Comparison of an iterative microcalorimetric method and dialysis equilibrium for calculating thermodynamic parameters of a binding protein which presents a weak affinity for its substrate

An original iterative microcalorimetric method is used to study the interaction of 5-fluorouracil, a cancer chemotherapeutic agent, with human serum albumin. Two equivalent binding sites are demonstrated with an association constant and an enthalpy variation equal to 370 ± 30 m^?1 and ?10 ± 0,5 kcal/mol, respectively. By means of a competitive microcalorimetric method, the partially competitive binding of dipotassium chlorazepate and 5-fluorouracil is shown. This iterative microcalorimetric method can be applied to all techniques involving the measurement of a phenomenon which is proportional to the concentration of the complex.     

Effect of berberine on Escherichia coli, Bacillus subtilis, and their mixtures as determined by isothermal microcalorimetry

The strong toxicity of pathogenic bacteria has resulted in high levels of morbidity and mortality in the general population. Developing effective antibacterial agents with high efficacy and long activity is in great demand. In this study, the microcalorimetric technique based on heat output of bacterial metabolism was applied to evaluate the effect of berberine on Escherichia coli, Bacillus subtilis, individually and in a mixture of both using a multi-channel microcalorimeter. The differences in shape of the power-time fingerprints and thermokinetic parameters of microorganism growth were compared. The results revealed that low concentration (20?μg/mL) of berberine began to inhibit the growth of E. coli and mixed microorganisms, while promoting the growth of B. subtilis; high concentration of berberine (over 100?μg/mL) inhibited B. subtilis. The endurance of E. coli to berberine was obviously lower than B. subtilis, and E. coli could decrease the endurance of B. subtilis to berberine. The sequence of half-inhibitory concentration (IC(50)) of berberine was: B. subtilis (952.37?μg/mL)?>?mixed microorganisms (682.47?μg/mL)?>?E. coli (581.69?μg/mL). Berberine might be a good selection of antibacterial agent used in the future. The microcalorimetric method should be strongly suggested in screening novel antibacterial agents for fighting against pathogenic bacteria.     

口蹄疫病毒感染BHK—21细胞的代谢热谱研究

本文应用高灵敏度的LKB2277生物活性检测仪测定FMDV感染BHK-21细胞的代谢热谱,并与传统方法测定的一步生长曲线进行比较,二者具有显著的相似性。结果表明,微量热法通过对细胞及其受病毒感染的细胞体系代谢热的测定,能有效地监测病毒在宿主细胞内增殖的过程。该方法还提供了一种动态连续分析病毒感染增殖的新手段。     

Cell growth experiments using a microcalorimetric vessel equipped with oxygen and pH electrodes.

A method is described for simultaneous measurements of heat production rate, oxygen activity and pH, using a 3-ml stirred microcalorimetric titration/perfusion vessel fitted with a polarographic oxygen sensor and a combination pH-electrode. Heat production rate (+/- 0.3 microW), pH (+/- 0.01) and oxygen concentration (+/- 4 mumol/l) could be measured without any cross interference between the three signals. Results from measurements on suspensions of T-lymphoma cells and E. coli are presented.     

Microcalorimetric characterization of seasonal metabolic trends in marine microcosms

A microcalorimetric and oximetric circulation system has been used to describe the emergence of seasonal adaptative changes occurring in marine microcosms at the water-sediment interface. The microcosms were prepared from three stations: one estuarine and two lagoonal. They were submitted to the same acute eutrophication, using peptone, and the evolutions shown by power-time and oxygen-time curves were compared over a 2-yr period. The metabolic heat productions and their oxidative counterparts are well correlated with seasonal trends. In summer, the microcalorimetric curves are typically unimodal, with very low dispersion, and a low thermodynamic steady state is attained in a few hours (40–50 μW), indicating an efficient, integrated regulatory mechanism; oxygen conditions evolute towards anoxia within 6 to 8 h. In winter, the power-time curves are typically sigmoid, with low dispersion; the thermodynamic steady state attained indicates a high maintenance energy requirement (140–150 μW), probably due to microbial populations less adapted to eutrophication stress. In winter, oxygen depletion is only partial and a plateau is attained within limits of 80 to 40% of initial saturation, and lasts 1 wk at least. Spring and autumn responses are very heterogeneous, varying from summer to winter types and displaying evolutive bimodal patterns. The sea-water-sediment interfaces from different estuarine and lagoonal habitats with distinct physical and chemical characteristics, but subjected to similar environmental and seasonal perturbations, show similar energetic patterns according to the seasons. It is proposed, as a working hypothesis, that the seasonal variations of the microcalorimetric response to experimental eutrophication are the expression of a global homeostatic adaptative memory, determined by natural environmental perturbations. Spring and autumn can be considered as intermediate periods within which a seasonal “adaptative memory” is replaced gradually by another one. The heterogeneity of the microcalorimetric response in spring and autumn microcosms could be related to an asynchronous maturation for the different components of the benthic ecosystem.     

不同灌溉方式下3种土壤微生物活性测定方法比较

探究不同灌溉方式下土壤微生物活性,对维持土壤稳定和提高水资源利用效率具有重要意义。以沈阳农业大学长期定位灌溉试验基地为平台,采用土壤酶活性、土壤呼吸和微量热法,研究节水灌溉组覆膜滴灌、渗灌及对照组沟灌下的土壤微生物活性并比较3种微生物活性测定方法。结果表明:不同灌溉方式下土壤脲酶、转化酶、脱氢酶活性无显著差异,土壤呼吸在3个主要生长季也没有明显变化规律;而微量热法得到的热功率时间曲线呈现了典型的微生物生长特征趋势,覆膜滴灌的生长速率较大,且与沟灌的总热量、最大热功率相差不大。因此,从可持续农业观点出发,覆膜滴灌是保证土壤微生物活性较高的一种节水灌溉方式;微量热法也为传统方法下不易检测的微生物活性提供了新思路。     

Microcalorimetry: A tool for the study of the biodegradability of straw by mixed bacterial cultures

Summary The biodegradability of straw by a mixed bacterial culture obtained from a pile of weeds was studied by microcalorimetry. All the cultures were grown at 30°C under anaerobic conditions in microcalorimetric vessels. The fermentation thermograms, obtained using well defined conditions, were very reproducible. The quantities of heat produced during straw degradation were found to be proportional to the quantity of straw introduced at the beginning of the fermentation.The recovered carbon was also found to be proportional to the initial quantity of straw. From both microcalorimetric and chemical analysis it was concluded that the limiting factor of the straw degradation was the cellulolytic activity of the mixed culture. This is supported by the fact that commercially available cellulase added to the growth medium increases the amount of straw degradation by about four times. The heat associated with fermentation of each cellulose monomer (C₆H₁₀O₅) was found to be 120 kJ, a value which is close to the heat associated with hexose fermentation by pure cultures. In conclusion, we propose that microcalorimetry can be used as a powerful tool for the analysis of the biodegradability of complex heterogeneous substrate by pure or mixed cultures.     

Microcalorimetry is a sensitive method for studying the effect of nucleotide mutation on promoter activity

Microcalorimetric method was successfully used to study the effect of nucleotide mutations on promoter activity and identify the important nucleotide necessary for the promoter function in Escherichia coli. The thermokinetic parameters, such as k, I and IC(50), were calculated from the metabolic power-time curves obtained by microcalorimetric measurement using the TAM air Isothermal Microcalorimeter (manufactured by Thermometric AB company of Sweden). Analysis of these data revealed that different nucleotide mutations in -10 box sequence of RM07 fragment had different effect on the promoter activity. Our research also suggest that the microcalorimetric method is a very sensitive and easily performed method for investigation of promoter mutation.     

Flow Microcalorimetric Studies of Yeast Growth: Fundamental Aspects

The importance of factors such as cultural conditions e.g. organism background, inoculum density and glucose concentration are explored and their effects on the experimental recording discussed. The relevance of these findings to microbial microcalorimetry in general and to microcalorimetric identification of organisms in particular is described. The conclusions drawn are (i) that microbial identification by microcalorimetry is not possible and (ii) that great caution must be exercised in interpreting metabolic data derived from microcalorimetric studies.     

Comparisons of microcarrier cell culture processes in one hundred mini-liter spinner flask and fifteen-liter bioreactor cultures

Microcarrier cell culture process can be used to culture anchorange-dependent cells in large bioreactor vessels. The process performance in large bioreactors is usually less prominent than that in spinner flask vessels and bench scale reactors. In this study we investigated the microcarrier cell culture processes in 100?ml spinner flask and 15-liter bioreactor cultures, including the kinetics for cell attachment, cell growth and the production of Japanese encephaltilis vaccine strain (Beijing-1) virus. Under a fixed concentration of microcarrier and cell density used in inoculations, the attachment kinetics of Vero cells on Cytodex 1 microcarrier in a 15-liter bioreactor vessel was 2 folds slower than with 100?ml spinner flask culture. Virus replication in 15-liter bioreactor culture also revealed an approximately one day lag-time compared to 100?ml spinner flask culture. Findings presented herein provide valuable information for designing and operating microcarrier cell culture processes in large bioreactor vessels.     

Heat production as a cell cycle monitoring parameter

A microcalorimetric method was applied to define the cell cycle by measuring heat production of mouse breast cancer cell line, FM3A. FM3A cells, were synchronised at the Go phase and produced 13.0 mu Watts (W) per 1 X 10(6) cells. Although the number of cells in fresh medium remained unchanged during the following 24 hour, a dramatic increase of heat production was observed and maximum heat (46.2 mu W) was produced by the cells at 24 hours when the cell cycle was presumably at the G2 phase. At 26 hours, although cell number increased, heat production decreased. Since the cells were not treated in any manner, this microcalorimetric method of measuring the cell cycle by monitoring heat production can be a very useful tool.     

Microcalorimetric evaluation of the biological activity of polyene complexes with divalent metal ions: Mg(II), Ca(II), Ni(II), Cu(II) and Zn(II)

Polyene complexes with Mg(II), Ca(II), Ni(II), Cu(II) and Zn(II) have been prepared and evaluated for biological activity in a flow microcalorimetric study. The bioactivities are all lower per g of complex than is the bioactivity of the patent polyene, nystatin. However extrapolation of the linear bioassay data suggests that because of enhanced solubilities the metal ion complexes may be able to yield higher overall bioactivity than can nystatin alone.     

Analysis of microcalorimetric curves for bacterial identification

A numeric method is suggested for the treatment of microcalorimetric curves of bacterial growth to provide a new tool for their automatic identification. In this method the microcalorimetric curves are searched against certain reference profiles (stored in a library) by means of a cross-correlation analysis and a parametric comparison. The matching between the new curve and each reference profile is evaluated by means of a specific identification coefficient which provides an objective criterion for the identification of each species. The reliability of the method is discussed.     

Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus Acer

? Vulnerability to cavitation and conductive efficiency depend on xylem anatomy. We tested a large range of structure-function hypotheses, some for the first time, within a single genus to minimize phylogenetic 'noise' and maximize detection of functionally relevant variation. ? This integrative study combined in-depth anatomical observations using light, scanning and transmission electron microscopy of seven Acer taxa, and compared these observations with empirical measures of xylem hydraulics. ? Our results reveal a 2 MPa range in species' mean cavitation pressure (MCP). MCP was strongly correlated with intervessel pit structure (membrane thickness and porosity, chamber depth), weakly correlated with pit number per vessel, and not related to pit area per vessel. At the tissue level, there was a strong correlation between MCP and mechanical strength parameters, and some of the first evidence is provided for the functional significance of vessel grouping and thickenings on inner vessel walls. In addition, a strong trade-off was observed between xylem-specific conductivity and MCP. Vessel length and intervessel wall characteristics were implicated in this safety-efficiency trade-off. ? Cavitation resistance and hydraulic conductivity in Acer appear to be controlled by a very complex interaction between tissue, vessel network and pit characteristics.     

Interaction of calcium ions with α-elastin: An equilibrium dialysis,circular dichroism,and microcalorimetric study

The interaction of calcium ions with α-elastin has been investigated by equilibrium dialysis, CD, and microcalorimetric techniques. Consistent with literature data, it was found that the interaction in water is very poor. In trifluoroethanol (TFE), equilibrium dialysis experiments showed that calcium ions bind to ～-elastin with an association constant of ～250 L × mol^?1. Such a figure is not consistent with highly specific, highly selective binding. It was also found that the CD response is not directly proportional to the amount of bound calcium but depends on the protein concentration. From microcalorimetric experiments it was found that the heat effect relative to the binding process is of the order of 1.9 kcal/g ion. From this figure and from the binding constant, a positive ΔS value of about 17 e.u. was evaluated, leading to the conclusion that the binding process is entropy driven. From microcalorimetric measurements a ΔH of 1.5 kcal/residue was found for the calcium-induced conformational transition of the protein.     

Microcalorimetric Study of Glucose Permeation in Microbial Cells

A microcalorimetric method for measuring the influence of extracellular glucose concentration on the rate of catabolism is described. This method has been applied to anaerobically growing cultures of Zymomonas mobilis and of a respiratory-deficient ("petite") mutant of Saccharomyces cerevisiae (strain YFa). The Michaelian kinetics recorded with both organisms were apparently related to glucose transport. With Z. mobilis, it was found that, in the range of glucose concentrations at which this organism was growing exponentially, cell activity was limited by the maximal rate of the catabolic enzymes; at lower concentrations, glucose transport was the rate controlling step. The metabolic activity of yeast always depended on external glucose concentration; when this was lowered under a threshold, a change of kinetics took place. The microcalorimetric method described seems to be widely applicable to kinetic studies of the permeation of metabolizable substrates in microorganisms.     

微量量热法在生命科学中的应用

介绍了微量量热系统的基本结构和工作原理,以及微量量热法在酶学、细胞生物学、微生物学、植物生理学研究中的应用。     

Microcalorimetric study on the growth and metabolism of microencapsulated microbial cell culture

The rate of heat output is one of the suitable measurements of metabolic activity of the organism or its parts, down to the cellular or even the sub-cellular levels. In this paper, microcalorimetry was first applied to study the metabolic activity of microbial in both alginate-polylysine-alginate and alginate-chitosan-alginate microencapsulated cultures as well as in free non-encapsulated culture. The organisms used for the measurements were Escherichia coli and Saccharomyces cerevisiae. As a result of this work, it was found that, despite E. coli cell in free non-encapsulated culture has the highest metabolic rate due to the highest value of heat output, the proliferation of the cells terminates quickly with a lowest biomass formed. And we found also an obviously longer stationary phase in microencapsulated culture. As far as S. cerevisiae was concerned, it was found that there was also the highest value of heat output in free non-encapsulated culture, but the cell density was lower than that in microencapsulated culture. On account of the microcalorimetric and metabolic measurements, it can be concluded that more substrate can be used to convert to biomass in microencapsulated culture which means a higher biomass yield existed.     

嗜盐古菌启动子DNA片段的功能检测

将来源于嗜盐古菌染色体DNA的启动子片段RM07或RM13插入到启动子探针载体pYLZ_2的报告基因lacZ之前,通过β_半乳糖苷酶酶活性的检测,进一步确证RM07和RM13片段在大肠杆菌(Escherichia coli)中的启动功能。同时用微量热技术检测了大肠杆菌DH5α及其重组菌株在LB培养基中37℃生长过程的热输出功率。T2(pYLZ_2)、TE07(pYL726)、TE07_2(pYL702)、TE131(pYL131)和TE132(pYL132)菌株的生长速率分别比大肠杆菌DH5α降低了6.5%、11%4、1.1%4、7.5%和42.7%。当启动子启动了基因表达时,菌株的生长速率显著降低,热力学参数与酶活性检测结果有较好的一致性。微量热结果表明基因的表达比质粒DNA的复制过程需要消耗更多的能量,对细菌的生理代谢有较大改变。微量热技术为检测基因的表达和转录调控提供了新的方法和思路。     

Use of microcalorimetry for the characterization of marine metabolic activity at the water-sediment interface

Metabolic processes occurring at the sea-water-sediment interface were studied using a circulation flow microcalorimeter. A methodology was developed to characterize rapid and global changes in metabolism and energy flow, not easily detectable with reductionist approaches. Sea water was pumped continuously, 5–10 mm above the sediment, in experimental microcosms; a 100-μm filter prevented passage of meiofauna. This “circulating interface” was taken through the microcalorimeter and from there to an oxygen electrode, and was returned to the microcosm. The microcosms were experimentally eutrophicated using peptone (4 mg·ml ^?1). The relationship between heat production and oxygen tension in the circulating interface has been compared with ATP production, ¹⁴CO₂ and [¹⁴C]particulate matter turnovers. Initial heat steady-state production rises to a peak of 130 to 180 μW·ml^?1 in 6 to 8 h after peptone treatment. The microcalorimetric peak is closely correlated with ¹⁴CO₂ turnover and partially correlated with micro-events on the pO₂ curve. ATP concentration and particulate-¹⁴C turnover increase constantly and then stabilize, with the establishment of a new heat production steady state. The approach provides an indication of the temporal behaviour of complex mixtures of microorganisms and ciliates at the water-sediment interface, and gives holistic measurements of energy flow after induced perturbation (eutrophication) of the ecosystem. Although many problems remain to be solved in this field, it is shown here that flow microcalorimetric measurements can be used to monitor the effects of addition of reagents like pollutants and nutrients.