Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10¹³ photons · cm^?2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10¹³–10¹⁶ photons · cm^?2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Modelling the acid/base <Superscript>1</Superscript>H NMR chemical shift limits of metabolites in human urine

Introduction

Despite the use of buffering agents the ¹H NMR spectra of biofluid samples in metabolic profiling investigations typically suffer from extensive peak frequency shifting between spectra. These chemical shift changes are mainly due to differences in pH and divalent metal ion concentrations between the samples. This frequency shifting results in a correspondence problem: it can be hard to register the same peak as belonging to the same molecule across multiple samples. The problem is especially acute for urine, which can have a wide range of ionic concentrations between different samples.

Objectives

To investigate the acid, base and metal ion dependent ¹H NMR chemical shift variations and limits of the main metabolites in a complex biological mixture.

Methods

Urine samples from five different individuals were collected and pooled, and pre-treated with Chelex-100 ion exchange resin. Urine samples were either treated with either HCl or NaOH, or were supplemented with various concentrations of CaCl₂, MgCl₂, NaCl or KCl, and their ¹H NMR spectra were acquired.

Results

Nonlinear fitting was used to derive acid dissociation constants and acid and base chemical shift limits for peaks from 33 identified metabolites. Peak pH titration curves for a further 65 unidentified peaks were also obtained for future reference. Furthermore, the peak variations induced by the main metal ions present in urine, Na⁺, K⁺, Ca²⁺ and Mg²⁺, were also measured.

Conclusion

These data will be a valuable resource for ¹H NMR metabolite profiling experiments and for the development of automated metabolite alignment and identification algorithms for ¹H NMR spectra.

     

A software complement to AMDIS for processing GC-MS metabolomic data

The software package AMDIS performs gas chromatography–mass spectrometry (GC–MS) peak deconvolution but tends to produce false positives and leaves missing values where peaks are found in only a proportion of a set of chromatograms. We have developed a software complement to AMDIS that (i) allows rapid manual inspection of chromatographic peaks across all samples to confirm data quality and (ii) for a given sample set, integrates peak areas across all samples even where AMDIS deconvolution would leave missing values. The freely available package runs within the commercial Matlab environment and is useful where GC–MS is used to profile complex mixtures.     

NMR confirmation of an alkali-insoluble glucan from Kluyveromyces marxianus cultivated on a lactose-based medium

Yeast cells of Kluyveromyces marxianus grown in a sterile lactose-based medium, were extracted with sodium hydroxide to give a fraction of alkali-insoluble yeast (AIY) glucan. From ¹H- and ¹³C-NMR characterization it was concluded that the glucan consisted mostly of (1 3) linkages and was similar to that obtained from Saccharomyces cerevisiae used in the preparations for skin revitalization and repair. The molecular weight of the glucan was determined to be approximately 3 × 10⁵ Da. Therefore, K. marxianus grown on a lactose-based medium similar to whey, a waste stream of the dairy industry, could be a commercial source of (1 3) glucans for use in the food and pharmaceutical industries.     

Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier

Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7.The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods. The yield of purified bioemulsifier from this strain of K. marxianus was 4–7% of the original dry cell weight. The purified product, at a concentration of 12 g l^–1, formed emulsions that were stable for 3 months when subjected to a range of pH (3–11) and NaCl concentrations (2–50 g l^–1). The composition of this mannoprotein was 90% carbohydrate (mannan) and 4–6% protein. These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source. Consequently K. marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.     

Translational Arrest Due to Cytoplasmic Redox Stress Delays Adaptation to Growth on Methanol and Heterologous Protein Expression in a Typical Fed-Batch Culture of Pichia pastoris

Results

We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation.

Conclusion

Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Intra-Operative Analysis of Scoliosis Surgery in 3-D

Scoliosis is a three-dimensional deformity characterized by coronal, sagittal and axial rotation of the spine. Surgical fusion of the spine is required in severe cases. Assessment of the surgical procedure requires enough accuracy and flexibility to allow planning of individual interventions or implant designs. Conventional 2-D radiography and even 3-D CT scanning have limitations for in-depth analysis of scoliosis that limit the ability to see the three-dimensional deformity and expose the patient to considerable doses of radiation, respectively. Our stereophotogrammetric analysis is able to provide accurate, intra-operative measurement of vertebral movement during surgical manuevres. Stereophoto pairs taken at each stage of the operation and robust statistical techniques can be used to determine rotation, translation, goodness of fit, and overall spinal contour before, during, and after the surgical instrumentation. A demonstration of data available from this system is included.