Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.     

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.     

A new method to evaluate the thermal stability of lyophilized enzymes

Summary The thermal inactivation of lyophilized chymotrypsin was studied at controlled water activities. At 60 °C the enzyme showed good stability except at aw 0.97, whereas at 75 °C considerable inactivation occured at most water activities. Increasing the amount of buffer on the preparation decreased the stability significantly. The optimal temperature of enzymatic activity was increased 14 °C, when the water activity was decreased from 1 to 0.5.     

Osmotic relations of the drought-tolerant shrub Artemisia tridentata in response to water stress

Turgor maintenance, solute content and recovery from water stress were examined in the drought-tolerant shrub Artemisia tridentata. Predawn water potentials of shrubs receiving supplemental water remained above ?2 MPa throughout summer, while predawn water potentials of untreated shrubs decreased to ?5 MPa. Osmotic potentials decreased in conjunction with water potentials maintaining turgor pressures above 0 MPa. The decreases in osmotic potentials were not the result of osmotic adjustment (i.e. solute accumulation). Leaf solute contents decreased during drought, but leaf water volumes decreased more than 75% from spring to summer, thereby passively concentrating solutes within the leaves. The maintenance of positive turgor pressures despite decreases in leaf water volumes is consistent with other studies of species with elastic cell walls. Inorganic ion, organic acid, and carbohydrate contents of leaves declined during drought. The only solutes accumulating in leaves of A. tridentata with water stress were proline and a cyclitol, both considered compatible solutes. Total and osmotic potentials recovered rapidly following rewatering of shrubs; solute contents did not change except for a decrease in proline. Maintaining turgor through the passive concentration of solutes may be advantageous compared to synthesis of new solutes for osmotic adjustment in arid environments.     

Dynamics of submerged macrophyte populations in response to biomanipulation

1. A 6‐year study (1992–97) of changes in submerged vegetation after biomanipulation was carried out in the eutrophicated Lake Finjasjön, Southern Sweden. Ten sites around the lake were revisited each year. At each site five samples of above‐ground biomass were taken at 10 cm water depth intervals. An investigation of the seed bank at the 10 sites, and a grazing experiment where birds and large fish were excluded was also conducted. 2. Between 1992 and 1996, in shallow areas (water depth < 3 m), vegetation cover increased from < 3 to 75% and above‐ground biomass from < 1 to 100 g DW m^–2. Mean outer water depth increased from 0.3 to 2.5 m. Elodea canadensis and Myriophyllum spicatum accounted for > 95% of the increase in biomass and plant cover. The following year (1997), however, cover and above‐ground biomass decreased, mainly attributable to the total disappearance of E. canadensis. Secchi depth increased after biomanipulation until 1996, but decreased again in 1997. 3. Total and mean number of submerged species increased after biomanipulation, probably as a result of the improved light climate. However, after the initial increase in species number there was a decrease during the following years, possibly attributed to competition from the rapidly expanding E. canadensis and M. spicatum. The lack of increase in species number after the disappearance of E. canadensis in 1997 implies that other factors also affected species richness. 4. A viable seed bank was not necessary for a rapid recolonization of submerged macrophytes, nor did grazing by waterfowl or fish delay the re‐colonization of submerged macrophytes. 5. Submerged macrophytes are capable of rapid recolonization if conditions improve, even in large lakes such as Finjasjön (11 km²). Species that spread by fragments will increase rapidly and probably outcompete other species. 6. The results indicate that after the initial Secchi depth increase, probably caused by high zooplankton densities, submerged vegetation further improved the light climate. The decrease in macrophyte biomass in 1997 may have caused the observed increase in phosphorus and chlorophyll a, and the decrease in Secchi depth. We suggest that nutrient competition from periphyton, attached to the macrophytes, may be an important factor in limiting phytoplankton production, although other factors (e.g. zooplankton grazing) are also of importance, especially as triggers for the shift to a clear‐water state.     

Arsenic Uptake by Lemna minor in Hydroponic System

Arsenic is hazardous and causes several ill effects on human beings. Phytoremediation is the use of aquatic plants for the removal of toxic pollutants from external media. In the present research work, the removal efficiency as well as the arsenic uptake capacity of duckweed Lemna minor has been studied. Arsenic concentration in water samples and plant biomass were determined by AAS. The relative growth factor of Lemna minor was determined. The duckweed had potential to remove as well as uptake arsenic from the aqueous medium. Maximum removal of more than 70% arsenic was achieved at initial concentration of 0.5 mg/l arsenic on 15th day of experimental period of 22 days. Removal percentage was found to decrease with the increase in initial concentration. From BCF value, Lemna minor was found to be a hyperaccumulator of arsenic at initial concentration of 0.5 mg/L, such that accumulation decreased with increase in initial arsenic concentration.     

The effect of relative humidity on osmoregulation in the squirrel monkey (Saimiri sciureus)

Osmoregulatory balance was studied in four young, tamed squirrel monkeys (Saimiri sciureus, Columbia) after acclimatization to relative humidities (rh) commonly used in laboratories (30% and 50%) and to higher humidities representative of the dry and wet seasons in their natural environment (75% and 95%). The temperature was constant at 25°C and the light-dark cycle was 12:12 hours. The animals were maintained in large metabolism cages and were free moving. Water consumption and urine flow rates increased at each higher humidity from 30% rh to 75% rh and then decreased at 95% rh (p<0.05). Fecal water loss was greater with higher humidity (p<0.05). Evaporative water loss remained relatively constant until a break between 75% and 95% rh, at which point it decreased dramatically (p<0.05). Expressed relative to total water intake, evaporative water loss demonstrated a progressive decrease with increasing humidity: 65%, 56%, 51%, and 42%, at 30%, 50%, 75%, and 95% rh. This indicates that as the humidity approaches maximum saturation, not only is the evaporative water loss component necessarily diminished, but also the apparent precipitation of alternate strategies of thermoregulation leads to a reduction in the overall water requirements of the animals. Potassium/sodium excretion ratios were relatively constant at 30%, 50%, and 95% rh but decreased significantly at 75% rh (p<0.05). As a reflection of adrenocorticosteroid activity, this suggests that at 25°C, 75% rh is an optimal humidity in the maintenance of squirrel monkeys. The significant osmoregulatory alteration occurring between 75% and 95% rh provides further evidence that relative humidity may be an important factor in the seasonal physiological cycles of the squirrel monkey. In conducting the research described in this report the investigators adhered to the “Guide for Laboratory Animal Facilities and Care,” as promulgated by the Committee on the Guide for Laboratory Animal National Academy of Sciences, National Research Council.     

Study of the formation and propagation of a leader channel in air

Results are presented from calculations of the initial stage of leader channel formation in air. It is shown that the channel forms in two stages: one occurring at an essentially constant gas density on time scales much shorter than the characteristic gas-dynamic time and another in which gas-dynamic rarefaction of the channel becomes important and thermal-ionizational instability develops. The leader propagation velocity is largely determined by the time of channel contraction. The dependence of the leader velocity on the current and initial pressure is calculated. The results of calculations agree with experiment for relatively low leader currents (I _L = 0.5–5 A) and for leader currents above tens of amperes. A comparative analysis of the leader propagation velocity in air at different initial pressures is given. In the current range I _L = 0.5–30 A, the leader velocity depends weakly on the pressure; however, for currents as high as I _L ≥ 50 A, it increases appreciably with pressure.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

干旱胁迫下发菜光合作用相关基因差异表达分析

该研究以不同失水处理的发菜为研究材料,以充分吸水状态的发菜为对照,利用高通量测序技术和qRT PCR技术检测了干旱胁迫下发菜光合作用相关基因差异表达规律,并对光合色素和酶活在干旱胁迫下的变化进行了研究。结果表明：(1)发菜在不同程度干旱胁迫下有113个光合作用相关基因差异表达,其中失水30%、75%和100%的发菜分别有44个、74个和91个光合作用相关基因差异表达。(2)随着干旱胁迫程度的加深藻胆素、叶绿素a和类胡萝卜素含量逐渐降低,Rubisco活性随着干旱胁迫程度的增强先上升后下降,GAPDH活性随着干旱胁迫的增强呈现下降的趋势。研究表明,发菜通过光合作用相关基因的差异表达调控光合作用以适应干旱胁迫。该研究结果对进一步研究发菜干旱胁迫响应机制及其耐旱光合机理奠定了基础。     

The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short‐term thermal stress of the alga

The dinoflagellate alga Symbiodinium sp., living in symbiosis with corals, clams and other invertebrates, is a primary producer in coral reefs and other marine ecosystems. The function of the carbon‐fixing enzyme ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in dinoflagellates is difficult to study because its activity is rapidly lost after extraction from the cell. We report procedures for the extraction of Rubisco from Symbiodinium cells and for stable storage. We describe a continuous assay for Rubisco activity in these crude cell extracts using the Mn²⁺ chemiluminescence of Rubisco oxygenase. Chemiluminescence time courses exhibited initial transients resembling bacterial Form II Rubisco, followed by several minutes of linearly decreasing activity. The initial activity was determined from extrapolation of this linear section of the time course. The activity of fast‐frozen cell extracts was stable at ?80 °C and, after thawing and storage on ice, remained stable for up to 1 h before declining non‐linearly. Crude cell extracts bound [¹⁴C] 2‐carboxy‐D‐arabitinol 1,5‐bisphosphate to a high molecular mass fraction separable by gel filtration chromatography. After pre‐treatment of Symbiodinium cell cultures in darkness at temperatures above 30 °C, the extracted Rubisco activities decreased, with almost complete loss of activity above 36 °C. The implications for the sensitivity to elevated temperature of Symbiodinium photosynthesis are assessed.     

根茎萌发与幼苗期的虉草水分生态幅

为揭示虉草根茎萌发和幼苗期的水分生态幅,该研究通过盆栽控制试验,设置8个水分梯度：(2±0.5)％、(10±0.5)％、(15±0.5)％、(20±0.5)％、(25±0.5)％、(30±0.5)％、(40±0.5)％(水分饱和)和淹水(2 cm),通过萌发率、各形态指标和生理指标对虉草水分生态幅进行了探讨。结果表明：(1)土壤水分含量过低和过高都会降低虉草根茎萌发率。(2)随土壤水分含量的增加,虉草幼苗株高和地上生物量都呈现先上升后下降的趋势,两者并呈指数函数关系y ＝0.2038e0.0486x(R2＝0.803)。(3)虉草通过调整自身结构特点,降低个体大小、减少叶片数量和叶面积来适应胁迫环境,土壤水分含量为10％~30％时虉草含水率能保持相对稳定的水平。虉草的光和色素( Chl a、Chl b、Car)和相对叶绿素( SPAD)含量均随土壤水分含量的增加呈现出先上升后下降的趋势,Chl a/b则相反,呈先下降后上升趋势。(4) Chl/Car各组之间差异不显著。综合上述研究,利用高斯模型分析得出虉草的水分生态幅为7.48％~52.20％,最适水分生态幅为18.99％~40.60％。     

Biocatalysis with hydroperoxide lyase in extracts from Penicillium camemberti in neat organic solvent media

Abstract

Biocatalysis with hydroperoxide lyase (HPL) in extracts from Penicillium camemberti, in neat organic solvent media has been investigated. The effects of reaction conditions including organic solvent mixtures, initial water activity (a_w) and reaction temperature as well as the effect of the lyoprotectants, KCl and dextran 1 kDa, on HPL activity were studied. The addition of KCl to the enzymatic extract (70:1 protein, w/w) prior to lyophilization, enhanced HPL activity 6.53-fold. In contrast, the presence of dextran at a ratio of 8:1 decreased the enzymatic activity. Using hexane as the reaction medium, with an initial a_w of 0.1 and 0.5, the HPL specific activity was determined to be as 6.3 and 65.9 nmol converted 10-HPOD/mg protein/min, for the enzymatic extract without and with KCl present, respectively. Although HPL enzymatic extract with KCl showed a relatively low optimum reaction temperature (45°C) compared to 55°C without KCl, it exhibited a 2.51- and 2.78-fold higher thermal stability at 60 and 80°C, respectively. The kinetic results indicated that the highest HPL catalytic efficiency, V_max/K_m, of 6.58 × 10^?2 mL/mg protein/min, was obtained in the presence of KCl.     

Native root xylem embolism and stomatal closure in stands of Douglas-fir and ponderosa pine: mitigation by hydraulic redistribution

Hydraulic redistribution (HR), the passive movement of water via roots from moist to drier portions of the soil, occurs in many ecosystems, influencing both plant and ecosystem-water use. We examined the effects of HR on root hydraulic functioning during drought in young and old-growth Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and ponderosa pine (Pinus ponderosa Dougl. Ex Laws) trees growing in four sites. During the 2002 growing season, in situ xylem embolism, water deficit and xylem vulnerability to embolism were measured on medium roots (2–4-mm diameter) collected at 20–30 cm depth. Soil water content and water potentials were monitored concurrently to determine the extent of HR. Additionally, the water potential and stomatal conductance (g_s) of upper canopy leaves were measured throughout the growing season. In the site with young Douglas-fir trees, root embolism increased from 20 to 55 percent loss of conductivity (PLC) as the dry season progressed. In young ponderosa pine, root embolism increased from 45 to 75 PLC. In contrast, roots of old-growth Douglas-fir and ponderosa pine trees never experienced more than 30 and 40 PLC, respectively. HR kept soil water potential at 20–30 cm depth above –0.5 MPa in the old-growth Douglas-fir site and –1.8 MPa in the old-growth ponderosa pine site, which significantly reduced loss of shallow root function. In the young ponderosa pine stand, where little HR occurred, the water potential in the upper soil layers fell to about –2.8 MPa, which severely impaired root functioning and limited recovery when the fall rains returned. In both species, daily maximum g_s decreased linearly with increasing root PLC, suggesting that root xylem embolism acted in concert with stomata to limit water loss, thereby maintaining minimum leaf water potential above critical values. HR appears to be an important mechanism for maintaining shallow root function during drought and preventing total stomatal closure.     

Methylglyoxal can completely replace the requirement of kinetin to induce differentiation of plantlets from some plant calluses

It is well known that cytokinins, a group of plant hormones are absolutely required for the differentiation of calluses for the regeneration of plantlets through organogenesis. In the present work, it had been observed that methylglyoxal could completely replace kinetin to initiate differentiation of plantlets from calluses of Solanum nigrum and Daucus carota. Moreover, the effect of methylglyoxal was more pronounced compared to that of kinetin and the optimum concentration for methylglyoxal had been determined to be 0.5Â mM. Parallel with the differentiation of calluses to plantlets, the chlorophyll contents increased whereas the endogenous level of methylglyoxal remained unchanged. This remarkable effect of methylglyoxal in plant differentiation had been found out to be specific because some related compounds such as pyruvate and -lactate could not replace the requirement for methylglyoxal in the differentiation process. The activities of several enzymes were monitored during both methylglyoxal and kinetin-induced differentiation. The activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (NADP-dependent) involved in energy generation process in photosynthesis, increased as the differentiation proceeded. Whereas, the activities of both glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent) decreased with differentiation. The activity of glyoxalase I, which catalyzes the conversion of methylglyoxal and glutathione to S--lactoylglutathione, decreased with differentiation. The endogenous level of glutathione showed an initial decrease followed by an increase. It appears from the results presented above, that the effect of kinetin and methylglyoxal are similar in nature, the significance of which has been discussed.     

Dinitrogen fixation in a water-stressed Alnus clone is limited by host xerotolerance

The osmotolerance, rather than the halotolerance, of the endosymbiont predicted the xerotolerance of acetylene reduction by Alnus nodulated withFrankia ARgP5 ^AG . Cloned plants ofAlnus glutinosa (L.) Gaertn. AG8022-16 were subjected to water stress under controlled conditions in an environmental growth chamber. Transpiration, stomatal conductance, and leaf water potential had decreased after successive 10 day periods of moderate (75% of water demand) and severe (50% of water demand) water stress. After severe stress had wilted the plants, reducing leaf water potential to –2.10 MPa, nitrogenase activity had fallen to 2.51 M per plant per hour. The reported rapid turnover of nitrogenase implies thatFrankia mycelium was metabolically active at this low water potential, a water potential at which no Alnus-derivedFrankia has been reported active. Although ARgP5 ^AG was similar to other such strains in halotolerance (lower limitca.–1.25 MPa), the low water potential limit for growth with glucose (a non-assimilated osmoticum) wasca.–2.53 MPa. Nitrogenase activity was apparently more limited by host xerotolerance than by endophyte xerotolerance.Journal article J-5400 of the Oklahoma Agriculture Experiment Station, Oklahoma State University, Stillwater, OK 74078, USA.     

Water relations, activities of antioxidants, ethylene evolution and membrane integrity of pigeonpea roots as affected by soil moisture

The plants of pigeonpea (Cajanus cajan L.) cv. H77-216 were subjected to moderate [soil moisture content (SMC) = 7.3 ± 0.5 %] and severe (SMC = 4.3 ± 0.5 %) drought by withholding the irrigation at vegetative stage (45 d after sowing). The control plants were maintained at SMC of 11.0 ± 0.5 %. Half of the stressed plants were re-irrigated and their recovery was studied after 2 d. Leaf water potential, osmotic potential, and relative water content of leaf and root decreased significantly while a sharp rise in proline and total soluble sugars contents were noticed. Drought induced a significant increase in 1-aminocyclopropane 1-carboxylic acid (ACC) content and ACC oxidase activity which caused a considerable increase in ethylene evolution. Malondialdehyde content and relative stress injury were increased under drought whereas reverse was true for ascorbic acid content. The membrane integrity of roots decreased during stress and recovered on rehydration. The specific activity of total superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione transferase decreased to 37 – 78 %, 17 – 62 %, 29 – 36 % and 57 – 79 % at moderate and severe drought, respectively. The increase in activity of catalase and peroxidase could not overcome the accumulation of H₂O₂ content in the roots.     

柚子皮黑曲霉的分离鉴定及产酶特性研究

对柚子皮上自然生长的黑曲霉进行分离鉴定,并探讨其产酶特性。以平板稀释法从柚子皮上分离出一株霉菌菌株,通过观察其形态特征和培养特征,对照《真菌鉴定手册》判定该菌株的种属;采用鉴定培养基法对其产酶特性进行分析。根据柚子皮的成分特性,以干柚子皮为主要原料,该菌为生产菌株,采用固态发酵法探究培养基的成分、柚子皮含量、培养基初始含水量及发酵时间4个因素对纤维素酶活力的影响。结果表明,该菌株为黑曲霉(Aspergillus nige),可产淀粉酶、蛋白酶、纤维素酶、果胶酶;固态发酵培养基中添加柚子皮12g,麸皮0.5 g和(NH_4)_2SO_40.5 g,培养基初始含水量保持在68.5 mL/100 g,培养时间控制在60 h左右时纤维素酶产量较高。     

Effect of tetracaine and sovcaine on the ATP-dependent calcium accumulation in sarcoplasmic reticulum vesicles of skeletal muscles

The tetracaine and cinchocaine in concentration less than 2 mM and 0.5 mM, respectively, stimulate ATP-dependent Ca-loading by enhancing the initial rate of Ca2+-accumulation, do not affect the Ca2+-binding and Ca-ATPase activity of sarcoplasmic reticulum vesicles. These data suggest blocking of Ca2+-efflux from vesicles which occurs during Ca-accumulation. Higher concentrations of the same compounds (above 2 mM and 0.5 mM for tetracaine and cinchocaine, respectively) caused inhibition of the Ca-ATPase activity and decreased the ability of SR vesicles to retain Ca2+, probably, due to their nonspecific lipophilic action.     

Glutaraldehyde-fixation of yeast cells: Kinetics of a model system for enzyme cytochemistry

The kinetics of glutaraldehyde inactivation of a protoplasmic (-fructofuranosidase) and an extracytoplasmic (acid phosphatase) enzyme inSaccharomyces rouxii cells were studied at pH 5.5 and 30°C. The effects of glutaraldehyde concentration (0.5–3%), pH value, and temperature were surveyed by varying the fixation conditions. Cells from 1- to 10-day cultures retained 50–75% of their acid phosphatase activity and 15–24% of their -fructofuranosidase activity after 1-h exposures to 0.5% glutaraldehyde. The surviving -fructofuranosidase activity remained physically cryptic and was revealed only after further membrane perturbation with ethyl acetate. This crypticity barrier disappeared after overnight incubation of the treated cells at 4°C, with or without added glutaraldehyde, during which time the enzyme was resistant to further inactivation. The velocity ratio for raffinose versus sucrose, as substrate, decreased in treated cells, and changes inV _max andK _m were indicative of frank destruction of some enzyme molecules as well as modification of survivors. A comparable set of changes was also generated by treating cell-free extract with glutaraldehyde. Glutaraldehyde (0.5%) killed all yeast cells at 30°C within 5 min; at 4°C survival rates were quite high—81% after 15 min and 65% after 1 h. The bearing of these examples of enzyme inactivation, permeability barrier abolition, and structural stabilization on the general problems of yeast cytochemistry is discussed.