周期性静水压对人软骨细胞活性的时间依赖性影响

目的：目前软骨细胞体外研究多为动物模型,本研究以正常成人软骨细胞研究对象,探讨在适当强度、类型的周期性静水压下不同持续时间对软骨细胞活性的影响,探究人软骨细胞体外培养、构建组织工程软骨合适的时间参数。方法：将体外培养的正常成人膝关节软骨第3代软骨细胞随机分为4组：4h组、8h组、12h组、对照组。应用高压恒温静水压加压系统,充入含有95％的空气和5％的CO2混合气体,以2MPa压力大小对3个实验组进行周期性加压,分别每天加压4h、8h、12h,三组加压时间均为10d。10d后倒置相差显微镜下观察4组细胞形态,甲苯胺蓝及II型胶原免疫细胞化学染色进行细胞鉴定,并对II型胶原免疫细胞化学染色行半定量分析。MTT法分析各组软骨细胞增殖情况,流式细胞术检测细胞凋亡率。结果：3个实验组细胞增殖均快于对照组（P〈0．05）,与对照组相比4h组、8h组均抑制凋亡,12h组促进凋亡。12h组第6代细胞开始细胞形态即逐渐发生改变。结论：软骨细胞的增殖和凋亡水平对静水压的作用时间具有依赖性。在2MPa静水压力下,8h组更适合细胞生长,细胞活性更强。为进一步构建组织工程软骨人类模型及组织工程软骨的临床应用提供了一定的实验基础。     

低温处理对北美冬青叶片抗氧化能力的影响

为了解低温对北美冬青(Ilex verticillata)生长的影响,对2年生扦插苗经低温处理(-6℃)后的生长和抗氧化能力进行了研究。结果表明,随着低温处理时间的延长,北美冬青叶片的叶绿素/类胡萝卜素比值和叶绿素b含量增加,而其他色素含量都呈下降趋势;相对电导率(REC)和H_2O_2含量(72 h除外)显著增加。低温处理对超氧化物歧化酶(SOD)活性影响不大,却显著降低CAT的活性。低温处理过氧化物酶(POD)活性显著提高,48 h活性达到最高;而抗坏血酸过氧化物酶(APX)活性在处理12 h达最高,然后下降。还原型抗坏血酸(As A)、还原型谷胱甘肽(GSH)含量均呈先升高再下降的趋势。因此,北美冬青在短时间内(≤24 h)能够忍受低温(-6℃),抗氧化防御系统在低温初期起关键的作用,但随时间延长(72 h)其抗氧化能力减弱。     

超声波对铜绿微囊藻与蛋白核小球藻生理特征及竞争生长的影响

铜绿微囊藻是常见的水华蓝藻,常常在湖泊中与蛋白核小球藻共存或竞争生长。超声波可用于藻华即时治理,能够降低藻类生理活性,影响藻类生长,还可能改变藻类种间竞争关系。为了探究超声胁迫(35 kHz,0.035 W·cm^-3)对铜绿微囊藻与蛋白核小球藻的生理特征及种间竞争的影响,本研究设置纯藻组和1:1混合组(细胞浓度比)进行试验。结果表明: 铜绿微囊藻对超声胁迫更加敏感。超声处理600 s后,铜绿微囊藻的光合活性(F_v/F_m)和酯酶活性存在显著变化,纯藻组和混合组的F_v/F_m分别降低了51.8%和64.7%。而各组中蛋白核小球藻的光合活性变化较小。同时,铜绿微囊藻释放的荧光溶解性有机物(类色氨酸、类酪氨酸、类富里酸物质)含量多于蛋白核小球藻。两种藻的细胞浓度对超声波的响应也不同,蛋白核小球藻变化较小,而铜绿微囊藻的细胞浓度出现不同程度的下降。尤其是600 s超声处理大幅降低了混合组中铜绿微囊藻的细胞浓度(-42.6%),在超声胁迫解除后的8 d内蛋白核小球藻占优势,种间关系由铜绿微囊藻单边抑制蛋白核小球藻转变为两者互相抑制。在超声处理后,铜绿微囊藻的活性能够逐渐恢复,为了提高控藻效果的持久性,建议在一周后再次进行超声处理。     

小球藻替代鱼粉对鲫生长、体组成、肝脏脂肪代谢及其组织学的影响

设计了5组等氮(粗蛋白约38%)的饲料, 饲料中小球藻的添加量分别为0(对照)、17%、34%、51%和68%, 替代0(对照组)、21.8%、43.6%、65.5%和87.3%的鱼粉, 探讨小球藻替代鱼粉对初始体重为(5.540.08) g的鲫幼鱼生长、体组成、肝脏组织学及脂肪代谢相关酶活性的影响, 实验期为8周。实验结果表明: 随着小球藻替代鱼粉水平的增加, 鲫的增重率呈现先增加后下降的趋势; 与此相反, 饲料系数呈现先下降后增加的趋势。随着小球藻替代比例的增加, 肝体比和脏体比有增大的趋势, 而各个处理组肠脂比差异不显著。小球藻替代鱼粉使鲫肌肉和肝脏的蛋白含量降低, 而肝脏的脂肪含量随着替代比例的增加先上升后下降。当饲料中小球藻替代鱼粉的水平从0增加到65.5%, 肝脏生脂酶如G6PD、ME和FAS酶的活性显著增加, 而当饲料小球藻替代水平更进一步增加到87.3%, 肝脏生脂酶如G6PD、ME和FAS酶的活性下降。各个处理组6PGD酶活性没有显著差异。小球藻替代鱼粉对鲫的肝脏组织结构产生不利影响, 替代组的肝细胞体积有所增大, 部分肝细胞出现细胞核溶解、核消失、肝细胞坏死。以增重率和饲料系数为因变量进行二次线性回归分析, 表明小球藻对鱼粉的最适替代比例分别为47.14%和49.88%。       

[OMIm]Br对蛋白核小球藻的致毒效应

通过急性毒性实验考察了溴化1-辛基-3-甲基咪唑([OMIm]Br)离子液体对蛋白核小球藻(Chlorella pyrenoidosa)的致毒作用。结果表明:经过[OMIm]Br处理96 h,藻细胞生物量(包括藻细胞密度、叶绿素和蛋白质含量)发生改变,呈现正相关的剂量-效应关系;[OMIm]Br抑制蛋白核小球藻生长的半最大效应浓度(EC50)为14.95 mg·L-1;藻细胞的抗氧化机制受到破坏,超氧化物歧化酶(SOD)和过氧化物酶(POD)活性均下降,膜脂质过氧化产物丙二醛(MDA)的含量增加,且SOD、POD、MDA三者的变化幅度与受试物剂量呈正相关;[OMIm]Br通过直接损伤蛋白质和破坏抗氧化机制系统等途径对蛋白核小球藻产生致毒机制,运用流式细胞仪等手段证实藻细胞周期被[OMIm]Br阻滞在S期和G2/M期,并导致藻细胞坏死,阻滞程度和坏死程度与[OMIm]Br剂量呈正相关。     

溶藻细菌胞外活性物质对蛋白核小球藻的毒性效应

为了探索分离到的溶藻细菌L7胞外活性物质对蛋白核小球藻的毒性效应和致毒机理, 采用不同质量浓度的L7胞外活性物质冻干粉(L7-LPEAC)处理蛋白核小球藻(Chlorella pyrenoidosa), 测定藻细胞的光合作用效率(EPR)以及蛋白质、叶绿素a和丙二醛(MDA)的含量。L7-LPEAC溶液浓度较低(0.80 g/L和1.25 g/L)时促进蛋白核小球藻的生长, 其96 h、120 h的EC50分别为5.75 g/L和2.55 g/L。当L7-LPEAC溶液浓度≥2.00 g/L时, 叶绿素a和蛋白质含量变化同步呈现先增加后减少的趋势; 处理72 h后, L7-LPEAC溶液浓度分别为2.00 g/L, 3.13 g/L, 4.90 g/L的浓度组中, 藻细胞MDA含量与对照组相比差异显著(P<0.05), 浓度组7.67 g/L和12.00 g/L则与对照组差异极显著(P<0.01); 处理120 h后, 各浓度组藻细胞叶绿素a含量的相对抑制率均大于60%。使用L7-LPEAC修复富营养化水体时, 选择适当的投加浓度, 既能杀灭引起水体富营养化的目标藻类, 又能避免对其他藻类产生抑制作用, 可以较好地维持水生生态系统的平衡。     

干热处理对甜瓜种子活力和细菌性果斑病抑制能力的影响

以厚皮甜瓜品种‘哈密绿’种子为材料,在70℃、75℃和80℃温度下分别处理24h、48h和72h,研究不同干热处理对甜瓜种子活力萌发和生理生化指标变化及细菌性果斑病的防治效果。结果显示:(1)随着处理温度的升高和时间的延长,干热处理甜瓜种子发芽指标和成苗率显著下降,而70℃处理24h和48h对种子发芽指标(活力指数除外)和成苗率无显著影响。(2)与对照相比,干热处理种子胚芽中的SOD活性、可溶性糖和脯氨酸含量升高,在同一温度处理下,SOD活性和可溶性糖含量随着处理时间的延长基本呈增长趋势,脯氨酸含量则随着处理时间的延长呈下降趋势,POD和APX活性以及可溶性蛋白含量变化随着温度的升高和处理时间的延长呈下降趋势;与对照相比,胚芽中CAT活性在70℃处理下降低,而在75℃和80℃处理下升高,但其随着处理时间的延长呈下降趋势;胚芽中O-·2产生速率在70℃和75℃处理下与对照接近,而在80℃处理下随着处理时间的延长呈上升趋势,且均显著高于对照。(3)随着处理温度的升高和时间的延长,甜瓜接菌种子幼苗细菌性果斑病发病率较对照显著降低。研究表明,适宜干热处理温度和时间诱导甜瓜种子中抗氧化酶活性增强,渗透调节物质含量增加,超氧阴离子产生速率降低,种子活力和出苗率有效提高,‘哈密绿’种子有效、安全的干热处理组合为70℃、48h。     

铜绿微囊藻与小球藻对低温和黑暗的响应与恢复

研究以水华蓝藻铜绿微囊藻(Microcystis aeruginosa PCC 7806)与绿藻小球藻(Chlorella sp. FACHB-31)为研究对象, 探讨低温和黑暗对其生长、色素含量、最大光化学效率(F_v/F_m)、丙二醛(MDA)含量及过氧化氢酶活性的变化。结果表明, 30d的低温和黑暗处理, 显著降低了铜绿微囊藻和小球藻的叶绿素a浓度, 增加了单位细胞类胡萝卜素含量。在低温黑暗条件下, 铜绿微囊藻的MDA含量及CAT活性均显著增加, 而小球藻变化不明显。30d低温黑暗处理, 铜绿微囊藻的存活率为54.6%, 显著高于小球藻的31.3%。当恢复正常温度与光照, 2种藻均迅速生长。这些结果表明低温黑暗影响了微囊藻和小球藻的生理特性。在低温黑暗处理下, 微囊藻的F_v/F_m显著降低, 而小球藻则保持较为恒定的F_v/F_m, 表明微囊藻通过降低自身光合活性来渡过冬季低温黑暗的条件, 而小球藻在低温黑暗条件下仍保持较高的光合活性。     

氯化钠处理对原壳小球藻生长及油脂积累的影响

采用Basal培养基,通过光学显微镜、电子显微镜、激光共聚焦显微镜以及尼罗红染色定量等方法研究了不同浓度氯化钠(0、150、300、600 mmol/L)对小球藻属原壳小球藻的生长状态、脂滴分布、总脂含量的影响。结果表明,添加不同浓度的氯化钠对原壳小球藻的生长有明显的影响,随着氯化钠浓度的增加,小球藻的生长速度受到明显的抑制,600 mmol/L氯化钠处理时生长几乎完全被抑制。在显微镜下观察,可见氯化钠浓度的增加会导致小球藻聚集成团,这种现象在150 mmol/L和300 mmol/L氯化钠培养下比较明显;通过电子显微镜下观察,可以发现培养初期,随着氯化钠浓度的增加,小球藻细胞壁增厚,脂滴增多。通过尼罗红染色对脂含量进行定量,处理初期脂滴的合成量在600 mmol/L时最高,但到后期,随着藻生物量的增加,150 mmol/L和300 mmol/L处理下脂合成量逐渐升高,而对照小球藻脂合成量基本不变。稳定期后,从生物量(干重)和脂总量来看,300 mmol/L氯化钠培养处理的小球藻虽然生物量只有对照的73.55%,但是总脂含量却是对照的2.22倍,可见一定浓度的氯化钠处理一定时间可显著提高原壳小球藻的油脂含量。     

大鼠脑缺血／再灌注损伤中一氧化氮和一氧化氮合酶的变化

目的:探讨脑缺血/再灌注损伤中脑组织一氧化氮和一氧化氮合酶的变化.方法:用线栓法建立大脑中动脉梗死(MCAO)模型,观察局灶性脑缺血30 min再灌注30 min、1 h、3 h、 6 h、12 h、24 h、48 h 、72 h、96 h、168 h NO含量和NOS活性的变化.结果:脑缺血/再灌注过程中NO含量和NOS活性呈"双峰样"改变.缺血/再灌注30 min后NO含量和NOS活性升高,再灌注3 h时NO含量和NOS活性下降,再灌注6 h、12 h、24 h、48 h 、72 h NO含量和NOS活性再次显著升高,与再灌注72 h达峰值.结论:NO和NOS通过多种途径参与了脑缺血/再灌注损伤的病理过程.     

beta-Glucanases of Geotrichum candidum

The formation of (1-4)-, (1-3)- and (1-6)-beta-glucanases and beta-glucosidases was studied during the growth of the fungus Geotrichum candidum under the conditions of submerged cultivation in a medium optimal for the production of cellulolytic enzymes. Endo-(1-4)-beta-glucanases and C1 enzyme, as well as (1-3)- and (1-6)-beta-glucanases appeared in the medium as soon as by the 45th hour of growth. However, the maximal concentration of the enzymes in the medium was observed at different periods of the fermentation: between 75th and 105th, 70th and 95th, 55th and 100th, 80th and 105th hours, respectively. The content of the enzymes abruptly decreased by the 160th hour of the growth. The activity of beta-glucosidases, which was low at the beginning of the growth, sharply increased by the 70th hour and remained at the same level by the 160th hour of the growth. The accumulation of beta-glucanases was an uneven process, consistent with irregular changes in the content of DNA and protein in the biomass. The isoelectric points of beta-glucanases and beta-glucosidases were studied in the filtrate of the cultural broth after 96 h of the cultivation. The high activity of endo-(1-4)-beta-glucanase was found at the pH 4.6, 4.1 and 3.8; its low activity was detected at the pH 6.4, 3.2, 1.6 and 1.3. Other glucanases behaved also as acid proteins. During isoelectric focusing, (1-3)-beta-glucanase showed the peaks of activity at the pH 4.4, 4.0, 3.8 and 2.9; (1-6)-beta-glucanase, at the pH 5.0, 3.7, 3.5, 3.1 and 2.0; beta-glucosidases were distributed over a broad pH range from 6.7 to 2.0, with the maximal activity at the pH 6.2, 4.8 and 3.7.     

A reliable method for precise initial rate determination of enzyme activity under high hydrostatic pressure with simple apparatus - Application to Aspergillus niger fructosyl-transferase activity measurements

An original procedure for initial rate accurate determination of enzyme activity under high hydrostatic pressure is reported. This method, adapted to most kinds of enzyme systems, is based on the use of the linear property of product formation during the initial phase of a reaction and does not require specific high pressure equipment. The reliability of the method was tested and illustrated with the study of Aspergillus niger fructosyl-transferase activity as a function of substrate concentration above 300 MPa. © Rapid Science Ltd. 1998     

Stability and activity of lipase in subcritical 1,1,1,2-tetrafluoroethane (R134a)

The stability and activity of commercial immobilized lipase from Candida antarctica (Novozym 435) in subcritical 1,1,1,2-tetrafluoroethane (R134a) was investigated. The esterification of oleic acid with glycerol was studied as a model reaction in subcritical R134a and in solvent-free conditions. The results indicated that subcritical R134a treatment led to significant increase of activity of Novozym 435, and a maximum residual activity of 300% was measured at 4 MPa, 30 °C after 7 h incubation. No deactivation of Novozym 435 treated with subcritical R134a under different operation factors (pressure 2–8 MPa, temperature 30–60 °C, incubation time 1–12 h, water content 1:1, 1:2, 1:5 enzyme/water, depressurization rate 4 MPa/1 min, 4 MPa/30 min, 4 MPa/90 min) was observed. While the initial reaction rate was high in subcritical R134a, higher conversion was obtained in solvent-free conditions. Though the apparent conversion of the reaction is lower in subcritical R134a, it is more practicable, especially at low enzyme concentrations desired at commercial scales.     

Effects of abscisic Acid on growth, RNA metabolism, and respiration in germinating bean axes

The effect of abscisic acid on growth, respiration, the ATP pool, and rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination has been measured in experiments where the duration of abscisic acid application and its concentration have been varied. At concentrations from 10(-7) to 10(-4)m, abscisic acid inhibits synthesis of RNA with maximal inhibition (80%) at 10(-5)m. RNA synthesis is inhibited by abscisic acid at all times examined (12, 18, and 24 hours), but the extent of inhibition is maximal at 18 hours. In 18-hour axes RNA synthesis is inhibited 42%, ATP pool size is reduced 3%, and O(2) consumption is decreased by 6% after 75 minutes of abscisic acid treatment. Inhibition of RNA synthesis is complete by 2 hours of treatment with abscisic acid, and recovery to near control levels occurs by the 3rd hour after removal from abscisic acid.     

COMBINED EFFECTS OF TEMPERATURE AND IRON ON THE GROWTH AND PHYSIOLOGY OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)

Phaeodactylum tricornutum Bohlin (Bacillariophyceae) was maintained in exponential growth under Fe‐replete and stressed conditions over a range of temperatures from 5 to 30° C. The maximum growth rate (GR) was observed at 20° C (optimal temperature) for Fe‐replete and ‐stressed cells. There was a gradual decrease in the GR decreasing temperatures below the optimum temperature; however, the growth rate dropped sharply as temperature increased above the optimum temperature. Fe‐stressed cells grew at half the growth rate of Fe‐replete cells at 20° C, whereas this difference became larger at lower temperatures. The change in metabolic activities showed a similar pattern to the change in growth rate temperature aside from their optimum temperature. Nitrate reductase activity (NRA) and respiratory electron transport system activity (ETS) per cell were maximal between 15 and 20° C, whereas cell‐specific photosynthetic rate (P_cell) was maximal at 20° C for Fe‐replete cells. These metabolic activities were influenced by Fe deficiency, which is consistent with the theoretical prediction that these activities should have an Fe dependency. The degree of influence of Fe deficiency, however, was different for the four metabolic activities studied: NRA > P_cell > ETS = GR. NRA in Fe‐stressed cells was only 10% of that in Fe‐replete cells at the same temperature. These results suggest that cells would have different Fe requirements for each metabolic pathway or that the priority of Fe supply to each metabolic reaction is related to Fe nutrition. In contrast, the order of influence of decreasing the temperature from the optimum temperature was ETS > P_cell > NRA > GR. For NRA, the observed temperature dependency could not be accounted for by the temperature dependency of the enzyme reaction rate itself that was almost constant with temperature, suggesting that production of the enzyme would be temperature dependent. For ETS, both the enzyme reactivity and the amount of enzyme accounted for the dependency. This is the first report to demonstrate the combined effects of Fe and temperature on three important metabolic activities (NRA, P_cell, and ETS) and to determine which activity is affected the most by a shortage of Fe. Cellular composition was also influenced by Fe deficiency, showing lower chl a content in the Fe‐stressed cells. Chl a per cell volume decreased by 30% as temperature decreased from 20 to 10° C under Fe‐replete conditions, but chl a decreased by 50% from Fe‐replete to Fe‐stressed conditions.     

Pressure-induced Shape Change of Phospholipid Vesicles: Implication of Compression and Phase Transition

A microscopic study has allowed the analysis of modifications of various shapes acquired by phospholipid vesicles during a hydrostatic pressure treatment of up to 300 MPa. Giant vesicles of dimyristoylphosphatidylcholine / phosphatidylserine (DMPC/PS) prepared at 40°C mainly presented a shape change resembling budding during pressure release. This comportment was reinforced by the incorporation of 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or by higher temperature (60°C) processing. The thermotropic main phase transition (Lα to Pβ′) of the different vesicles prepared was determined under pressure through a spectrofluorimetric study of 6-dodecanoyl-2-dimethylamino-naphtalene (Laurdan) incorporated into the vesicles’ bilayer. This analysis was performed by microfluorescence observation of single vesicles. The phase transition was found to begin at about 80 MPa and 120 MPa for DMPC/PS vesicles at, respectively, 40°C and 60°C. At 60°C the liquid-to-gel transition phase was not complete within 250 MPa. Addition of DMPE at 40°C does not significantly shift the onset boundary of the phase transition but extends the transition region. At 40°C, the gel phase was obtained at, respectively, 110 MPa and 160 MPa for DMPC/PS and DMPC/PS/DOPE vesicles. In comparing volume data obtained from image analysis and Laurdan signal, we assume the shape change is a consequence of the difference between lateral compressibility of the membrane and bulk water. The phase transition contributes to the membrane compression but seems not necessary to induce shape change of vesicles. The high compressibility of the Lα phase at 60°C allows induction on DMPC/PS vesicles of a morphological transition without phase change.     

Effects of high pressure on the viability,morphology, lysis,and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.     

Uterine growth responses of the mature castrate rat to estradiol-17B

To examine estrogen-stimulated uterine growth we have monitored changes in uterine DNA synthesis, ornithine decarboxylase (ODC) activity and protein content as well as luminal epithelial (LE) cell mitotic index and ultrastructural changes. We have utilized this model to examine castrate mature rat uterine growth as a function of time between 18 and 40 hours following a single injection of 25.0 ug of estradiol-17B. LE cell mitotic index and protein content increases were maximally elevated as early as 18 hours postinjection while uterine ODC activity was maximal at 28 hours; uterine DNA synthesis increases continued throughout the experiment. In addition, the infusion of either 1 or 2 ug E2 plus progesterone over a 24 hour period, stimulated elevated ODC activity under both treatment regimens and LE cell mitotic index which was inversely related to E2 dose.     

Inactivation of creatine kinase by high pressure may precede dimer dissociation.

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1-200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.     

Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.