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.     

Discriminant analysis of microcalorimetric data of bacterial growth

In this work a bacterial classification method based on the discriminant analysis of the microcalorimetric data provided by the growth power-time (p-t) curves is developed. This method is applied to classify several species of Enterobacteria of different origins, and the results are compared with those obtained by conventional techniques. The proposed analysis allows us to classify bacteria into species and discriminate among strains of the same species. The classification is carried out using one run of each isolate after standardization of inocula and growth conditions. The discrimination power of available microcalorimetric data is also discussed, and the most discriminant set of data is proposed as the input variables of the analysis. Finally, the advantages of microcalorimetry as a taxonomical technique are discussed.     

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.     

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。     

Characterization of commercial yeast strains by flow microcalorimetry

A flow microcalorimetric procedure has been developed for the characterization of representative strains of Saccharomyces cerevisiae and Sacch. uvarum (carlsbergensis) used in the food industry. Growth in a chemically defined medium containing single and mixed sugars gave reproducible and characteristic power-time curves. The technique is proposed as a rapid alternative method for the characterization of yeast strains selected for commercial use.     

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.     

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.     

Studies on the growth metabolism of Bacillus thuringiensis and its vegetative insecticidal protein engineered strains by microcalorimetry

The metabolic power-times curves of Bacillus thuringiensis and its vegetative insecticidal protein engineered strains were determined at 30 degrees C by using a thermal activity monitor air Isothermal Microcalorimeter, ampoule method. From the power-times curves, the maximum power (Pmax) in the log phase, the growth rate constant (k), the generation times (tG), the time of the maximum power (tmax), the heat effects (Qlog) for log phase, and the total heat effect in 45 h (Qtotal) of B. thuringiensis strains can be obtained. The results indicate that their power-times curves are different. The relationship between their metabolic power-times curves and character of bacteria metabolism, and thermokinetics and gene expression were analyzed and discussed. The character of the bacteria power-times curves reflected the physiologic character of gene expression. The microcalorimetric method proved to be a reliable and sensitive tool for the assessment of the growth metabolism, the heat output in bacteria and its engineered strains. The determination of the thermokinetic character is beneficial to the control of fermentation.     

用胆固醇代谢的房室模型速率系数来分析动脉粥样硬化程度

理论上认为胆固醇逆向转运的速率与动脉粥样硬化程序呈负相关。但目前尚无完善的测试血浆脂蛋白－胆固醇体内代谢的方法。我们运用同位素＾３Ｈ－胆固醇示踪方法,建立房室模型,选取健康兔与ＡＳ兔对照,研究血浆脂蛋白转运胆固醇能力的差异,并结合ＡＳ兔主动脉斑块程度对比,结果验证了上述理论,此法如改用短半衰期同位素或稳定性同位素标记的胆固醇,就可用于人体,这可为临床判断ＡＳ程度提供一种无创性的新方法。     

细菌生长的热谱图测定

The fundamental growth thermograms of bacteria have been determined by using the microcalorimetric method. These perfect thermogram curves reflect the changes of bacterial growth patterns (including the lag phase of growth, log growth, stationary phase and the decline phase of growth). In our experiments, highly characteristic and reproducible growth patterns are observed under the same condition, therefore one can use these thermograms as "finger print" to discriminate bacteria. On the other hand, there thermogram curves contain ample information, which are very significant for the studies on microorganism metabolism, bio-thermokinetic and clinical fields.     

Application and Validation of PFGE for Serovar Identification of Leptospira Clinical Isolates

Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars.     

A microcalorimetric method for studying the biological effects of La(3+) on Escherichia coli

A microcalorimetric technique based on the bacterial heat-output was explored to evaluate the stimulatory effect of La(3+) on Escherichia coli. The power-time curves of the growth metabolism of E. coli and the effect of La(3+) on it were studied using an LKB-2277 BioActivity Monitor, stopped-flow method, at 37 degrees C. For evaluation of the results, the maximum power (P(max)), the growth rate constants (k) and the heat effects (Q(LOG), Q(STAT)) for the log phase, the stationary phase and the total heat effect (Q(T)) for E. coli were determined. The microcalorimetric method agreed with the conventional methods, such as cell numbers and biomass. La(3+) in the concentration ranges of 0-400 microg/ml has stimulatory effects on E. coli, while La(3+) ion of higher concentrations (>400 microg/ml) can inhibit the growth. This phenomenon is very similar to those observed from the in vitro cells and tissues from animals, plants and some microorganisms by other methods.     

Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: a contribution to the study of these free-living nitrogen-fixing bacteria

A 16S rRNA gene-based fingerprinting method was developed for the identification of Azotobacteraceae and tested onto 48 soil isolates and 28 reference strains belonging to the free-living nitrogen-fixing bacterial group and to the most common species found in soil samples. According to this method, the 16S rRNA gene was amplified using universal primers for Eubacteria and PCR products were subsequently digested with RsaI, HhaI, HpaII, FnuDII, and AluI. The analysis of the restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the eight Azotobacteraceae species tested. Cluster analysis was successfully employed for the identification of members of the family Azotobacteraceae, being assignation into species of the isolates confirmed by means of partial 16S rRNA gene sequencing.     

Microcalorimetric study of the action of Ce(III) ions on the growth of E. coli

A microcalorimetric technique based on bacterial heat output was used to evaluate the action of Ce(III) ions on the growth of Escherichia coli. The power-time curves of the growth metabolism of the bacteria were studied in the presence and absence of Ce(III) by means of a LKB-2277 Bioactivity Monitor, by a stopped-flow method at 37°C. For evaluation of the results, the maximum power, P _max, the growth rate constant k, and the heat effects Q _log, Q _stat, and Q _tot for the log phase, the stationary phase, and total heat output, respectively, were determined. For comparison, a spectrophotometer was used to estimate the number of cells in the liquid culture. The shape of the bacteria was examined by electron microscopy. We concluded that the presence of cerium ions at concentrations below 350 μg/mL have a stimulatory effect on the growth of E. coli, whereas concentrations at or above 400 μg/mL may have an inhibitory effect.     

A microcalorimetric vessel for monitoring cytotoxic reactions in the micro-submicrowatt region

A microcalorimetric vessel for monitoring the initial phases of cytotoxic reactions in the micro-submicrowatt region has been designed and tested. The vessel is intended to be used with a multichannel microcalorimetric system from Thermometric, J?rf?lla, Sweden and can be built up stepwise in a modular way. The different functions of perfusion, stirring and addition of small amounts of soluble immune reactants can be used separately but also in combination. This is accomplished by the use of a vertical stirring mechanism and a reaction vessel of low volume, 200 microliters. The simplicity of the vessel permits an identical vessel to be used on the reference side, handled in parallel with the measuring vessel. No gas phase is present.     

Microcalorimetric study of the anaerobic growth of Escherichia coli: growth thermograms in a synthetic medium.

A microcalorimetric technique was used for studying the growth of Escherichia coli during anaerobiosis. The growth thermograms obtained are complex and the shape of curves is dependent on the hydrogen lyase activity of the cells. Fermentation balances are given for different culture conditions, and simple growth thermograms are obtained when the hydrogen lyase activity is inhibitied.     

Microcalorimetric investigation on the growth model and the protein yield of Bacillus thuringiensis

A novel microcalorimetric technique based on the bacterial heat output was applied to evaluate the special growth model, the protein expression and the generation time of Bacillus thuringiensis for the first time. The thermogenic curves of the aerobic metabolism of B. thuringiensis strains YBT-833, YBT-1520 and YBT-833-2-1 were determined by using an LKB-2277 BioActivity Monitor. The analysis of the thermogenic curves indicated both the mutant strain and the wild-type strains followed the same linear growth model during sporulation. The metabolism heat output revealed heat output was correlated to the yield of the insecticidal crystal proteins (ICPs) very well, the more protein product, and the less heat output. Based on the data acquired, we proposed that this method could be a useful tool in monitoring the fermentation of B. thuringiensis.     

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.     

Thermodynamic features of bile salt-human serum albumin interaction

Interaction with human serum albumin is responsible for important aspects of the physiological behaviour of bile salts, although this factor has not been adequately examined. The nature of the binding is investigated here by means of thermodynamic parameters determined by equilibrium dialysis and microcalorimetric measurements. The positive enthalpy and entropy values obtained indicate the presence of a poorly specific hydrophobic bonding.     

Characterisation and metabolic studies of Saccharomyces cerevisiae and Kluyveromyces fragilis by flow microcalorimetry

The use of microcalorimetry in the routine identification of microorganisms is critically discussed and assessed. By use of flow microcalorimetric studies on Saccharomyces cerevisiae and Kluyveromyces fragilis the role of physical parameters and that of oxygen tension are discussed. The conclusion reached is that identification of microorganisms by microcalorimetry and subsequent discussion of metabolic events revealed by the thermogram, except under restrictive conditions, is inappropriate. However flow microcalorimetry, in contrast to batch microcalorimetry which has been used in the published material on microorganism identification, may allow characterization of yeasts suitable for particular industrial processes.