Small-angle X-ray scattering (SAXS) patterns from breast tissue samples are compared with their histology. Formalin fixed human breast tissue specimens containing ductal and lobular carcinoma were studied. Histo-pathological information is compared with the scattering data, and there is a clear spatial correlation. Supra-molecular organisation of collagen fibrils is modelled and the model is used to create scattering maps. The model parameters include the axial periodicity (d-spacing), radius and packing of the fibrils, and these are derived from comparison with the experimental scattering patterns. The d-spacing is to 0.5% larger in malignant zones of the tumours than in the healthy zones. There are also characteristic differences in the fibril diameter and packing.

M. Fernández

J. Keyriläinen

M.-L. Karjalainen-Lindsberg

M. Leidenius

K. von Smitten

S. Fiedler

P. Suortti

Human breast tissue characterisation with small-angle X-ray scattering

Parvez I. Haris

Second International Conference on Biomedical Spectroscopy&#x3a; From the Bench to the Clinic

The application of high pressure in bioscience and biotechnology has become an intriguing field in un/refolding and misfolding processes of proteins. NMR spectroscopy is the only generally applicable method to monitor pressure–induced structural changes at the atomic level in solution. Up to now the application of most of the multidimensional NMR experiments is impossible due to the restricted volume of the high pressure glass cells which causes a poor signal–to–noise ratio. Here we present high strength single crystal sapphire cells which double the signal–to–noise ratio. This increased signal–to–noise ratio is necessary to perform, for example, phophorus NMR spectroscopy under variable pressures. To understand the effect of pressure on proteins, we need to know the pressure dependence of 1H chemical shifts in random coil model tetrapeptides. The results allow distinguishing structural changes from the pressure dependence of the chemical shifts. In addition, the influence of pressure on the buffer system was investigated. Since high pressure was shown to populate intermediate amyloidogenic states of proteins the investigation of pressure effects on proteins involved in protein conformational disorders like Alzheimer's Disease (AD) and Transmissible Spongiform Encephalopathies (TSE) is of keen interest. 1H–15N–TROSY–spectra were acquired to study the effects of pressure and temperature on chemical shifts and signal volumes of the human prion protein. These measurements show identical pressure sensitivity of huPrP(23–230) and huPrP(121–230). First results suggest a folding intermediate for the human prion protein which can be populated by high hydrostatic pressure.

W. Kremer

M. R. Arnold

N. Kachel

H. R. Kalbitzer

The use of high–sensitivity sapphire cells in high pressure NMR spectroscopy and its application to proteins

There are few but important problems in magnetic resonance (MR) diagnosis of the brain tumours such as predicting the grade, exact definition of the tumour borders, differentiation of the cystic tumours from abscess, the tumoral core from peritumoral oedema, and the tumour recurrence from radiation necrosis. MR spectroscopy (MRS) can add more information to MR imaging (MRI) in solving many of these problems. Widespread usage of faster MRS applications with higher signal‒to‒noise ratio (SNR) and spatial resolution, allows us to detect functional metabolic changes, which provides more data to understand the exact nature of the tumour and the morphological and physiological changes occurring in the surrounding brain parenchyma.

Nail Bulakbasi

Clinical applications of proton MR spectroscopy in the diagnosis of brain tumours

Investigators using mono channel near infrared spectroscopy (NIRS) have reported different patterns of change in cytochrome c oxidase (Cyt) in similar studies of tissue ischaemia. We investigated whether there were distinctive differences in NIRS signals obtained simultaneously from different sampling sites during the same imposed physiological intervention within the same subject. Methods: Subjects were 36, healthy, 10 kg, commercial swine undergoing cardiopulmonary bypass to initiate 3 to 7 periods of 7.5 minutes of circulatory arrest. Each arrest was initiated at one of 81 combinations of high, normal, or low levels of core temperature, haematocrit, pH, and serum glucose. Each combination was repeated twice, yielding 162 NIRS data sets. Results: Six distinct patterns of change of Cyt were found. Typically, brain Cyt quickly became reduced shortly after the start of arrest, muscle Cyt did not start becoming reduced until after 3½ minutes of arrest, and spinal cord Cyt either did not change status or became gradually reduced throughout the period of arrest. The brain response may reflect strong oxygen dependence, while the muscle response may indicate a dependency buffered by myoglobin stores, and the spine response may indicate a low concentration of available Cyt that is too diffuse to be rapidly influenced by changes in oxygen availability. Conclusion: Multi‒channel NIRS is needed for systemic evaluation of respiration at the cellular level in clinical settings. Distinctive Cyt patterns of change occur in different organs at the same time, in response to circulatory arrest.

Roy E. Gagnon

Andrew J. Macnab

Jacques G. LeBlanc

Patterns of change in cytochrome c oxidase redox status

Mass spectrometry (MS) techniques have spear‒headed the field of proteomics. Recently, MS has been used to structurally analyse carbohydrates. The heparin/heparan sulfate‒like glycosaminoglycans (HLGAGs) present a special set of difficulties for structural analysis because they are highly sulfated and heterogeneous. We have used a matrix‒assisted laser desorption/ionization time of flight mass spectrometry (MALDI‒MS) technique in which heparin fragments are non‒covalently bound to basic peptides of a known mass, so as to limit in‒source desulfation and hence afford an accurate mass. We examined a range of different sized fragments with varying degrees of sulfation. The potential of combining the MALDI‒MS technique with enzymatic digestion to obtain saccharide sequence information on heparin fragments was explored. A disaccharide analysis greatly assists in determining a sequence from MALDI‒MS data. Enzymatic digestion followed by MALDI‒MS allows structural data on heparin fragments too large for direct MALDI‒MS to be obtained. We demonstrate that synthetic sulfated oligosaccharides can also be analysed by MALDI‒MS. There are advantages and limitations with this methodology, but until superior MS techniques become readily accessible to biomedical scientists the MALDI‒MS method provides a means to structurally analyse HLGAG fragments that have therapeutic potential because of their ability to bind to and functionally regulate a host of clinically important proteins.

Warren C. Kett

Deirdre R. Coombe

A structural analysis of heparin‒like glycosaminoglycans using MALDI‒TOF mass spectrometry

1H NMR spectroscopy has previously been employed to detect and monitor changes in the lipid metabolism of neuroblastoma cells upon cytotoxic treatment. Here, we addressed the question whether altered growth conditions, by presence or absence of serum, would impact on the metabolites detectable with 1H NMR spectroscopy. Chronic serum deprivation of SH‒SY5Y human neuroblastoma cells resulted in a decrease in the intracellular content of several metabolites, in particular total choline. This metabolic effect was paralleled by significant growth inhibition. In addition, we investigated the potential functional origin of intracellular 1H NMR visible lipids in SH‒SY5Y cells. A drop in lipid methylene protons could be observed shortly after serum‒withdrawal. Contrary, removal of lipoproteins from the serum led to a pronounced increase in intracellular lipids, as did inhibition of de novo sterol synthesis by lovastatin. In conclusion, we demonstrate that intracellular total choline in neuroblastoma cells in vitro is highly dependent on the availability of growth factors. Furthermore, we show that 1H NMR visible lipids decrease upon serum‒withdrawal but are accumulated when cholesterol supply is abrogated. The biological and potential clinical implications of these findings are discussed.

Magnus Lindskog

Jüri Jarvet

Astrid Graslund

Per Kogner

Monitoring intracellular metabolites in neuroblastoma with 1H NMR spectroscopy: effects of growth factor withdrawal and modulation of lipid metabolism

Vibrational spectroscopy is an important tool in mineralized tissue research. This review focuses on recent applications of Raman and IR spectroscopies as contrast enhancement agents, tools for studying the chemical nature of changes in bone tissue with age and disease and understanding the molecular nature of changes in bone tissue during mechanical loading and fracture.

Michael D. Morris

William F. Finney

Recent developments in Raman and infrared spectroscopy and imaging of bone tissue

The use of dimethylsulfoxide (DMSO) as a cryoprotectant to reduce cellular injury during freezing is well known, however the intermolecular interactions between this amphiphilic molecule and biological membranes that form the basis of this protection are unknown. DMSO&#x2013;dipalmitoylphosphatidylcholine (DPPC) vesicle interactions were investigated in pulsed-field gradient NMR (PFGNMR) experiments and spectra analysis allowed for the determination of self-diffusion coefficients for each species present. The mole fraction of DMSO associated with the DPPC vesicles was then calculated from the diffusion coefficients&#x3a; the mole fraction increased from 14&#x25; to 42&#x25; as the membrane was heated from below to above the main phase transition temperature.

Anthony Kennedy

Carla J. Long

Peter J. Hmel

Rickey Hicks

Thomas J. Reid

The association of dimethylsulfoxide and model membranes studied by pulse-field gradient NMR

The change in the secondary and tertiary structure of bovine serum albumin (BSA) induced by the interaction with spherical polyelectrolyte brushes (SPB) has been investigated using fluorescence and circular dichroism (CD) spectroscopy. The SPB consist of poly(acrylic acid) chains grafted to a poly(styrene) core. The colloidal SPB represent a new substrate for protein immobilization because their protein binding capacity can be controlled by the ionic strength of the solution: SPB bind large amounts of BSA at low ionic strength (pH=6.1), but they are largely protein resistant at moderate salt concentrations of 500 mM. The conformation of BSA which was labeled with the environmentally sensitive dansyl fluorophore was studied before adsorption to the SPB, in the adsorbed state, and after desorption from the SPB. In the adsorbed state the obtained fluorescence spectrum is red-shifted which indicates a hydration of the dansyl fluorophores due to a distortion of the tertiary structure of BSA. Fluorescence and CD spectroscopic analysis of BSA that was desorbed from the SPB shows that the adsorption-induced conformational changes are largely reversible. Convex constraint analysis of the observed CD spectra of BSA yield α-helix fractions of 68% and 57% before adsorption to and after desorption from the SPB, respectively. In a general view, the results of this study demonstrate that spherical polyelectrolyte brushes are suitable for a controlled immobilization and release of proteins without major conformational changes.

G. Jackler

A. Wittemann

M. Ballauff

C. Czeslik

Spherical polyelectrolyte brushes as carrier particles for proteins: An investigation of the structure of adsorbed and desorbed bovine serum albumin

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Journal of Spectroscopy publishes research into the theory and application of spectroscopy across all disciplines, including biology, chemistry, engineering, earth sciences, medicine, materials science, physics, and space science.

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Chief Editor&nbsp;Dr Daniel Cozzolino&nbsp;is based at the University of Queensland, Australia. His research focuses on the developments of chemometric and spectroscopic methods for use in agriculture and food applications.

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Journal of Spectroscopy publishes research into the theory and application of spectroscopy across all disciplines, including biology, chemistry, engineering, earth sciences, medicine, materials science, physics, and space science.

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