were compared for their effectiveness as cryoprotectants for in vitro-matured bovine oocytes. Survival, defined as the number of morphologically normal oocytes, after freeze-thawing in 1,2-propanediol (49.8%) was significantly higher (P<0.01) than in glycerol (28.6%) or dimethylsulfoxide (32.6%). After insemination, the fertilization rate of morphologically normal oocytes frozen-thawed in 1,2-propanediol (57.9%) was higher (P<0.05) than that of those frozen in dimethylsulfoxide (38.3%). However, the proportion of surviving oocytes developing to blastocysts was not different among the 3 groups.     

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.     

Activation of aspartase by glycerol

Aspartase [EC 4.3.1.1] of Escherichia coli, which exhibits a sigmoidicity in the substrate saturation profile at alkaline pH, was markedly activated by 10–20% glycerol at low substrate concentrations and pH 8.5. In contrast, no activation, but an inhibition was observed at pH 7.0 throughout the substrate concentrations tested. The activation profile of the enzyme as a function of glycerol concentration was considerably influenced by L-aspartate concentration. Neither alteration of the cooperative nature of the enzyme nor subunit dissociation was associated with the activation. Besides glycerol, ethylene glycol, propylene glycol, dimethylsulfoxide, and dioxane also activated the enzyme.     

Improved methods for freeze preservation of chimpanzee sperm

Freeze preservation of human and nonhuman semen has been used effectively for a number of years; however, the application of freezing to preserve nonhuman primate sperm has been less successful. This study compares five freeze methods and various concentrations of the cryoprotectants glycerol and dimethylsulfoxide (DMSO) for cryopreservation of chimpanzee (Pan troglodytes) sperm. The different methods were compared using quantitative analysis of sperm function and, by an indirect measure of fertilizing capacity, the denuded hamster oocyte penetration assay (SPA). The best results were obtained using an extender comprising Ham's F10 medium containing 15% heat inactivated human cord serum and 15.6% glycerol. Semen extended 1 to 1 by this method, for a final glycerol concentration of 7.8%, and frozen using a computer programmed freezer maintained approximately 65% of its original viability and up to 70% of its initial motility. The extended semen was used to initiate pregnancy in two female chimpanzees.     

Block of endplate channels by permeant cations in frog skeletal muscle

Motor endplates of frog semitendinosus muscles were studied under voltage clamp. Current fluctuations induced by iontophoretic application of acetylcholine were analyzed to give the elementary conductance, gamma , and mean open time, tau , of endplate channels. Total replacement of the external Na+ ion by several other metal ions and by many permeant organic cations changed both gamma and tau . Except with NH4+ ions, the gamma values with foreign test ions were all smaller than expected from the independence relation and their previously measured permeability ratios. The more hydrophobic ions gave the smallest gamma values. Foreign permeant cations also depress gamma when mixed with Na+ ions. These effects could be interpreted in terms of binding of ions to a saturable site within the endplate channel as they pass through. The site for organic ions would have a hydrophobic component. Similar evidence is given for a metal ion binding site on the cytoplasmic end of the channel accessible to internal ions. Most foreign cations also shortened tau when applied externally. The changes of gating did not seem to be correlated with changes in gamma . Thus there is no evidence for control of tau by ions bound within the pore.     

Refolding kinetics of denatured-reduced lysozyme in the presence of folding aids

The refolding kinetic behavior of denatured-reduced lysozyme in the presence of folding aids (acetamide, acetone, thiourea, L-arginine or glycerol) was studied utilizing a simplified model describing the competition between first-order folding reaction and third-order aggregation. It was found that the protein folding aids could be categorized into two groups. One of them at proper concentrations, such as acetamide, acetone, thiourea and L-arginine, stabilized unfolded protein or folding intermediates. In the presence of these additives, the folding rate decreased with increasing their concentration, and there existed a concentration where the aggregation rate constant was minimized. So, there was an optimum concentration for the folding aids to produce a high yield. The other group was protein stabilizers such as glycerol. In the presence of this kind of folding aids, both the refolding rate and yield were enhanced by increasing their concentration to a proper value. Moreover, their effect on improving protein refolding was additive to those of the first group. So the cooperative application of the two kinds of folding aids could result in favorable refolding rate and yield of protein.     

Studies on the vitality of human bone marrow during collection and deep-freeze preservation

The cryoprotective effect of dimethylsulfoxide, glycerol and polyethylenglycol during freezing and thawing of human bone marrow was investigated by eosin staining test, an acridinorange fluochrome staining test and by RNA- and DNA-synthesis tests. In these tests the overall yield of vital nucleated cells, referred to the number in the absence of cryoprotectants and freezing and thawing, amounted to 50% with dimethylsulfoxide, 30% with glycerol, and 10% with polyethylenglycol. With dimethylsulfoxide and glycerol the loss of vital nucleated cells is almost entirely due to the addition of cryoprotectants. Polyethylenglycol freezing and thawing also leads to a great loss of vital nucleated cells. The results with dimethylsulfoxide show that the currently employed techniques of punction, preparation, freezing and thawing of bone marrow are suitable for clinical application.     

Cryopreservation of Sheep Red Blood Cells

The protection of sheep erythrocytes at freezing temperatures was investigated using glycerol, dimethylsulfoxide (DMSO), glucose and four different types of polyvinylpyrrolidone (PVP) as cryoprotective agents. Depending on type (molecular weight) and concentration good protection was obtained with PVP, whereas glycerol, DMSO and glucose were unsatisfactory. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 1–2 × 109 cells/ml. No cells tolerated freezing at −20 °G. Best results were obtained when the cells were frozen directly in liquid nitrogen (−196°G).     

Survival of Solanum tuberosum suspension cultures to –14°C: The mode of action of proline

Normally suspension cultures of potato, Solanum tuberosum , can survive only to a freezing temperature of –2°C. The treated cultures were able to survive at much lower freezing temperature after the addition of l -proline of 0.43 M or more to the suspension medium and treatment for 1.5 h. Cultures could survive to -14°C with 0.87 M proline treatment. Treatments with higher concentration than 0.87 M resulted in no additional freezing survival. Microscopical observations of the treated cultures before freezing showed that proline induced plasmolysis, which was initiated at the 0.43 M level and reached maximum at 0.87 M. The mode of action of proline, appears to be due to the removal of excessive intracellular water by the osmotic gradient. Sucrose at about the same concentration acted in the same way as proline. The cryoprotective role of proline and sucrose is discussed in relation to the action of the permeant dimethylsulfoxide.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

无明胶布氏菌活疫苗冻干稳定剂的筛选

目的为皮上划痕人用布氏菌活疫苗筛选存活率高、无明胶冻干稳定剂。方法以冻干活菌存活率为指标,对甘油、甘露醇、蔗糖、葡萄糖、乳糖、谷氨酸钠、甘氨酸、谷氨酸、脯氨酸和硫脲等10种稳定剂通过单因素筛选法,筛选出冻干存活率高的4种单因素稳定剂成分;将4种单因素稳定剂成分进行正交试验优化,筛选出最优稳定剂组合。结果单因素试验结果显示,甘油、葡萄糖、谷氨酸钠和硫脲4种稳定剂成分冻干后活菌存活率较高,对布氏菌活疫苗具有良好保护效果。通过正交试验筛选出最优稳定剂配方中四组分的质量分数分别为甘油1.5%、葡萄糖5%、硫脲1.5%、谷氨酸钠1.0%,该配方的冻干存活率可达81.5%。结论无明胶冻干稳定剂对布氏菌活疫苗具有较好的保护作用。     

Oxidation of ethanol by cumene hydroperoxide in the presence of cytochrome P-450 LM2 and hemoglobin

Ethanol oxidation by cumene hydroperoxide (CHP) with participation of cytochrome P-450 LM-2 (pH 7.4) and hemoglobin (pH 7.0) was studied at 37 degrees C in phosphate buffer. Both hemoproteins form complexes with CHP that are decomposed with the liberation of the RO2., RO. and HO. radicals, thus initiating the chain oxidation of ethanol. Ethanol oxidation catalyzed by cytochrome P-450 LM-2 and hemoglobin occurs only through a radical formation and is competitively inhibited by the radical scavenging agents, e.g., 1-naphthol, thiourea, mannitol and dimethylsulfoxide (DMSO). The values of effective inhibition constants were determined for all antioxidants whose activity decreases in the following order: 1-naphthol greater than thiourea greater than mannitol greater than DMSO. The non-inhibited oxidation of ethanol in "CHP-hemoproteins" systems is characterized by low ethanol conversion because of bimolecular termination of radicals and biocatalyst destruction.     

Synthesis of novel steroidal heterocyclic derivatives as antibacterial agents

This study aimed at the synthesis of novel structurally promising steroidal heterocycles and to elucidate the potential role of these compounds as antibacterial agents. Epi-androsterone 1 reacted with CS2 and sodium hydride in dimethylsulfoxide to yield alpha-oxoketene dithiodisodium salt 2. The non-isolable salt 2 reacted with acetyl chloride, benzoyl chloride, phenacyl bromide and iodomethane to afford the corresponding alpha-oxodithioacetal derivatives 4a,b, 6 and 7, respectively. Interaction of 2 with the alkyl halide reagents 8a-d yielded the corresponding thiophene derivatives 10a-d. Alpha-oxoketene dithioacetal 7 reacted with urea and thiourea to furnish the pyrimidinoandrostane derivatives 12a,b. Compound 7 also reacted with ortho-phenylene diamine and ortho-aminophenol 13a,b to produce the dinucleophilic adducts 15a,b. The in vitro antibacterial evaluation of some newly prepared compounds showed that all compounds have high significant antibacterial activity against the used strains of gram positive and gram negative bacteria.     

Plasmolysis shape in relation to freeze-hardening of cabbage plants and to the effect of penetrating solutes*

Abstract In conformity with earlier results, low-temperature hardening of cabbage seedlings lowered the osmotic potential and increased the permeability to thiourea of the petiole cells. It also decreased the time required for rounding-up of the protoplasts in cells plasmolysed in 1. 5 × isotonic (or higher) glucose or CaCl₂ solutions. Solutions of dimethylsulphoxide (DMSO), thiourea, urea, and glycerol each accelerated the rate of rounding-up of protoplasts in plasmolysed cells, compared to the rate in glucose solution of the same hypertonicity. Each also penetrated the cell membranes as indicated by deplasmolysis. Only in the case of DMSO, in which there was very rapid deplasmolysis (5–6 min), was this rounding-up due to protoplast expansion. In the case of thiourea (deplasmolysis within 30–60 min) rounding-up occurred almost immediately (less than 2 min), before protoplast expansion was sufficient to induce it. It was concluded that the accelerated rounding-up was due to a rapid osmotic adjustment in the protoplasm by the penetrating solution, which increased its water content and decreased its viscosity.     

Permeability of cryptands through dihexadecyl phosphate bilayer membranes

The leakage of Ru(bpy)3(2+) across a membrane of dihexadecyl phosphate (DHP) vesicles was compared when induced by (2.1.1), (2.2.1), (2.2.2.), (2.2.1.)C10 and (2.2.2.)C10-cryptands, and by (2.2.) and (2.2.)-bishydroxyethyl, i.e., ionizable macrobicyclic and monocyclic amino polyethers. Ru(bpy)3(2+) leakage increased as the permeant concentrations rose and was much higher for the very lipophilic cryptands. It also increased as the pH fell, and was lower and less dependent on the permeant concentration when induced by addition of partially titrated than by non-pretitrated cryptand. The efficiency of the permeant decreased as the alkali cation concentration rose and was independent of the cation type. It also varied with the membrane type: the efficiency of the (2.2.2.)C10-cryptand was higher on permeation of the membrane of DHP vesicles made from dihexadecyl phosphate than that of large unilamellar vesicles (LUV) composed of alpha-phosphatidylcholine, alpha-phosphatidic acid and cholesterol in an 8:1:1 molar ratio. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of the permeating agents and of the membranes.     

Cellular test for RNA-synthesis in rat bone marrow and its use in testing cryoprotective and toxic agents]

A system for the measurement of the RNA-synthesis of bone marrow cells of the rat has been developed and the incorporation of [3H]-uridine into the cellular RNA has been standardized with respect to the time of incubation, the concentration of [3H]-uridine and the number of cells. A plateau of the incorporation of [3H]-uridine into the RNA is reached after 20 min of incubation and is interpreted as the expression of a steady state in synthesis and degradation of the cellular RNA. A constant labelling of the RNA is reached above 8.3 with 10(-6)M [3H]-uridine. The optimal cell number in the 500 mul standard assay is 4 with 10(6). Actinomycin D inhibits the RNA-synthesis to 94% in a concentration of 1.2 with 10(2) mug/ml. The cryoprotectants dimethylsulfoxide, polyethylene-oxide and glycerol and the potential haematotoxic substances dichlorodiphenyltrichloroethane and gamma-hexane were tested in this system. 5% dimethylsulfoxide and 10% polyethylen-oxide in Eagle's-medium with ethylendiamintetra-acetate do not influence the RNA-synthesis. 5% glycerol reduces the incorporation of [3H]-uridine into the cellular RNA to about 30%.