bluuebunny/crossref_metadata_2025 huggingface dataset:
The Use of the Time‐Resolved X‐Ray Solution Scattering for Studies of Globular Proteins
Authors: Kuwajima Kunihiro , Arai Munehito , Inobe Tomonao , Ito Kazuki , Nakao Masaharu , Maki Kosuke , Kamagata Kiyoto , Kihara Hiroshi , Amemiya Yoshiyuki
Published: March 2012

In order to improve the low signal‐to‐noise ratio of the time‐resolved small‐angle X‐ray scattering, we have used a two‐dimensional X‐ray detector with a beryllium‐windowed X‐ray image intensifier and a charge‐coupled device as an image sensor, and applied this to studies on (1) the kinetic folding reaction of α‐lactalbumin, which accumulates the molten globule‐like intermediate at an early stage of refolding and (2) the cooperative conformational transition of Escherichia coli chaperonin GroEL induced by ATP, which occurs in an allosteric manner between the close and open conformational states. In the α‐lactalbumin reaction, we have firmly established the equivalence between the kinetic intermediate and the equilibrium molten globule state, and obtained further information about dehydration from the highly hydrated folding intermediate during a late stage of refolding. In the chaperonin study, we have successfully observed the kinetics of the allosteric transition of GroEL that occurs with a rate constant of about 3–4 s−1 at 5°C. The combination of the time‐resolved X‐ray scattering with other spectroscopic techniques such as circular dichroism and intrinsic fluorescence is thus very effective in understanding the conformational transitions of proteins and protein complexes.
An alternative method for rapid quantification of protein secondary structure from FTIR spectra using neural networks
Authors: Hering Joachim A. , Innocent Peter R. , Haris Parvez I.
Published: March 2012

Lack of reliable methods for accurate estimation of protein secondary structure from infrared spectra of proteins is a major barrier in its widespread use in protein secondary structure characterisation. Here we report a method for protein secondary structure estimation, from FTIR spectra of proteins, based on a multi‒layer feed‒forward neural network approach using an enhanced “resilient backpropagation” learning algorithm. The method utilises a database consisting of infrared spectra of 18 proteins, with known X‒ray structure, as the reference set. Our study revealed that providing the neural network analysis with only part of the amide I region from empirically determined structure sensitive regions in combination with appropriate pre‒processing of the spectral data produced the best overall results. This lead to a standard error of prediction (SEP) of 4.47% for α‒helix, an SEP of 6.16% for β‒sheet, and an SEP of 4.61% for turns. Compared to a previous factor analysis study by Lee et al., using the same set of 18 FTIR spectra of proteins, the error in prediction of α‒helix and β‒sheet was improved by 3.33% and 3.54% respectively, with minor increase for turns by 0.31%. Generally, our neural network analysis achieved comparable, in most cases even better prediction accuracy than most of the alternative pattern recognition based methods that were previously reported indicating the significant potential of this approach.
A bioinformatics approach for mass spectrometry data processing: Applications to proteomics and small molecule analysis
Authors: Sonderegger Martin , Staniszewski Kristin , Meyers Andrew , Siuzdak Gary
Published: March 2012

We have developed a web‒based software system, JULIAN, that simplifies the process of relaying mass spectral information for chemists, protein chemists, biochemists and all others performing mass spectrometry experiments through a centralized mass spectrometry laboratory. JULIAN allows for relative ease in submitting compound information as well as instant access to analysis results from any networked computer equipped with a web browser. Compound information is centralized in a Microsoft Access database and results are available in Adobe′s portable document format (PDF) from an NT4 server. This gives researchers the ability to easily obtain data and allows the analysts in the mass spectrometry lab to browse analysis results when assisting researchers with their inquiries. Due to this web‒based design JULIAN is independent of the mass spectrometers′ hardware and operating system. Approximately seven hundred on‒site and off‒site users have utilized JULIAN transmitting over 40,000 analyses. The conversion from paper to electronic mass spectrometry data processing has enabled our Center to receive compound information, perform analysis, and relay the results four times faster than required previously.
Phosphoinositide metabolism, lithium and manic depressive illness
Authors: Yildiz Aysegul
Published: March 2012

Physiology underlying manic depressive illness and treating effects of its most commonly used remedy – “lithium” is yet to be elucidated. Recent years of psychopharmacology research witnessed sparkling developments in our understanding of the mechanisms underlying lithium’s mood stabilizing effects. Recent data on molecular biology and in vivo magnetic resonance spectroscopy suggest that some of the initial actions of lithium may occur through the inhibition of the enzyme inositol monophosphatase (IMPase) and reduction of myo–inositol, which in turn initiate a cascade of events at different levels of signal transduction process and gene expression in brain; such as the effects on protein kinase C, myristoylated alenine rich C kinase substrate protein, glycogen synthase kinase 3β, B cell lymphoma–2 protein, and activator protein–I. It is likely that the enzyme IMPase other that being the key point in initiating lithium’s therapeutic effects, may also play a critical role in the physiology underlying manic depressive illness.
Physical aspects of the interaction of pyrimidine and purine bases with proteins
Authors: Sulkowska A.
Published: March 2012

The study of complexation of purine and pyrimidine derivatives with two kind of serum albumin using 1H NMR and spectrofluorescence technique are performed in order to obtain model interactions nucleic acid bases–protein.

The order of binding strength is as follows: 5mC > 5mU(T) > 3mU > U for pyrimidines and N6mAde > N6Ado > 1mAde > ATP > Ado > Ade for adenine derivatives.

The effect of temperature on the hydrophobic interaction between the nitrogen bases and protein has been studied. The destabilization of protein structure causes loss of tertiary structural contacts, indicating that protein conformation is crucial for binding.
Near–infrared spectroscopy for objectifying cerebral effects of needle and laserneedle acupuncture
Authors: Litscher Gerhard , Schikora Detlef
Published: March 2012

Near infrared spectroscopy (NIRS) has been successfully used in this study to objectify cerebral alterations in oxyhemoglobin and desoxyhemoglobin, due to manual needle acupuncture and laserneedle acupuncture, in 88 healthy volunteers mean age 25.7±4.0 (
±SD) years (19–38 years). Results from Traditional Chinese Acupuncture, Korean and Chinese hand acupuncture, ear acupuncture, combinations of the different acupuncture methods and placebo needling are presented. NIRS seems to be able to shed some light upon the functioning of the different acupuncture methods.
NIRS Measurement of Peripheral Fractional Oxygen Extraction (FOE) after Cardiopulmonary Bypass
Authors: Yoxall Charles William , Menon Kusum , Macnab Andrew John , Gagnon Roy Everett , LeBlanc Jacques Gerard
Published: March 2012

Objectives: To compare peripheral fractional oxygen extraction (FOE), as measured by near infrared spectroscopy
(NIRS), with conventional indicators of tissue perfusion in haemodynamically stable and unstable children after cardiopulmonary
bypass. Design: Observational study. Setting: Paediatric Intensive Care Unit of a large teaching hospital. Patients: 17 children immediately after cardiopulmonary bypass. Male : female = 9 : 8, median age 7 months (range, newborn to 16 years).
Methods: On admission, children were classified as “stable” or “unstable” based on the haemodynamic support they needed. Peripheral venous oxyhaemoglobin saturation (SvO2) was measured non‐invasively using NIRS with venous occlusion. FOE was calculated from SvO2 and arterial saturation measured by pulse oximetry. Repeated measurements of peripheral SvO2 were made for up to 8 hours. In 5 children who had pulmonary artery catheters, simultaneous mixed SvO2 measurements were recorded. Results: Median FOE was 7.9% higher in the unstable group than in the stable group (p = 0.013). Peripheral SvO2 and mixed SvO2 were correlated (R2 = 0.65, p < 0.0001). Conclusions: Peripheral FOE is higher in unstable children. Changes in peripheral SvO2 are related to changes in mixed SvO2. These measurements may provide useful information about haemodynamic status in critically ill children. Further evaluation of the technique is warranted.
FT‐Raman spectroscopy study for skin cancer diagnosis
Authors: Nunes Lilian de Oliveira , Martin Aírton Abrahão , Silveira Landulfo , Zampieri Marcelo
Published: March 2012

Early detection of cancer is still a great challenge in clinical oncology. Recently, Raman spectroscopy has been used for skin lesion detection. FT‒Raman spectroscopy is a modern analytical tool and its use for cancer diagnosis will lead to several advantages for the patient as, for example, real time and less invasive diagnosis. The primary objective of this work was to use FT‒Raman spectroscopy to detect spectral changes between benign and malignant (basal cell carcinoma – BCC) skin tissues. Those spectral changes can provide important information about the biochemical alterations between these two types of tissues. We have analyzed by FT‒Raman eight set of samples histopathologically diagnosed as BCC and made a comparison with five set of samples diagnosed as benign tissue. We have found that the main spectral differences between these samples were in the shift region of 1220–1300 cm–1 and 1640–1680 cm–1. The vibration bands in these regions correspond to the amide III and to the amide I vibrations, respectively. Principal components analysis applied over all 13 samples could identify tissue type with 100% of sensitivity and specificity.
Folding mechanism of canine milk lysozyme studied by circular dichroism and fluorescence spectroscopy
Authors: Nakao Masaharu , Arai Munehito , Koshiba Takumi , Nitta Katsutoshi , Kuwajima Kunihiro
Published: March 2012

We have studied the guanidine hydrochloride‒induced equilibrium unfolding and the kinetics of refolding of canine milk lysozyme by circular dichroism and fluorescence spectroscopy. The thermodynamic analysis of the equilibrium unfolding measured by circular dichroism and fluorescence has shown that unfolding is represented by a three‒state mechanism and that the intermediate state of canine milk lysozyme is remarkably more stable than the intermediates observed in other lysozyme and α‐lactalbumin. In the kinetic refolding of this protein, there are at least two kinetic intermediates; a burst=phase intermediate accumulated within the dead time (4 ms) of the measurement and an intermediate that has been observed during the kinetics with a rate constant of 10–20 s–1 after the burst phase. This result is apparently in contrast with those previously observed in the kinetic refolding of α‒lactalbumin and equine lysozyme that show only the burst‒phase intermediate. The relationship between the extraordinarily stable equilibrium molten globule and the kinetic folding intermediates will be discussed.
Unusual Rearrangement of Dihalocyclopropanes
Authors: Jankowski Christopher K. , Laouz Antoun Bou , Lesage Denis , Diaz T. Eduardo
Published: March 2012

Dihalocyclopropanation of the double bond of some olefins, leading to dihalocyclopropanes, offered an opportunity to perform their rearrangement to dihalomethylvinyl with Hiyama type reagents, in presence of cationic system such as Cr2+/Cr3+. The chain elongation of alkenes via the gem‒dihalocyclopropanes produced α,β–unsaturated aldehydes and acids.
Parallel folding pathway of proline‐free staphylococcal nuclease studied by the stopped‐flow double‐jump method
Authors: Kamagata Kiyoto , Kuwajima Kunihiro
Published: March 2012

The folding mechanism of proline‒free staphylococcal nuclease (SNase (pro−)) (P11A, P31A, P42A, P47T, P56A, P117G) was investigated using the double‒jump stopped‒flow method (interrupted refolding). This method has enabled us to specifically monitor the amount of the native molecules during the refolding. The results indicate that the middle and slow phases observed in the refolding kinetics represent the formation of the native state (IM→N, IS→N) and that the folding mechanism of SNase (pro−) is not represented by a single sequential pathway, but at least two parallel pathways are required for interpreting the results.
LC/MS/MS warfarin assay − An emerging tool for the early detection of cytochrome P450‐associated drug−drug interactions in drug discovery
Authors: Zhang Zhi-Yi
Published: March 2012

The LC/MS/MS warfarin assay, combining stereo‐ and regioselective cytochrome P450 (CYP) form‐specific warfarin hydroxylation with sensitive and specific LC/MS/MS detection technique, is emerging to be a promising tool for the study of CYP‐associated drug−drug interactions for new chemical entities (NCEs) during drug discovery process.
NMR structural analysis of vascular endothelial growth factor in complex with a phage‐derived peptide antagonist
Authors: Pan Borlan , Fairbrother Wayne J.
Published: March 2012

Vascular endothelial growth factor (VEGF) is a covalently linked homodimeric protein that functions as an endothelial cell‐specific mitogen, and is an important mediator of pathological angiogenesis. Phage display has been used to select three different classes of novel disulfide‐constrained peptides that bind to VEGF and disrupt receptor binding with IC50 values between 0.2–10 µM. Mapping of peptide induced nuclear magnetic resonance (NMR) chemical shift changes shows that they target a region of the VEGF receptor‐binding domain that overlaps with the contact surfaces of the receptors, Flt‐1 and KDR. The structure of one of these 28‐kDa VEGF/peptide complexes was determined by NMR spectroscopy. The structure is based on a total of 4416 internuclear distance and dihedral angle restraints derived from data obtained using samples of the complex containing either 13C/15N‐labeled peptide or protein. Incorporation of residual dipolar coupling restraints improved both the precision and accuracy of the structure (as judged by comparison with crystal structures of VEGF). Comparison with the structure of a different VEGF/peptide complex reveals different peptide binding modes that each resemble those of natural protein ligands (an anti‐VEGF antibody and the VEGF‐receptor Flt‐1). Prospects for the development of small‐molecule antagonists of VEGF, based on the VEGF‐bound peptide structures, are discussed.
Raman Spectroscopy of Regulatory Protein Omega from Streptococcus pyogenes Plasmid pSM19035 and Complexes with Operator DNA
Authors: Dostál Lubomίr , Misselwitz Rolf , Laettig Stefan , Alonso Juan C. , Welfle Heinz
Published: March 2012

pSM19035‐encoded homodimeric ω protein (ω2) regulates transcription of genes required for control of plasmid copy number and stable inheritance. ω2 belongs to the MetJ/Arc structural superfamily of repressors forming a ribbon‐helix‐helix (RHH) DNA binding motif, and binds specifically to operator regions containing at least two consecutive copies of heptad sequences 5′‐A/TATCACA/T‐3′ in direct or inverted orientation. Solution properties of a double stranded 19 base‐pairs oligonucleotide designed to model an operator DNA binding site of ω2 (top strand 5′‐GCG AATCACA TGTGATT GG‐3′), ω2, and the ω2:19‐bp DNA complex were analysed by Raman spectroscopy. The Raman data indicate a sequence specific induced fit of both interacting macromolecules with ω2 binding to the major groove of the DNA, large perturbations of the DNA attributable to base unstacking, changes in vibrational modes of deoxyribose moieties, and protein‐induced DNA bending. Protein marker bands indicate that α‐helices are preserved, whereas amino acid side chains are largely perturbed, and unordered structures and turns become extensively restructured. Raman difference bands are consistent with interactions of thymine, adenine and cytosine with ω2 side chains. The results suggest that the central TCA/TGA stretch of the heptads might be the main target site for ω2 binding to operator DNA.
Surface plasmon resonance: principles, methods and applications in biomedical sciences
Authors: Englebienne Patrick , Hoonacker Anne Van , Verhas Michel
Published: March 2012

Surface plasmon resonance (SPR) is a phenomenon occuring at metal surfaces (typically gold and silver) when an incident light beam strikes the surface at a particular angle. Depending on the thickness of a molecular layer at the metal surface, the SPR phenomenon results in a graded reduction in intensity of the reflected light. Biomedical applications take advantage of the exquisite sensitivity of SPR to the refractive index of the medium next to the metal surface, which makes it possible to measure accurately the adsorption of molecules on the metal surface and their eventual interactions with specific ligands. The last ten years have seen a tremendous development of SPR use in biomedical applications. The technique is applied not only to the measurement in real‐time of the kinetics of ligand–receptor interactions and to the screening of lead compounds in the pharmaceutical industry, but also to the measurement of DNA hybridization, enzyme–substrate interactions, in polyclonal antibody characterization, epitope mapping, protein conformation studies and label‐free immunoassays. Conventional SPR is applied in specialized biosensing instruments. These instruments use expensive sensor chips of limited reuse capacity and require complex chemistry for ligand or protein immobilization. Our laboratory has successfully applied SPR with colloidal gold particles in buffered solution. This application offers many advantages over conventional SPR. The support is cheap, easily synthesized, and can be coated with various proteins or protein–ligand complexes by charge adsorption. With colloidal gold, the SPR phenomenon can be monitored in any UV‐vis spectrophotometer. For high‒throughput applications, we have adapted the technology in an automated clinical chemistry analyzer. This simple technology finds application in label‐free quantitative immunoassay techniques for proteins and small analytes, in conformational studies with proteins as well as in the real‐time association‐dissociation measurements of receptor–ligand interactions, for high‐throughput screening and lead optimization.
Nuclear magnetic resonance studies of ribonucleic acids
Authors: Ohlenschläger Oliver , Wöhnert Jens , Ramachandran Ramadurai , Sich Christian , Görlach Matthias
Published: March 2012

Ribonucleic acids (RNA) and RNA−protein complexes are essential components of biological information transfer, catalytic processes and are associated with regulatory functions. This broad range of biological functions is paralleled at the conformational level by a large number of non‐canonical structural elements or sequences with non‐standard backbone conformations, e.g., loops, bulges, pseudo‐knots and complex tertiary folds. NMR spectroscopy has evolved to a powerful tool for the determination of ribonucleic acid structures of up to 20 kDa. Uniform or selective stable isotope labelling aids in solving assignment problems arising from the inherently limited chemical shift dispersion and overlap of resonances for larger nucleotide sequences. Recent developments of multi‐dimensional heteronuclear NMR pulse sequences allow e.g., to directly observe the hydrogen bonding pattern of canonical Watson−Crick base pairs as well as of unusual types of base pairs, thereby opening up a fast access to secondary structure screening of RNA. Detailed conformational descriptions are obtained using conventional NOE and J coupling‐derived data, nowadays supplemented by information from residual dipolar couplings. The latter method also provides a new means for the probing of dynamical features of ribonucleic acids.
A frequency Domain Phase/Modulation Technique for Intracellular Multicomponent Fluorescence Analysis: Technical Approach and Pharmacological Applications
Authors: Praus P. , Sureau F. , Kocisova E. , Rosenberg I. , Stepanek J. , Turpin P. Y.
Published: March 2012

A UV confocal laser microspectrofluorimeter prototype has been adapted for fluorescence lifetime measurements by using a frequency‐domain phase/modulation method (modulation frequency 1 to 200 MHz, lifetime resolution: tenth of a ns). This technique enables excited state lifetimes of several fluorescent components to be resolved and determined. Through a global analysis, specific spectral contribution of each species can be monitored with no need to use model spectra. This approach is efficient to distinguish strongly overlapping components (e.g., intracellular multicomponent fluorescence signal) which otherwise cannot easily be discriminated from each other. Experimental set‒up is first described. Application dealing with an antisense oligonucleotide (a synthetic dT15 oligomer analogue containing isopolar, non‐isosteric, phosphonate [3′‐O‐P‐CH2‐O‐5′] internucleotide linkages) bound to a fluorescent label (tetramethylrhodamine dye), in various solutions and interacting with living cells, is then presented. This is of a major interest for antisense and/or antigene strategies which have recently been developed as efficient ways to cure viral and/or malignant diseases. The frequency‐domain phase/modulation technique enabled oligonucleotide stability inside the cells to be checked.
Total body water and extracellular water measurements through in vivo dilution of D2O and bromide as tracers
Authors: Sergi Giuseppe , Bertani Roberta , Calliari Irene , Lupoli Lucia , Berton Adriano , Volpato Stefania , Enzi Giuliano
Published: March 2012

Large interest is into the determination of body fluids because of changes in body water in a wide variety of physiological and pathological conditions. Here methods based on dilution of tracers, are reported, for the extracellular water (ECW) and total body water (TBW) determinations on the same sample. As for ECW, bromide is used as tracer and its concentration is determined by energy dispersive X‐ray spectrometry; while as for TBW, D2O is used as tracer and HOD is determined by FT IR. Both methods represent significant improvements with respect the procedures described in the literature in terms of availability, reproducibility and accuracy.
Tautomerism in 1‐hydroxy‐2‐naphthaldehyde Schiff bases: Calculation of tautomeric isomers using carbon‐13 NMR
Authors: Salman Salman R. , Kamounah Fadhil S.
Published: March 2012

Tautomeric equilibria in intramolecularly hydrogen bonded Schiff bases is studied on the basis of solution 13C NMR chemical shifts. NMR spectroscopic data and comparison with two anils model compound, namely, salicylideneaniline (1A) and naphthalylidinequinoline (2A) were used to represent the enol (A) and the keto (H) forms. The 13C NMR chemical shifts data of the novel series 4A (compounds, 4‐12) were used together with the values 13C chemical shifts data of compounds 1A and 2A to calculate the % keto form. The calculated % keto form of 3A was compared to that calculated for series 4A compounds.
NIRS monitoring of brain and spinal cord — detection of adverse intraoperative events
Authors: Macnab Andrew J. , Gagnon Roy E. , Gagnon Faith A. , LeBlanc Jacques G.
Published: March 2012

Near infrared spectroscopy (NIRS) monitors changes in oxygenated haemoglobin (HbO2), and redox status of cytochromeaa3 (cyt) continuously and non‐invasively in living tissue. We present examples where clinically relevant changes in HbO2 and/or cyt were detected in real time, allowing intervention to avert potentially harmful hypoxic‐ischaemic damage to the brain and/or spinal cord. Brain monitoring: In children undergoing surgery on cardiopulmonary bypass, observations include that: atrial fibrillation (cardiac arrhythmia) lowered cerebral HbO2 concentration; concealed haemorrhage decreased cerebral HbO2 concentration; inadequate level of anaesthetic resulted in spikes of changes in volume with interventions such as suturing; circulatory arrest reduced brain HbO2 and cyt redox status; and bypass pump problems compromised cerebral blood flow. Spinal cord monitoring: In the experimental animal, we observed that NIRS detected ischaemic change immediately following aortic compression, spinal column distraction (instrumentation to separate the vertebrae), and hypoxia. In an infant requiring release of a congenitally tethered spinal cord, we observed that traction on the spinal cord of the infant resulted in decreased total haemoglobin concentration. Summary: NIRS brain monitoring probably represents the “standard of care” during cardiac surgery because adverse events can be detected and quantified. Similarly, spinal cord monitoring could reduce ischaemic spinal cord damage in spinal cord surgery and aortic aneurysm repair.
Rationalisation of the activities of phenolic (vitamin E‐type) antioxidants
Authors: Rhodes Christopher J. , Tran Thuy T. , Denton Philip , Morris Harry
Published: March 2012

Using Transition‐State Theory, experimental rate constants, determined over a range of temperatures, for reactions of vitamin E type antioxidants are analysed in terms of their enthalpies and entropies of activation. It is further shown that computational methods may be employed to calculate enthalpies and entropies, and hence Gibbs Free Energies, for the overall reactions. Within the Linear Free Energy Relationship (LFER) assumption, that the Gibbs Free Energy of activation is proportional to the overall Gibbs Free Energy change for the reaction, it is possible to rationalise, and even to predict, the relative contributions of enthalpy and entropy for reactions of interest, involving potential antioxidants.
Structural studies on the antimicrobial peptide Brevinin 1E by spectroscopic methods
Authors: Son Woo-Sung , Kim Ji-Sun , Kim Hyung-Eun , Park Sang-Ho , Lee Bong-Jin
Published: March 2012

Skin extracts of frogs are a rich source of pharmacologically active peptides such as caeruleins, tachykinins, bradykinins, thyrotropin‐releasing hormone, bombesin‐like and opioid peptides. A large variety of antimicrobial peptides has been isolated from Rana species. These peptides, grouped in several families on the basis of differing length and distinct activity, were found to have one structural motif in common: an intramolecular disulfide bridge located at the C‐terminal end, forming a seven‐member ring, which was designated ‘Rana box’. Brevinin 1E is a 24‐residue antimicrobial peptide isolated from the skin of a frog, Rana brevipoda. This peptide shows a broad range of antimicrobial activity against prokaryotic cells but shows very much hemolytic activity against human red blood cells. The solution structure of Brevinin 1E was studied by using CD (circular dichroism) and NMR (nuclear magnetic resonance) spectroscopy. CD investigation revealed that Brevinin 1E adopts random structure in aqueous solution but adopts mainly α‐helical structure in TFE/water (6 : 4, v/v) solution. The three‐dimensional structure of Brevinin 1E was determined in 60% TFE/water solution using homonuclear NMR spectroscopy. This peptide showed mainly an α‐helical structure with amphipathic property. Its three‐dimensional structure is similar to those of other peptides such as magainin, nigrocin and ranalexin. Therefore, Brevinin 1E can be classified into the family of antimicrobial peptides containing a single linear α‐helix that interact with target microbial membrane, leading to cell death through disruption of membrane integrity.
Cardiac Bypass Pump Flow Management via NIRS Monitoring
Authors: Macnab Andrew J. , Gagnon Roy E. , Gagnon Faith A. , Blackstock Derek , LeBlanc Jacques G.
Published: March 2012

During cardiac surgery, bypass pumps rely on pressure monitors to evaluate flow. We studied whether it would be possible to optimize pump flow by monitoring changes in cerebral cytochrome a,a3 using NIRS to maintain cyt redox status at its pre‐bypass level. Method: 18 healthy 7–45 kg swine were placed on bypass for repeated cycles of cooling and re‐warming from 36 to 15 to 36°C in 3°C steps. Between each cycle, the swine′s bypass pump blood flow rate was adjusted to restore cytochrome redox status to its pre‐bypass value. Results: In all swine trials, the number of pump flow alterations imposed by NIRS monitoring ranged from 0 to 42, the average being 14 per trial. The best trial had 22 pump flow adjustments during which the range of cytochrome redox status change was 0.50±0.06 μmol l–1. The average trial had a range of cytochrome redox status change of 1.50±0.22 μmol l–1. Conclusion: NIRS‐driven alterations in pump flow rate to maintain pre‐bypass cytochrome redox status can be achieved successfully in the animal model.
Study of Thermal Aggregation and Gelation of Oat Globulin by Raman Spectroscopy
Authors: Ma C. Y. , Rout M. K. , Phillips D. L.
Published: March 2012

Thermal aggregation and gelation of oat globulin were studied by FT‐NIR Raman spectroscopy. The buffer‐soluble aggregates exhibited a Raman spectrum similar to that of the unheated control, whereas the insoluble aggregates showed intensity increases in the tryptophan, C–H bending and C–H stretching bands, and a decrease in the tyrosine doublet (I850 /I830), suggesting protein denaturation. However, analysis of the amide I region using Raman Spectral Analysis Package (RASP) program revealed marked decreases in α‐helical and increases in β‐sheet structure in both soluble and insoluble aggregates. Similar conformational changes were also observed in the heat‐induced oat globulin gels, and may be attributed to realignment of molecular segments and formation of intermolecular β‐sheet structures. Thermal gelation under the influence of different chaotropic salts showed some shifts in band positions and changes in band intensity, following the lyotropic series of anions. Several protein structure perturbants, including sodium dodecyl sulfate, dithiothreitol, urea and sodium laurate, were found to affect the Raman spectral characteristics of oat globulin gels. The data suggest that changes in gelling properties of oat globulin by these chemicals may be related to conformational changes of the protein during gelation.
Spectroscopic evaluation of carbohydrate levels in cell line treated with red microalgal polysaccharides
Authors: Erukhimovitch Vitaly , Talyshinsky Marina , Souprun Yelena , Huleihel Mahmoud
Published: March 2012

Sulfated polysaccharides obtained from red microalgae proved to be as potent antiviral agents against various viruses. The huge molecular weight of these polysaccharides contributed much to the difficulty in explaining and understanding the mechanism of their activity against internal steps in the replication cycle of these viruses. In the present study we used microscopic Fourier‐Transform Infrared Spectroscopy (FTIR) to investigate: (a) possible penetration of these polysaccharides into the treated cells, (b) effect of the polysaccharides treatment on some metabolic changes in these cells. Our results show a rapid accumulation of carbohydrates in the treated Vero cells. The carbohydrates level in these cells increases continuously after treatment during the entire experiment time period. In addition, we noted a gradual shift of PO2‐ionized symmetric stretching peak of Vero cells to the position found in primary cells as a result of the continuous treatment with the microalgal polysaccharides.
Structure and Behaviour of Proteins, Nucleic Acids and Viruses from Vibrational Raman Optical Activity
Authors: Barron Laurence D. , Blanch Ewan W. , McColl Iain H. , Syme Christoper D. , Hecht Lutz , Nielsen Kurt
Published: March 2012

On account of its sensitivity to chirality Raman optical activity (ROA), which may be measured as a small difference in vibrational Raman scattering from chiral molecules in right‐ and left‐circularly polarized incident light, is a powerful probe of structure and behaviour of biomolecules in aqueous solution. Protein ROA spectra provide information on the secondary and tertiary structure of the polypeptide backbone, hydration, side chain conformation and structural elements present in denatured states. Nucleic acid ROA spectra provide information on the sugar ring conformation, the base stacking arrangement and the mutual orientation of the sugar and base rings around the C–N glycosidic link. The ROA spectra of intact viruses provide information on the folds of the coat proteins and the nucleic acid structure. The large number of structure‐sensitive bands in protein ROA spectra is especially favourable for fold determination using pattern recognition techniques. This article gives a brief account of the ROA technique and presents the ROA spectra of a selection of proteins, nucleic acids and viruses that illustrate the applications of ROA spectroscopy in biomolecular research.
Secondary Structure as a Conformational Determinant in Processing of Peptide Precursors at Dibasic Amino Acids
Authors: Lazar Noureddine , Brakch Noureddine , Panchal Maï , Cohen Paul , Rholam Mohamed
Published: March 2012

The aim of this work was to establish the functional role of selected secondary structure motifs of peptide hormone precursors in their selective recognition by the corresponding converting endoproteases. The strategy was based on the use of synthetic peptides either reproducing or mimicking the sequences of the cleavage regions of two distinct models; i.e. pro‐ocytocin–neurophysin and prosomatostatin. Both prohormones were capable to release their biologically active sequences either by cleavage at a dibasic stretch or by proteolysis at a monobasic site.

Both kinetic and thermodynamic parameters of peptide cleavage by various convertases were measured. They were examined in light of structural data on preferred conformations adopted by these substrates, which were obtained by a combination of spectroscopical techniques including CD, FT‐IR and proton NMR. In the case of prosomatostatin, these approaches were in addition paralleled by site‐directed mutagenesis experiments.

The wealth of collected data point toward the conclusion that β‐turns and/or loops, favored by sequences bearing basic residues, constitute a key feature in the specification of those peptide loci which are proteolytically processed in vivo. They will be discussed with respect of other processing mechanisms where these mechanisms were also shown.
FTIR Spectroscopic Investigation of Mineral Structure of Streptozotocin Induced Diabetic Rat Femur and Tibia
Authors: Boyar Handan , Turan Belma , Severcan Feride
Published: March 2012

Diabetes mellitus (DM) can be accepted as a heterogenous multi organ disorder that can affect various systems of the human body. Disorders include retinopathy, neuropathy, cardiomyopathy, musculoskeletal abnormalities such as diminished bone formation and bone healing retardation. Low bone mineral density is often mentioned as a complication for patients with insulin dependent diabetes mellitus (type I DM). Streptozotocin (STZ) induced diabetic rats are good models for investigation of the complications of insulin dependent diabetes. In the present study, the effects of STZ induced diabetes on the mineral environment of rat bones namely femur and tibia were studied by Fourier transform infrared (FTIR) spectroscopic technique. The results revealed that mineral crystal sizes increased and carbonate content decreased for diabetic femur and tibia. These changes can be due to the formation of osteoporosis which is widely seen in diabetic patients.
Atomic force microscopy and Raman scattering spectroscopy studies on heat‐induced fibrous aggregates of β‐lactoglobulin
Authors: Ikeda Shinya
Published: March 2012

Nanometer‐thick fibrous aggregates of β‐lactoglobulin alone and its mixture with other globular proteins were formed by heating aqueous solutions at pH 2 with maintaining an effective level of electrostatic repulsion among denatured protein molecules. In atomic force microscopy (AFM) images, these fibrous aggregates appeared to be fairly uniform in width and height and composed of strings of globular elements. Fibrous aggregates formed in β‐lactoglobulin individual systems were only slightly thicker than the size of the native β‐lactoglobulin monomer, while those formed in the presence of other globular proteins were more than twice thicker, suggesting that different species of globular proteins were incorporated into each individual fibrous aggregate in the mixed systems. At neutral pH, aggregates were generally composed of ellipsoidal primary particles much larger than the size of the monomer, suggesting that aggregation proceeds in two steps at neutral pH. Molecular structural changes probed by Raman scattering spectroscopy revealed that considerable fractions of β‐sheet structures remained to be folded during the formation of fibrous aggregates but α‐helix structures were partially lost. It was also suggested that a limited extent of hydrophobic interactions among heat‐denatured protein molecules is required for the fibrous aggregation.
Phage display‐derived ligands provide structural insight into insulin‐like growth factor I function
Authors: Skelton Nicholas J. , Schaffer Michelle L. , Deshayes Kurt , Blandl Tamas , Runyon Steven , Nakamura Gerald , Sidhu Sachdev S.
Published: March 2012

Insulin–like growth factor–I (IGF–I) is a central mediator of cell growth, differentiation and metabolism. Structural characterization of the protein has been hampered by a combination of internal dynamics and self–association that prevent crystallization and produce broad NMR resonances. To better characterize the functions of IGF–I, we have used phage display to identify peptides that antagonize the binding of IGF–I to its plasma binding proteins (IGFBPs) and cell–surface receptor (IGF–R). Interestingly, binding of peptide improves dramatically the quality of the NMR resonances of IGF–I, and enables the use of triple–resonance NMR methods to characterize the complexes. One such peptide, designated IGF–F1–1, has been studied in detail. In the complex, the peptide retains the same loop–helix motif seen in the free state whilst IGF–I contains three helices, as has been seen previously in low–resolution structures in the absence of ligand. The peptide binds at a hydrophobic patch between helix 1 and 3, a site identified previously by mutagenesis as a contact site for IGFBP1. Thus, antagonism of IGFBP1 binding exhibited by the peptide occurs by a simple steric occlusion mechanism. Antagonism of IGF–R binding may also be explained by a similar mechanism if receptor binding occurs by a two–site process, as has been postulated for insulin binding to its receptor. Comparisons with crystallographic structures determined for IGF–I in other complexes suggest that the region around helix 1 of IGF–I is conformationally conserved whereas the region around helix 3 adopts several different ligand–induced conformations. The ligand–induced structural variability of helix 3 appears to be a common feature across the insulin super–family. In the case of IGF–I, exchange between such conformations may be the source of the dynamic nature of free IGF–I, and likely has functional significance for the ability of IGF–I to recognize two signaling receptors and six binding proteins with high affinity.
Investigation of protein–protein interactions by isotope‒edited Fourier transformed infrared spectroscopy
Authors: Li Tiansheng
Published: March 2012

Recent advance in FTIR spectroscopy has shown the usefulness of 13C uniform isotope labeling in proteins to study protein–protein interactions. 13C uniform isotope labeling can significantly resolve the spectral overlap in the amide I/I′ region in the spectra of protein–protein complexes, and therefore allows more accurate determination of secondary structures of individual protein component in the complex than does the conventional FTIR spectroscopy. Only a limited number of biophysical techniques can be used effectively to obtain structural information of large protein–protein complex in solution. Though X‒ray crystallography and NMR have been used to provide structural information of proteins at atomic resolution, they are limited either by the ability of protein to crystallize or the large molecular weight of protein. Vibrational spectroscopy, including FTIR and Raman spectroscopies, has been extensively employed to investigate secondary structures and conformational dynamics of protein–protein complexes. However, significant spectral overlap in the amide I/Iʹ region in the spectra of protein–protein complexes often hinders the utilization of vibrational spectroscopy in the study of protein–protein complex. In this review, we shall discuss our recent work involving the application of isotope labeled FTIR to the investigation of protein–protein complexes such as cytokine–receptor complexes. One of the examples involves G‒CSF/receptor complex. To determine unambiguously the conformations of G‒CSF and the receptor in the complex, we have prepared uniformly 13C/15N isotope labeled G‒CSF to resolve its amide Iʹ band from that of its receptor in the IR spectrum of the complex. Conformational changes and structural stability of individual protein subunit in G‒CSF/receptor complex have then been investigated by using FTIR spectroscopy (Li et al., Biochemistry 29 (1997), 8849–8859). Another example involves BDNF/trkB complex in which 13C/15N uniformly labeled BDNF is complexed with its receptor trkB (Li et al., Biopolymers 67(1) (2002), 10–19). Interactions of 13C/15N uniformly labeled brain‒derived neurotrophic factor (BDNF) with the extracellular domain of its receptor, trkB, have been investigated by employing FTIR spectroscopy. Conformational changes and structural stability and dynamics of BDNF/trkB complex have been determined unambiguously by FTIR spectroscopy, since amide I/Iʹ bands of 13C/15N labeled BDNF are resolved from those of the receptor. Together, those studies have shown that isotope edited FTIR spectroscopy can be successfully applied to the determination of protein secondary structures of protein complexes containing either the same or different types of secondary structures. It was observed that 13C/15N uniform labeling also affects significantly the frequency of amide IIʹ band, which may permit the determination of hydrogen–deuterium exchange in individual subunit of protein–protein complexes.
Mass spectrometry of some arylidene thioxobenzyl or thioxobiphenyloxoethyl thiazolidinone compounds
Authors: Guarda V. L. M. , Bosso C. , Galdino S. L. , Albuquerque J. F. C. , Lima M. C. A. , Silva J. B. P. , Leite L. F. C. , Pitta I. R.
Published: March 2012

This paper reports the results of a comparative study of the electron impact mass spectrometry data for a series of previously synthetized 5‐arylidene‐3‐(4‐chloro‐benzyl)‐4‐thioxo‐thiazolidin‐2‐one, and 5‐arylidene‐3‐(2‐biphenyl‐4‐yl‐2‐oxo‐ethyl)‐4‐thioxo‐thiazolidin‐2‐one derivatives. Theoretical calculations of molecular mechanics, MOPAC‒AM1, were used to explain the fragmentations observed.
The structure of 1,1,3‐trimethyl‐Δ2‐ pyrazolinium perchlorate: An X‐ray crystallographic and GIAO/DFT multinuclear NMR study
Authors: Yap Glenn P. A. , Alkorta Ibon , Elguero José , Jagerovic Nadine
Published: March 2012

The crystal and molecular structure of 1,1,3‐trimethyl‐Δ2‐pyrazolinium perchlorate has been determined and compared with those of other pyrazolinium salts (both 1,1 and 1,2‐substituted). Reported 13C and 15N chemical shifts for a series of related pyrazolines have been compared with GIAO/DFT calculations, with excellent agreement. The combination of the biological properties of pyrazolines with those of the perchlorate anion in the same molecule will be discussed.
Clinical applications of proton MR spectroscopy in the diagnosis of brain tumours
Authors: Bulakbasi Nail
Published: March 2012

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.
Continuous assays of glutaminyl cyclase: from development to application
Authors: Schilling Stephan , Demuth Hans-Ulrich
Published: March 2012

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyses the formation of pyroglutamyl residues from glutamine at the N‐terminus of peptides and proteins. In previously applied assays, QC activity was determined by either analysing the products formed using HPLC coupled with photometric or fluorometric detection, radioimmunoassay, or by detecting the release of ammonia spectrophotometrically. Although these methods are sensitive, they are all discontinuous and therefore time‐consuming and laborious. To conduct a detailed kinetic investigation of QC catalysis, we developed coupled continuous assays suitable for microplates which allow now convenient determination of QC activity. The methods either use pyroglutamyl aminopeptidase or glutamate dehydrogenase as auxiliary enzymes, which results in the liberation of chromophores or fluorophores such as pNA, AMC, βNA or in the conversion of the chromophore NADH/H+ into NAD+, respectively.

The assays were applied in various enzyme isolation and characterisation studies, using crude protein solutions as well as purified enzyme in pH‐dependence, substrate and inhibitor specificity investigations. Depending on the respective analytical task, both assays complement each other. Therefore, different enzymatic properties could be explored in more detail. Since the employed strategy of assay development could be of interest also for the analysis of other enzymes, the methods are described here in a comprehensive manner.
1H and 13C NMR spectroscopic studies of half‐esters from monohydrolysis of dialkyl bicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboxylates
Authors: Hiraga Yoshikazu , Niwayama Satomi
Published: March 2012

The structures of the half‐esters from the monohydrolysis of (exo,exo)‐, (endo,endo)‐, and (endo,exo)‐dialkyl bicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboxylates were determined by 1H and 13C NMR as well as 2D NMR spectra, and the complete spectral assignment has been made. After conversion of one of the carboalkoxy groups to a carboxyl group, different tendencies were observed for the differences in 1H and 13C NMR chemical shifts between half‐esters and the corresponding diesters.
Fluorescence anisotropy of oligomeric proteins
Authors: Czajkowsky Daniel M.
Published: March 2012

Previous studies of protein oligomerization using time‐resolved fluorescence anisotropy assumed a single fixed probe per oligomeric complex and an identical probe orientation in complexes of different stoichiometry. However, an oligomer consisting of “n” singly labeled monomers must necessarily have “n” probes. Moreover, in the expression for the anisotropy decay, the molecular axes from which the probe orientation is defined are different for complexes that differ in stoichiometry. Here, we derive an expression for the decay of the anisotropy for molecules with any number of fixed probes, and show how an explicit understanding of the probe orientation is necessary to properly assess oligomerization.