Spectroscopy is an analytical technique used to measure the interaction between matter and electromagnetic radiation. In equine research, spectroscopy is applied to assess various biological and chemical properties of horses. Techniques such as infrared spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry are utilized to analyze tissues, fluids, and other biological samples. These methods provide insights into metabolic processes, nutritional status, and disease states in horses. Spectroscopy aids in the identification and quantification of biomolecules, contributing to the understanding of equine physiology and pathology. This page compiles peer-reviewed research studies and scholarly articles that explore the application, methodology, and findings of spectroscopic techniques in equine science.
Kramer KJ, Kagan IA, Lawrence LM, Smith SR.Ethanol-soluble carbohydrates (ESCs) of cool-season grasses include mono- and disaccharides and sometimes short-chain fructans, which may exacerbate the risk of pasture-associated laminitis. A calibration for prediction of ESC concentrations by near-infrared reflectance spectroscopy (NIRS) was developed from 323 samples of four cool-season grass species (orchardgrass, Kentucky bluegrass, tall fescue, and perennial ryegrass) across 10 cultivars collected in central Kentucky in the morning and afternoon over two growing seasons. The calibration, which had accuracy above 95%, was used to predict ...
Sarin JK, Te Moller NCR, Mohammadi A, Prakash M, Torniainen J, Brommer H, Nippolainen E, Shaikh R, Mäkelä JTA, Korhonen RK, van Weeren PR, Afara IO....To assess the potential of near-infrared spectroscopy (NIRS) for in vivo arthroscopic monitoring of cartilage defects. Sharp and blunt cartilage grooves were induced in the radiocarpal and intercarpal joints of Shetland ponies and monitored at baseline (0 weeks) and at three follow-up timepoints (11, 23, and 39 weeks) by measuring near-infrared spectra in vivo at and around the grooves. The animals were sacrificed after 39 weeks and the joints were harvested. Spectra were reacquired ex vivo to ensure reliability of in vivo measurements and for reference analyses. Additionally, cartilage thickn...
Borunova SF, Tkachev N, Iolchiev B, Artyushina Z, Abramov P, Nikitina M, Silanteva A, Khusnetdinova N, Serezhenkov V.Diseases of the gastrointestinal tract of horses are caused by many factors and have a complex pathogenesis. Developing effective methods of differential diagnostics is of high fundamental and applied importance. The pathogenesis of diseases of the digestive tract of horses accompanied by the development of inflammation and oxidative stress, can be associated with a lack of the nitrogen monoxide which controls many signaling pathways in the body. The level of the nitric oxide (NO) is involved in the regulation of the immune and nervous systems, the tone of all the blood vessels, and the course...
Acri G, Testagrossa B, Giudice E, Arfuso F, Piccione G, Giannetto C.Raman spectroscopy is a rapid qualitative and quantitative technique that allows the simultaneous determination of several components, both biomolecules both chemical compounds, in the biological fluids to assess the metabolic status. In this study, the serum composition was evaluated in regularly trained athletic horses using Raman spectroscopy to identify biomarkers of sports performance. Five clinically healthy and regularly trained Italian Saddle horses were subjected to a standardized obstacle course (350 m/minute; eleven 1.25 high jumps) preceded by a warm-up. On the collected sera, at ...
McCrae P, Guigand C, Jones KB, Thueson ES, Troudt TA, Warlick LM, Sides RH, Jones JH, Bayly WM, Léguillette R.High pulmonary blood pressure contributes to exercise-induced pulmonary hemorrhage. The objective of this study was to use bioimpedance spectroscopy to assess body fluid compartment volumes under 3 conditions in 6 racehorses: i) Pre- and post-supramaximal treadmill exercise (control); ii) Exercise 4 hours after furosemide (0.5 mg/kg body weight, IV); iii) Exercise, removal of ~14 L of blood and subsequent reinfusion of the blood. Statistical analysis used linear mixed effects models. Body compartment volumes did not change during the control runs. Total body water (TBW) ( = 0.007, = 0.007), e...
Ekruth J, Gottschalk C, Ulrich S, Gareis M, Schwaiger K.Stachybotrys (S.) chartarum is a cellulolytic mould with the ability to produce highly cytotoxic macrocyclic trichothecenes. Two chemotypes are defined according to their ability to produce either atranones or satratoxins. S. chartarum has been well known as the causative agent of the lethal disease stachybotryotoxicosis in horses. Further investigations revealed that this disease is strictly correlated with the presence of macrocyclic trichothecenes. Furthermore, their occurrence in water-damaged buildings has been linked to adverse health effects such as the sick building syndrome. As the ch...
Beachler TM, Scott Bailey C, Gracz HS, Morgan DR, Von Dollen KA, Ellis KE, Gadsby JE, Lyle SK.The amniotic and allantoic fluid compartments in the mare serve essential roles throughout pregnancy and parturition. Although the global metabolomic profile of amniotic fluid in women has been extensively characterized, current data for equine fetal fluids are limited. Therefore, the goal of this study was to characterize the global metabolomic profile of equine allantoic and amniotic fluid through nuclear magnetic resonance spectroscopy. Fetal fluids were collected between 270 and 295 days of gestation from 12 pregnancies through ultrasound-guided transabdominal puncture. A total of 24 samp...
Nery IHAV, Araújo Silva RAJ, Souza HM, Arruda LCP, Monteiro MM, Seal DCM, Silva GR, Silva TMS, Carneiro GF, Batista AM, Câmara DR, Guerra MMP.l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through β-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In...
Anderson JR, Phelan MM, Foddy L, Clegg PD, Peffers MJ.Osteoarthritis is an age-related degenerative musculoskeletal disease characterized by loss of articular cartilage, synovitis, and subchondral bone sclerosis. Osteoarthritis pathogenesis is yet to be fully elucidated with no osteoarthritis-specific biomarkers in clinical use. equine cartilage explants ( = 5) were incubated in tumor necrosis factor-α (TNF-α)/interleukin-1β (IL-1β)-supplemented culture media for 8 days, with the media removed and replaced at 2, 5, and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent one-d...
Journal of proteomicsAugust 5, 2020
Volume 228 103927 doi: 10.1016/j.jprot.2020.103927
Chiaradia E, Pepe M, Sassi P, Mohren R, Orvietani PL, Paolantoni M, Tognoloni A, Sforna M, Eveque M, Tombolesi N, Cillero-Pastor B.Osteochondrosis is a developmental orthopedic disease affecting growing cartilage in young horses. In this study we compared the proteomes of equine chondrocytes obtained from healthy and osteochondrotic cartilage using a label-free mass spectrometry approach. Quantitative changes of some proteins selected for their involvement in different functional pathways highlighted by the bioinformatics analysis, were validated by western blotting, while biochemical alterations of extracellular matrix were confirmed via Raman spectroscopy analysis. In total 1637 proteins were identified, of which 59 wer...
Bazzano M, Laghi L, Zhu C, Lotito E, Sgariglia S, Tesei B, Laus F.In the present study, data related to the metabolomics of saliva and serum in trained standardbred horses are provided for the first time. Metabolomic analysis allows to analyze all the metabolites within selected biofluids, providing a better understanding of biochemistry modifications related to exercise. On the basis of the current advances observed in metabolomic research on human athletes, we aimed to investigate the metabolites' profile of serum and saliva samples collected from healthy standardbred horses and the relationship with physical exercise. Twelve trained standardbred horses we...
Cakir-Koc R, Budama-Kilinc Y, Ustun E, Babur C.Toxoplasma gondii is a parasite that causes severe health problems in the world. Toxoplasmosis, an infection caused by T. gondii, leads to high risk of mortality in patients with immunodeficiency, transplantation, and cancer. Besides that, it causes miscarriages in pregnancy, various abnormalities such as hydrocephalus in infants and congenital diseases. Because the clinical indication of the disease is not specific, it is confused with many diseases, and this leads to the necessity of directly detecting the presence of the toxoplasmosis. Therefore, various diagnostic assays are needed for th...
Li N, Dou S, Feng L, Zhu Q, Lu N.In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the analyte is usually distributed unevenly throughout the sample spot. The area with aggregated analyte molecules contributing abundant signal, is termed as "sweet spot", which results in poor detection reproducibility and makes it impossible to quantify analytes without internal standards. We proposed a strategy to eliminate sweet spot in MALDI-MS by using a hydrophobic ordered structure as target. The target is fabricated by creating a hydrophobic silicon nanopillar array and subsequently decorating it uniformly wi...
Fernández-Hernández P, Sánchez-Calabuig MJ, García-Marín LJ, Bragado MJ, Gutiérrez-Adán A, Millet Ó, Bruzzone C, González-Fernández L....Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the ...
Beachler TM, Gracz HS, Morgan DR, Bembenek Bailey SA, Borst L, Ellis KE, Von Dollen KA, Lyle SK, Nebel A, Andrews NC, Koipalli J, Gadsby JE, Bailey CS.Metabolomics may represent an avenue for diagnosis of equine ascending placentitis. Objective: To characterise the plasma metabolomic profile in healthy mares and mares with induced ascending placentitis, with the goal of identifying metabolites with potential clinical value for early diagnosis of placentitis. Methods: Controlled in vivo experiment. Methods: Placentitis was induced in 10 late-term pregnant pony mares via Streptococcal equi subsp. zooepidemicus inoculation in five mares between days 285 and 290 of gestation, while five mares served as healthy controls. Repeated ultrasound exami...
Kramer KJ, Kagan IA, Lawrence LM, Goff BM, Smith SR.An excess of water-soluble carbohydrates (WSCs) can present a risk for horses prone to pasture-associated laminitis or some other metabolic conditions. Determining WSC concentrations in commonly grazed cool-season grasses, at different times of day and under different fertilization treatments, can help optimize grazing times and management strategies. The goals of this study were to develop a near-infrared reflectance spectroscopy (NIRS) calibration curve for WSC and to apply it to four cool-season grass species. Tall fescue, Kentucky bluegrass, orchardgrass, and perennial ryegrass (10 cultiva...
X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ -oxo)tris[(μ -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the l...
Atkinson F, Evans R, Guest JE, Bavin EP, Cacador D, Holland C, Guest DJ.Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo e...
Yin NH, Parker AW, Matousek P, Birch HL.The lack of clinical detection tools at the molecular level hinders our progression in preventing age-related tendon pathologies. Raman spectroscopy can rapidly and non-invasively detect tissue molecular compositions and has great potential for in vivo applications. In biological tissues, a highly fluorescent background masks the Raman spectral features and is usually removed during data processing, but including this background could help age differentiation since fluorescence level in tendons increases with age. Therefore, we conducted a stepwise analysis of fluorescence and Raman combined s...
González-Fernández L, Sánchez-Calabuig MJ, Calle-Guisado V, García-Marín LJ, Bragado MJ, Fernández-Hernández P, Gutiérrez-Adán A....A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF sample...
Graham RJTY, Anderson JR, Phelan MM, Cillan-Garcia E, Bladon BM, Taylor SE.Palmar osteochondral disease (POD) is a common cause of lameness in competition horses. Magnetic resonance imaging (MRI) is the most sensitive diagnostic modality currently available, however it may not be financially or logistically practical for routine screening of POD. There is increasing interest in the use of metabolomics for diagnosis prior to progression to irreversible damage. Objective: To determine metabolite levels in synovial fluid (SF) of horses with a clinical diagnosis of POD based on diagnostic analgesia and MRI, with the hypothesis that metabolomic profiles differ between dis...
Sarin JK, Torniainen J, Prakash M, Rieppo L, Afara IO, Töyräs J.Near infrared (NIR) spectroscopy is a well-established technique that is widely employed in agriculture, chemometrics, and pharmaceutical engineering. Recently, the technique has shown potential in clinical orthopaedic applications, for example, assisting in the diagnosis of various knee-related diseases (e.g., osteoarthritis) and their pathologies. NIR spectroscopy (NIRS) could be especially useful for determining the integrity and condition of articular cartilage, as the current arthroscopic diagnostics is subjective and unreliable. In this work, we present an extensive dataset of NIRS meas...
Terzi A, Gallo N, Bettini S, Sibillano T, Altamura D, Campa L, Natali ML, Salvatore L, Madaghiele M, De Caro L, Valli L, Sannino A, Giannini C.The aim of this work is to evaluate the effects of different extraction and material processing protocols on the collagen structure and hierarchical organization of equine tendons. Wide and Small Angle X-ray Scattering investigations on raw powders and thin films revealed that not only the extraction and purification treatments, but also the processing conditions may affect the extent of the protein crystalline domain and induce a nanoscale "shield effect." This is due to the supramolecular fiber organization, which protects the atomic scale structure from the modifications that occur during f...
Gingold BM, Killos MB, Griffith E, Posner L.Maintaining adequate muscle tissue oxygenation is of paramount importance during equine general anesthesia. The objectives of this study were to assess the feasibility, reliability and repeatability of near-infrared spectroscopy (NIRS) muscle oximetry using the Inspectra m650 in conscious healthy adult horses. Methods: Prospective, observational study. Methods: A group of 30 healthy client-owned adult horses admitted to the equine hospital between July 2017 and July 2018. Methods: The probe of an Inspectra m650 NIRS tissue oximeter was placed on the hairless surface of five muscle sites (omotr...
Heales CJ, Summers IR, Fulford J, Knapp KM, Winlove CP.The aetiology of bone marrow oedema-like abnormalities (BMOA) seen on magnetic resonance imaging (MRI) is as yet not fully understood. The current study aimed to investigate the potential of projection radiography and Raman microspectroscopy to provide information regarding the underlying physiological changes associated with BMOA in equine bone samples. Methods: MRI was used to assess 65 limbs from 43 horses. A subset of 13 limbs provided 25 samples, 8 with BMOA present and 17 as controls; these were examined with projection radiography to assess bone mineral density and Raman spectroscopy to...
Le Moyec L, Robert C, Triba MN, Bouchemal N, Mach N, Rivière J, Zalachas-Rebours E, Barrey E.Endurance racing places high demands on energy metabolism pathways. Metabolomics can be used to investigate biochemical responses to endurance exercise in humans, laboratory animals, and horses. Although endurance horses have previously been assessed in the field (i.e., during races) using broad-window Nuclear Magnetic Resonance metabolomics, these studies included several different race locations, race distances, age classes, and race statuses (finisher or elimination). The present NMR metabolomics study focused on 40 endurance horses racing in three race categories over 90, 120, or 160 km. T...
Paraskevaidi M, Hook PD, Morais CLM, Anderson JR, White R, Martin-Hirsch PL, Peffers MJ, Martin FL.Reliable and validated biomarkers for osteoarthritis (OA) are currently lacking. Objective: To develop an accurate and minimally invasive method to assess OA-affected horses and provide potential spectral markers indicative of disease. Methods: Observational, cross-sectional study. Methods: Our cohort consisted of 15 horses with OA and 48 without clinical signs of the disease, which were used as controls. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to investigate serum samples (50 μL) collected from these horses. Spectral processing and multivariat...
Radunsky D, Blumenfeld-Katzir T, Volovyk O, Tal A, Barazany D, Tsarfaty G, Ben-Eliezer N.Multi-echo spin-echo (MESE) protocol is the most effective tool for mapping T relaxation in vivo. Still, MESE extensive use of radiofrequency pulses causes magnetization transfer (MT)-related bias of the water signal, instigated by the presence of macromolecules (MMP). Here, we analyze the effects of MT on MESE signal, alongside their impact on quantitative T measurements. Study used 3 models: in vitro urea phantom, ex vivo horse brain, and in vivo human brain. MT ratio (MTR) was measured between single-SE and MESE protocols under different scan settings including varying echo train lengths, n...
Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CFCOH along with one- and two-dimensional H and C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-gluc...
Bazzano M, Laghi L, Zhu C, Magi GE, Serri E, Spaterna A, Tesei B, Laus F.The present work characterized the metabolomic profile of tracheal wash (TW) and exhaled breath condensate (EBC) in healthy horses and horses with respiratory disease. Six asthma-affected horses (group A) and six healthy controls (group H) underwent clinical, endoscopic and cytologic examinations of upper airways to confirm the active phase of asthma. TW and EBC samples were collected from each animal and investigated by proton nuclear magnetic resonance (H-NMR) metabolomic analysis. A total of ten out of 38 metabolites found in the TW were significantly different between the groups (p < ...
Thorpe CT, Stark RJ, Goodship AE, Birch HL.Damage to the flexor tendons, particularly the superficial digital flexor tendon (SDFT), is one of the most common musculoskeletal injuries sustained by horses competing in all disciplines. Our previous work has shown that SDFTs from different individuals show a wide variation in mechanical strengths; this is important clinically as it may relate to predisposition to injury. The high mechanical strength of tendon relies on the correct orientation of collagen molecules within fibrils and stabilisation by the formation of chemical cross-links between collagen molecules. It is not known whether t...
Bazzano M, Laghi L, Zhu C, Magi GE, Serri E, Spaterna A, Tesei B, Laus F.The present work characterized the metabolomic profile of tracheal wash (TW) and exhaled breath condensate (EBC) in healthy horses and horses with respiratory disease. Six asthma-affected horses (group A) and six healthy controls (group H) underwent clinical, endoscopic and cytologic examinations of upper airways to confirm the active phase of asthma. TW and EBC samples were collected from each animal and investigated by proton nuclear magnetic resonance (H-NMR) metabolomic analysis. A total of ten out of 38 metabolites found in the TW were significantly different between the groups (p < ...
Oinas J, Ronkainen AP, Rieppo L, Finnilä MAJ, Iivarinen JT, van Weeren PR, Helminen HJ, Brama PAJ, Korhonen RK, Saarakkala S.Articular cartilage undergoes structural and biochemical changes during maturation, but the knowledge on how these changes relate to articular cartilage function at different stages of maturation is lacking. Equine articular cartilage samples of four different maturation levels (newborn, 5-month-old, 11-month-old and adult) were collected (N = 25). Biomechanical tensile testing, Fourier transform infrared microspectroscopy (FTIR-MS) and polarized light microscopy were used to study the tensile, biochemical and structural properties of articular cartilage, respectively. The tensile modulus ...
Le Moyec L, Robert C, Triba MN, Billat VL, Mata X, Schibler L, Barrey E.During long distance endurance races, horses undergo high physiological and metabolic stresses. The adaptation processes involve the modulation of the energetic pathways in order to meet the energy demand. The aims were to evaluate the effects of long endurance exercise on the plasma metabolomic profiles and to investigate the relationships with the individual horse performances. The metabolomic profiles of the horses were analyzed using the non-dedicated methodology, NMR spectroscopy and statistical multivariate analysis. The advantage of this method is to investigate several metabolomic path...
Zhu C, Faillace V, Laus F, Bazzano M, Laghi L.Metabolomics has been recognized as a powerful approach for disease screening. In order to highlight potential health issues in subjects, a key factor is the possibility to compare quantitatively the metabolome of their biofluids with reference values from healthy individuals. Such efforts towards the systematic characterization of the metabolome of biofluids in perfect health conditions, far from concluded for humans, have barely begun on horses. The present work attempts, for the first time, to give reference quantitative values for the molecules mostly represented in the urine metabolome of...
Celeste CJ, Deschene K, Riley CB, Theoret CL.Wound repair in horse limbs is often complicated by the development of exuberant granulation tissue (EGT) and excessive scarring while body wounds tend to repair uneventfully. EGT resembles the human keloid. While the events leading to keloid formation are not fully elucidated, tissue hypoxia has been proposed as a major contributing factor. The objective of this study was to investigate tissue oxygen saturation in healing full-thickness wounds created on the horse limb and body, using near-infrared spectroscopy. Spectroscopic reflectance data were collected from both anatomic sites at specifi...
Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojević V, Schleucher J, Morozova-Roche LA.Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed...
Sakakura M, Yamaguchi S, Hirota N, Terazima M.The energetics and structural volume changes after photodissociation of carboxymyoglobin are quantitatively investigated by laser-induced transient grating (TG) and photoacoustic calorimetric techniques. Various origins of the TG signal are distinguished: the phase grating signals due to temperature change, due to absorption spectrum change, and due to volume change. We found a new kinetics of approximately 700 ns (at room temperature), which was not observed by the flash photolysis technique. This kinetics should be attributed to the intermediate between the geminate pair and the fully dissoc...
Choi RK, Smith MM, Martin JH, Clarke JL, Dart AJ, Little CB, Clarke EC.Both mechanical and structural properties of tendon change after injury however the causal relationship between these properties is presently unclear. This study aimed to determine the extent of biomechanical change in post-injury tendon pathology and whether the sulphated glycosaminoglycans (glycosaminoglycans) present are a causal factor in these changes. Equine superficial digital flexor tendons (SDF tendons) were surgically-injured in vivo (n=6 injured, n=6 control). Six weeks later they were harvested and regionally dissected into twelve regions around the lesion (equal medial/lateral, pr...
Yang X, Chasteen ND.Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike th...
Hagen SJ, Latypov RF, Dolgikh DA, Roder H.Time-resolved spectroscopic studies of unfolded horse iron(II) cytochrome c have suggested that the imidazole side chains of His26 and His33 bind transiently to the heme iron on microsecond time scales, after photodissociation of a carbon monoxide ligand from the heme. Our studies of four variants of cytochrome c (horse wild type, horse H33N, horse H33N/H26Q, and tuna wild type), unfolded in guanidine hydrochloride at pH 6.5, demonstrate that these side chains are responsible for the observed microsecond spectral changes. As His33 and then His26 are eliminated from the horse wild-type sequence...
Nielsen SB, Wilhelm K, Vad B, Schleucher J, Morozova-Roche LA, Otzen D.The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells. ...
Gasa S, Makita A, Kinoshita Y.The chemical structure of an equine hematoside, which contained an ester group and comprised 72% of the total erythrocyte gangliosides, was determined by means of nondestructive and destructive procedures. A 400-MHz nuclear magnetic resonance spectrum of the ganglioside in perdeuterodimethyl sulfoxide demonstrated three protons due to a methyl group of an acetyl moiety, as well as amide and anomeric protons which were compatible with those of the ordinary hematoside. The spin decoupling difference spectroscopy of the ganglioside revealed the presence of the following structures. [formula: see ...
Svistunenko DA, Sharpe MA, Nicholls P, Blenkinsop C, Davies NA, Dunne J, Wilson MT, Cooper CE.The paramagnetic species in human metHb and horse metmyoglobin (metMb) have been studied at low temperature using EPR spectroscopy. The high-spin (HS) haem signal in aquometMb has a greater rhombic distortion than the HS metHb signal. Nevertheless, the individual line width (g=6) is smaller in metMb than in metHb, consistent with non-identical signals from the alpha and beta Hb subunits. Three low-spin (LS) haem forms are present in metHb, while metMb has only two. The major LS form in both proteins is the alkaline species (with OH(-) at the sixth co-ordination position). The minor LS forms ar...
Dillon PF, Root-Bernstein R, Robinson NE, Abraham WM, Berney C.Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. Objective: Extending this work to beta 2 adrenergic systems in vitro and in vivo. Methods: Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuter...
Gunther MR, Sturgeon BE, Mason RP.The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)...
Kreusch S, Schwedler S, Tautkus B, Cumme GA, Horn A.An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 microg of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mgxcm) for eight different proteins with a CV of 5.6% o...
Terzi A, Gallo N, Bettini S, Sibillano T, Altamura D, Campa L, Natali ML, Salvatore L, Madaghiele M, De Caro L, Valli L, Sannino A, Giannini C.The aim of this work is to evaluate the effects of different extraction and material processing protocols on the collagen structure and hierarchical organization of equine tendons. Wide and Small Angle X-ray Scattering investigations on raw powders and thin films revealed that not only the extraction and purification treatments, but also the processing conditions may affect the extent of the protein crystalline domain and induce a nanoscale "shield effect." This is due to the supramolecular fiber organization, which protects the atomic scale structure from the modifications that occur during f...
Salamon Z, Tollin G.Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, ...
Taddei N, Stefani M, Magherini F, Chiti F, Modesti A, Raugei G, Ramponi G.Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were...
Van Dael H, Haezebrouck P, Morozova L, Arico-Muendel C, Dobson CM.Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly d...
Pereira A, Braekman JC, Dumont JE, Boeynaems JM.The incorporation of iodide into proteins (PBI) and lipids (LBI) of horse thyroid slices was measured in various conditions. Their dependency on the concentration of extracellular iodide was strikingly different. For PBI the relationship was biphasic with a decrease above 10 microM, likely to correspond to the Wolff-Chaikoff effect. On the contrary, LBI increased as a function of iodide concentration up to 100 microM. Methimazole (MMI) inhibited the incorporation of iodide into both LBI and PBI, but higher concentrations of MMI were required to depress LBI as compared to PBI. The inhibition of...
Mikami T, Kashiwagi M, Tsuchihashi K, Daino T, Akino T, Gasa S.Equine brain gangliosides were isolated and their structures were characterized, to examine whether equine brain has N-glycolyl neuraminic acid in gangliosides, since other mammals predominantly possess N-acetyl neuraminic acid in brain gangliosides, and equine erythrocytes and organs except the brain have gangliosides exclusively containing N-glycolyl neuraminic acid. The gangliosides purified from the brain were identified by proton NMR spectroscopy and mass spectrometry, as well as GLC, resulting in their identification as GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Of these gangliosides, GM3...
Bauminger ER, Harrison PM, Nowik I, Treffry A.Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...
Morozova-Roche LA, Jones JA, Noppe W, Dobson CM.The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alph...
Qureshi SH, Moza B, Yadav S, Ahmad F.The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation X (intermediate) conformation and the second denaturation phase, X conformation D (denatured) conformation are...
Fernández-Hernández P, Sánchez-Calabuig MJ, García-Marín LJ, Bragado MJ, Gutiérrez-Adán A, Millet Ó, Bruzzone C, González-Fernández L....Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the ...
Stock SR, Deymier-Black AC, Veis A, Telser A, Lux E, Cai Z.Dentin contains 1-2μm diameter tubules extending from the pulp cavity to near the junction with enamel. Peritubular dentin (PTD) borders the tubule lumens and is surrounded by intertubular dentin (ITD). Differences in PTD and ITD composition and microstructure remain poorly understood. Here, a (∼200nm)(2), 10.1keV synchrotron X-ray beam maps X-ray fluorescence and X-ray diffraction simultaneously around tubules in 15-30μm thick bovine and equine specimens. Increased Ca fluorescence surrounding tubule lumens confirms that PTD is present, and the relative intensities in PTD and ITD correspon...
Garcia T, Hornof WJ, Insana MF.The strong dependence of tendon echogenicity on insonation angle is explored by analyzing echo spectra. Combining echo spectra with high-resolution images from several modalities reveals that fluid spaces surrounding fascicles and bundles are likely sources of ultrasonic scatter. Mathematical models of tendon structure are proposed to explain how the anisotropic microstructure of tendon gives rise to angle-dependent echogenicity. Echo spectra from spontaneously damaged equine tendon samples were compared with normal equine tendon and found to exhibit a dramatic decrease in anisotropic properti...
