Characterization of the membrane sensor PenJ for β-lactam antibodies from Bacillus licheniformis by amino acid substitution

Abstract The beta scanner and pattern matching software integrated in the automated microbiology identification system (AMBIS) were used to assess the relatedness of isolates of Colletotrichum acutatum, C. kahawae, C. fragariae, C. gloeosporioides and C. musae based on mitochondrial DNA restriction fragment length polymorphisms. The dendrograms generated reflected the intra-specific variation and inter-specific relationships of these species as determined by other previously reported methods. The adaptability of AMBIS as a rapid method for assessing genetic relatedness of fungal species based on DNA polymorphisms is discussed.     

Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2)

Background

Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.

Results

A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-β superfamily members TGF-β 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.

Conclusion

The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.     

The use of LipidGreen2 for visualization and quantification of intracellular Poly(3-hydroxybutyrate) in Cupriavidus necator

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490–520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.     

Statistical quantification of methylation levels by next-generation sequencing

Background/Aims

Recently, next-generation sequencing-based technologies have enabled DNA methylation profiling at high resolution and low cost. Methyl-Seq and Reduced Representation Bisulfite Sequencing (RRBS) are two such technologies that interrogate methylation levels at CpG sites throughout the entire human genome. With rapid reduction of sequencing costs, these technologies will enable epigenotyping of large cohorts for phenotypic association studies. Existing quantification methods for sequencing-based methylation profiling are simplistic and do not deal with the noise due to the random sampling nature of sequencing and various experimental artifacts. Therefore, there is a need to investigate the statistical issues related to the quantification of methylation levels for these emerging technologies, with the goal of developing an accurate quantification method.

Methods

In this paper, we propose two methods for Methyl-Seq quantification. The first method, the Maximum Likelihood estimate, is both conceptually intuitive and computationally simple. However, this estimate is biased at extreme methylation levels and does not provide variance estimation. The second method, based on Bayesian hierarchical model, allows variance estimation of methylation levels, and provides a flexible framework to adjust technical bias in the sequencing process.

Results

We compare the previously proposed binary method, the Maximum Likelihood (ML) method, and the Bayesian method. In both simulation and real data analysis of Methyl-Seq data, the Bayesian method offers the most accurate quantification. The ML method is slightly less accurate than the Bayesian method. But both our proposed methods outperform the original binary method in Methyl-Seq. In addition, we applied these quantification methods to simulation data and show that, with sequencing depth above 40–300 (which varies with different tissue samples) per cleavage site, Methyl-Seq offers a comparable quantification consistency as microarrays.     

Nitrogen management in a maize-groundnut crop rotation of humid tropics: effect on N2O emission

Development of appropriate land management techniques to attain sustainability and increase the N use efficiency of crops in the tropics has been gaining momentum. The nitrous oxides (N2Os) affect global climate change and its contribution from N and C management systems is of great significance. Thus, N transformations and N2O emission during maize-groundnut crop rotation managed with various N sources were studied. Accumulation of nitrate (NO3- ) and its disappearance happened immediately after addition of various N sources, showing liming effect. The mineral N retained for 2-4 weeks depending on the type and amount of N application. The chicken manure showed rapid nitrification in the first week after application during the fallow period, leading to a maximum N2O flux of 9889 g N2O-N m(-2) day(-1). The same plots showed a residual effect by emitting the highest N2O (4053 microg N2O-N m(-2) day(-1)) during maize cultivation supplied with a half-rate of N fertilizer. Application of N fertilizer only or in combination with crop residues exhibited either lowered fluxes or caused a sink during the groundnut and fallow periods due to small availability of substrates and/or low water-filled pore space (<40%). The annual N2O emission ranged from 1.41 to 3.94 kg N2O-N ha(-1); the highest was estimated from the chicken manure plus crop residues and half-rate of inorganic N-amended plots. Results indicates a greater influence of chicken manure on the N transformations and thereby N2O emission.     

Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis

Background

Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.

Methodology/Principal Findings

We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs ∼250-fold for generating 10⁶ ligand sequences.

Conclusions/Significance

Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.     

A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

Background

Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.

Principal Findings

We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.

Significance

We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.     

Determination of dextromethorphan and its metabolite dextrorphan in human urine using high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry: a study of selectivity of a tandem mass spectrometric assay

Analytical method for the simultaneous determination of dextromethorphan (1) and dextrorphan (2) in urine, based on solid-phase extraction of drug from acidified hydrolyzed biological matrix, were developed. The analytes (1 and 2) and the internal standard (levallorphan, 3, IS) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in positive ionization mode using a heated nebulizer (HN) probe and monitoring their precursor-->product ion combinations of m/z 272-->215, 258-->201, and 284-->201 for 1, 2, and 3, respectively, in multiple reaction monitoring mode. The analytes and IS were chromatographed on a Keystone Prism reverse phase (50 mm x 2.0 mm) 5 microm column using a mobile phases consisting of a 35/65 or 27/73 mixtures of methanol/water containing 0.1% TFA adjusted to pH 3 with ammonium hydroxide pumped at 0.4 ml/min for 1 and 2, respectively. The limits of reliable quantification of 1 and 2 were 2 and 250 ng/ml, respectively, when 1 ml of urine was processed. The absence of matrix effect was demonstrated by analysis of neat standards and standards spiked into urine extracts originating from five different sources. The linear ranges of the assay were 2-200 and 250-20,000 ng/ml for 1 and 2, respectively. Assay selectivity was evaluated by monitoring the "cross-talk" effects from other metabolites into the MS/MS channels used for monitoring 1, 2, and 3. In addition, an interfering peak originating from an unknown metabolite of 1 into the quantification of dextromethorphan was detected, requiring an effective chromatographic separation of analytes from other metabolites of 1. The need for careful assessment of selectivity of the HPLC-MS/MS assay in the presence of metabolites, and the assessment of matrix effect, are emphasized.     

Pesticide emission modelling and freshwater ecotoxicity assessment for Grapevine LCA: adaptation of PestLCI 2.0 to viticulture

Purpose

Consumption of high quantities of pesticides in viticulture emphasizes the importance of including pesticide emissions and impacts hereof in viticulture LCAs. This paper addresses the lack of inventory models and characterization factors suited for the quantification of emissions and ecotoxicological impacts of pesticides applied to viticulture. The paper presents (i) a tailored version of PestLCI 2.0, (ii) corresponding characterization factors for freshwater ecotoxicity characterization and (iii) result comparison with other inventory approaches. The purpose of this paper is hence to present a viticulture customized version of PestLCI 2.0 and illustrate the application of this customized version on a viticulture case study.

Methods

The customization of the PestLCI 2.0 model for viticulture includes (i) addition of 29 pesticide active ingredients commonly used in vineyards, (ii) addition of 9 viticulture type specific spraying equipment and accounting the number of rows treated in one pass, and (iii) accounting for mixed canopy (vine/cover crop) pesticide interception. Applying USEtox?, the PestLCI 2.0 customization is further supported by the calculation of freshwater ecotoxicity characterization factors for active ingredients relevant for viticulture. Case studies on three different vineyard technical management routes illustrate the application of the inventory model. The inventory and freshwater ecotoxicity results are compared to two existing simplified emission modelling approaches.

Results and discussion

The assessment results show considerably different emission fractions, quantities emitted and freshwater ecotoxicity impacts between the different active ingredient applications. Three out of 21 active ingredients dominate the overall freshwater ecotoxicity: Aclonifen, Fluopicolide and Cymoxanil. The comparison with two simplified emission modelling approaches, considering field soil and air as part of the ecosphere, shows that PestLCI 2.0 yields considerable lower emissions and, consequently, lower freshwater ecotoxicity. The sensitivity analyses reveal the importance of soil and climate characteristics, canopies (vine and cover crop) development and sprayer type on the emission results. These parameters should therefore be obtained with site-specific data, while literature or generic data that are acceptable inputs for parameters whose uncertainties have less influence on the result.

Conclusions

Important specificities of viticulture have been added to the state-of-the-art inventory model PestLCI 2.0. They cover vertically trained vineyards, the most common vineyard training form; they are relevant for other perennial or bush crops provided equipment, shape of the canopy and pesticide active ingredients stay in the range of available options. A similar and compatible model is needed for inorganic pesticide active ingredients emission quantification, especially for organic viticulture impacts accounting.

     

A study of Pseudomonas fluorescens biovars using the Automated Microbiology Identification System (AMBIS)

The AMBIS is a system which can determine the relationships between microbial strains by comparing the profiles produced after their radiolabelled intracellular proteins are subjected to SDS PAGE. This system was used to compare the profiles of strains representing the five biovars of Pseudomonas fluorescens , a species implicated in food spoilage. The three strains of biovar I, three strains of biovar III and two strains of biovar IV segregated into three distinct clusters with correlation coefficients (cc) of 0·85, 0·85 and 0·87 respectively. Although two of the biovar II strains studied clustered together (cc = 0·74) one of the remaining biovar II strains linked (cc = 0·83) with the cluster of biovar IV strains while the other was linked with biovar I and V strains (cc = 0·68). Biovar V strains (three in total) also failed to form a single cluster which was expected since this biovar is known to be heterogeneous. The findings are in substantial agreement with more comprehensive taxonomic studies of this species. AMBIS may be a useful tool in taxonomic studies of micro-organisms.     

Enzyme-linked bridging assay method for the quantification of oligonucleotide-based drugs in biological matrices

With ongoing efforts to develop oligonucleotide-based (ODN-based) therapeutics, there is a need for a sensitive, high-throughput method of quantification of ODN-based drugs in biological matrices. To overcome the insufficient sensitivity and time-consuming sample extraction procedures involved in conventional capillary gel electrophoresis (CGE) and high-performance liquid chromatography (HPLC), we developed a nucleic acid hybridization-based enzyme-linked bridging assay (ELBA), which shows significant advantages over CGE methods in evaluating ODN-based drugs in plasma and tissue: (1) It has higher sensitivity; (2) it involves easier sample extraction procedures; (3) it is suitable for many ODN-based drugs, even those with different secondary structures and modifications, including phosphorothioate oligonucleotide (PSODN), mixed backbones with 2'-O-Me (MBO), locked nucleic acid (LNA) modifications, and B- and C-type CpG sequences; and (4) it is highly selective, even during simultaneous quantification, with regard to intact ODNs and their 3'-metabolites. This universal design produces a rapid, sensitive, specific assay with minimal method development time. It is well suited to high-throughput analysis of various ODN-based drugs.     

2001-2009年中国碳排放与碳足迹时空格局

碳排放引发的全球变暖给自然环境及人类社会都带来了显著影响,而碳足迹可以衡量自然生态系统对人类活动碳排放的响应。为研究自然-社会二元系统碳动态,基于MODIS(Moderate Resolution Imaging Spectroradiometer)数据和统计资料计算2001—2009年中国陆地植被净初级生产力、能源消费碳排放、碳足迹和碳赤字;在GIS(Geographic Information System)技术支持下,运用空间自相关分析方法讨论其时空格局;据此划分生态经济区。结果表明:(1)2001—2009年全国植被净初级生产力(Net Primary Production,NPP)平均值为3.32 Pg C/a(1 Pg=1015g),呈西南地区东南沿海华中、华东地区东北、华北地区西北地区的空间格局;(2)2001—2009年全国能源消费碳排放逐年增加,年均增长率16.7%,多年平均值2.53 Pg C/a,呈东部中部西部的空间格局;(3)2001—2009年全国碳足迹逐年增加,年均增长率14.7%,多年平均值6.98×106km2;具有正碳赤字(即碳源)的省份为山西、环渤海地区各省、长三角地区各省、广东;相邻省份碳赤字的相对大小由于互相影响而改变;(4)全国分为中东部、南部、北部、西部四个生态经济大区。研究结果直观揭示了中国碳排放和碳足迹的时空动态,为实现自然-社会二元系统的可持续发展提供科学依据。     

Rapid analysis for metabolites of C-labelled drugs: fate of [C]-S-4-(tert.-butylamino-2-hydroxypropoxy)-benzimidazol-2-one in the dog

Positron emission tomography (PET) requires the use of compounds labelled with short-lived, positron-emitting isotopes (e.g., t_1/2 of ¹¹C 120 min). As the concentration of unbound, non-metabolised drug is required as the input function for modeling, this presents particular problems for the study of the kinetics and metabolism of such compounds. We have now developed a rapid extraction procedure, followed by high-performance liquid chromatography using a short analytical column coupled to an on-line γ-detector to determine the metabolism and kinetics of a non-selective β-adrenergic antagonist of high affinity, S-4-(tert.-butylamino-2-hydroxypropoxy)benzimidazol-2-one. This antagonist is potentially well suited to the non-invasive localisation of β-receptors in vivo. The ligand was rapidly taken up into the β-receptor pool or excreted in urine, with less than 5% of the drug converted to metabolites. Plasma protein binding was only 16%. No significant metabolism of the ligand was observed in the anaesthetised dog, and, therefore, no correction for blood metabolite concentration is required for kinetic analysis of the ¹¹C-labelled ligand during PET studies in this species. The analytical method reported here should be widely applicable: quantification of metabolites enables accurate estimation of the input function and is critical to the interpretation of PET data.     

Bioelectrochemical immunoassay for human chorionic gonadotrophin in serum using an electrode-immobilised capture antibody

An amperometric technique for the quantification of an enzyme immunoassay which utilises a capture antibody covalently attached to a carbon electrode is described. The electrode is used both to separate the assay and to monitor the activity of the bound enzyme label. A ‘two-site’ immunometric assay with monoclonal antibodies directed against human chorionic gonadotrophin (HCG) was used as the model system. The activity of the enzyme bound to the electrode is determined electrochemically by the use of an electron transfer mediator (dimethylaminomethyl ferrocene) permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the assay is 9mIU HCG ml⁻¹ in serum (1st International Reference Preparation). The correlation between the amperometric measurement of serum HCG and data for an immunoradiometric assay was r = 0·988. The assay is rapid requiring a total assay time of 20 min per sample, which includes 15 min for antibody—antigen binding.     

Validated method for rapid inhibition screening of six cytochrome P450 enzymes by liquid chromatography-tandem mass spectrometry

In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytochrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis-Menten constant (K(m)) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0-2000 ng/ml for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng/ml. The limit of detection (LOD) for the tested analytes was 0.48 ng/ml or better based on signal-to-noise ratio >3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC(50) values) measured by this method demonstrated high precision and are consistent with the literature values.     

Life cycle inventory practices for major nitrogen,phosphorus and carbon flows in wastewater and sludge management systems

Purpose

Nitrogen, phosphorus and carbon originating from wastewater and sludge can, depending on their partitioning during wastewater treatment, either become available as potential resources or leave as emissions. Several reviews have highlighted the dependence of life cycle assessment (LCA) results on the inventory data. To provide a foundation for future assessments of systems in which resources are utilised from wastewater or sludge, this paper identifies common practice and highlights deficiencies in the selection and quantification of nitrogen, phosphorus and carbon containing flows.

Methods

Inventories of major direct flows containing nitrogen, phosphorus and carbon in 62 studies on wastewater and sludge management operations have been reviewed. A special focus was put on flows of nitrogen, phosphorus and carbon originating from the wastewater and sludge and on how these are either leaving the system as emissions and hereby contributing to environmental impacts, or how potential resource flows of these elements are accounted for, in particular when sludge is used in agriculture.

Results and discussion

The current study shows a large variation between studies regarding what resource and emission flows were included in inventories on wastewater and sludge treatment, the type of data used (primary or secondary data) and, when flows have been modelled rather than measured, how the modelling has been done. Except for nitrogen and phosphorus emissions via the effluent, which were generally quantified using measured data or data modelled to represent the specific situation, direct emissions to air from the water and sludge lines at the wastewater treatment plant were mostly estimated using secondary data, sometimes of poor data quality. In systems where resources were recovered through agricultural application of sludge, studies often credited the system for avoided use of mineral fertiliser, but the considered replacement ratio differed.

Conclusions

The current review identified increased completeness and specificity in the modelling of the evaluated flows as particularly relevant for future studies and highlighted a need for improved transparency of data inventories. The review can be used as a support for LCA analysts in future studies, providing an inventory of common practices and pinpointing deficiencies, and can thereby support more conscious and well-motivated choices as regard which flows to include in assessments and on the quantification of these flows.

     

Rate of acclimation of the capacity for isoprene emission in response to light and temperature

Isoprene emission from plants accounts for nearly half of all non‐methane hydrocarbons entering the atmosphere. Light and temperature regulate the instantaneous rate of isoprene emission but there is increasing evidence that they also affect the capacity for isoprene emission (i.e. the rate measured under standard conditions). We tested the rate of acclimation of the capacity for isoprene emission following step changes in growth conditions. Acclimation to new growth temperatures was very rapid, with most of the change occurring within a few hours and complete adjustment occurring within a day. Acclimation to new light levels was more complicated. Following a switch from low‐light growth conditions to standard assay conditions (30 °C and 1000 µmol photons m^?2 s^?1), there was a rapid (5–10 min) and a slightly slower (10–50 min) acclimation of the capacity for isoprene emission. After accounting for these short‐term changes, there was also a small, long‐term (4–6 d) acclimation of the isoprene emission capacity to the light level of growth conditions. We found no effect of growth conditions on the coefficients used to describe the instantaneous light and temperature response of isoprene emission. Therefore, current models of isoprene emission will only need to be altered to account for changes in the capacity for isoprene emission.     

Gluconeogenesis in the kidney cortex. Flow of malate between compartments

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.