Beef Heart Research Articles

Toward the prevention of childhood undernutrition: diet diversity strategies using locally produced food can overcome gaps in nutrient supply.

Chronic undernutrition is prevalent in Mozambique, where children suffer from stunting, vitamin A deficiency, anemia, and other nutrition-related disorders. Complete diet formulation products (CDFPs) are increasingly promoted to prevent chronic undernutrition. Using linear programming, to investigate whether diet diversification using local foods should be prioritized in order to reduce the prevalence of chronic undernutrition. Market prices of local foods were collected in Tete City, Mozambique. Linear programming was applied to calculate the cheapest possible fully nutritious food baskets (FNFB) by stepwise addition of micronutrient-dense localfoods. Only the top quintile of Mozambican households, using average expenditure data, could afford the FNFB that was designed using linear programming from a spectrum of local standard foods. The addition of beef heart or liver, dried fish and fresh moringa leaves, before applying linear programming decreased the price by a factor of up to 2.6. As a result, the top three quintiles could afford the FNFB optimized using both diversification strategy and linear programming. CDFPs, when added to the baskets, were unable to overcome the micronutrient gaps without greatly exceeding recommended energy intakes, due to their high ratio of energy to micronutrient density. Dietary diversification strategies using local, low-cost, nutrient-dense foods can meet all micronutrient recommendations and overcome all micronutrient gaps. The success of linear programming to identify a low-cost FNFB depends entirely on the investigators' ability to select appropriate micronutrient-dense foods. CDFPs added to food baskets are unable to overcome micronutrient gaps without greatly exceeding recommended energy intake.

Redox equilibration after one-electron reduction of cytochrome c oxidase: Radical formation and a possible hydrogen relay mechanism

Kinetic studies using UV/visible and EPR spectroscopy were carried out to follow the distribution of electrons within beef heart cytochrome c oxidase (CcO), both active and cyanide-inhibited, following addition of reduced cytochrome c as electron donor. In the initial one-electron reduced state the electron is shared between three redox centers, heme a, CuA and a third site, probably CuB. Using a rapid freeze system and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) a protein radical was also detected. The EPR spectrum of the DMPO adduct of this radical was consistent with tyrosyl radical capture. This may be a feature of a charge relay mechanism involved in some part of the CcO electron transfer system from bound cytochrome c via CuA and heme a to the a3CuB binuclear center.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Structure Determination of Functional Membrane Proteins using Small-Angle Neutron Scattering (SANS) with Small, Mixed-Lipid Liposomes: Native Beef Heart Mitochondrial Cytochrome c Oxidase Forms Dimers

The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59×70×120)Å with uncertainties, respectively, (11, 12, 20Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410±124)kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59×101×129)Å with fitting uncertainties, respectively, (2, 3, 3Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.

Replacement of moist ingredients in the feed training of carnivorous fish

The study evaluated the replacement of bovine heart by gelatin in the feed training of carnivorous fish, using giant trahira (Hoplias lacerdae) as an experimental model. A completely randomized design with four treatments and five repetitions was employed. The treatments were composed of wet ingredients beef heart (control), gelatin diluted in water, gelatin diluted in beef heart broth, and gelatin diluted in water mixed with fish meal. The fish (3.22±0.03 cm and 0.57±0.01 g) were conditioned to accept industrialized diets by the technique of gradual feed ingredients transition in the diet. Gains in weight and length, efficiency of feed training, specific growth rate, cannibalism, mortality and survival rates were evaluated. There was significant difference in weight and length gains and specific growth rate, whereby the use of bovine heart gave the best results. Greater efficiency of feed training was observed for fish fed diets containing beef heart and gelatin diluted in water mixed with fish meal. The high survival rates and the absence of significant differences among treatments for rates of cannibalism, mortality and survival indicate the feasibility of using gelatin as a moist ingredient in the feed training of carnivorous fish.

An unusual phosphatidylethanolamine-utilizing cardiolipin synthase is discovered in bacteria

Cardiolipin (also known as diphosphatidylglycerol) is a four-chained anionic membrane phospholipid composed of two phosphatidyl moieties joined by a glycerol link (Fig. 1). It was first isolated from beef heart as a serologically active phospholipid (1), hence the name cardiolipin. In animals, plants, and lower eukaryotic organisms (e.g., yeast), cardiolipin is a component of the inner mitochondrial membrane bilayer, where it plays an important role in the function of enzyme complexes involved in energy transduction and ATP synthesis (2, 3). It is found in the membranes of Gram-positive and Gram-negative bacteria, where it also functions in energy production (2, 3). The nature (i.e., chain length and degree of saturation) of the four acyl groups and the hydrophilic head group of this phospholipid have an impact on mitochondrial membrane structure and function, and, by extension, the roles this organelle plays in cell physiology (2, 4). Thus, how cardiolipin is synthesized, remodeled, and metabolized are important areas of investigation. The importance of cardiolipin in mitochondrial function is further emphasized by the fact that defects in tafazzin-mediated remodeling of the fatty acyl composition of cardiolipin is the molecular basis for human Barth syndrome, a disease characterized by heart failure, myopathy, neutropenia, and growth retardation in children (3, 4).

Isolation, Biological Activity Evaluation, Structure Elucidation, and Total Synthesis of Eliamid: A Novel Complex I Inhibitor

Eliamid is a secondary metabolite isolated from two bacterial strains. This molecule features a linear polyketide backbone terminated by a tetramic acid amide moiety. Among other biological activities, eliamid shows a high and specific cytostatic action on human lymphoma and cervix carcinoma cell lines. The 2,4-anti relative configuration of the C-2,C-4-dimethyl substituted amide fragment was assigned by means of Breit's rule. The absolute configuration of all stereocenters was determined by a combination of degradation methods, structural similarity analysis and total synthesis. The stereogenic centers were introduced by vinylogous Mukaiyama aldol reaction and two consecutive Myers alkylations. The use of pentafluorophenyl ester as acylation agent allowed the efficient formation of tetramic acid amide. The longest linear sequence in the synthesis consist of 13 steps and proceeds with 12% overall yield. Differential spectroscopy experiments with beef heart submitochondrial particles established that eliamid is a potent inhibitor of the NADH-ubiquinone oxidoreductase complex. Additionally, biosynthesis of eliamid was investigated by feeding experiments with (13)C-labeled precursors.

MALDI-TOF Mass Spectrometry Analysis of Amphipol-Trapped Membrane Proteins

Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a β-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Effects of 4-hydroxy-2-nonenal on beef heart mitochondrial ultrastructure, oxygen consumption, and metmyoglobin reduction

The effects of 4-hydroxy-2-nonenal (HNE) on mitochondria isolated from bovine hearts (n=5) were assessed using ultrastructure, oxygen consumption, membrane permeability, HNE binding, and metmyoglobin reduction in vitro. Pre-incubation (pH 5.6 and 7.4 at 25°C) of mitochondria with HNE decreased oxygen consumption compared with samples without HNE (P<0.05). Electron microscopy revealed that HNE-treated mitochondria were swollen and had increased membrane permeability at pH 7.4, compared with ethanol controls. Conversely, mitochondria incubated with HNE at pH 5.6 had decreased volume and permeability. Fluorescence studies indicate that HNE binds to the membrane of mitochondria isolated from bovine cardiac muscle (at pH 5.6 and 7.4). HNE-treated mitochondria at both pH 5.6 and 7.4 had lower metmyoglobin reduction and NADH dependent metmyoglobin reductase activity compared with control mitochondria without HNE (P<0.05). In addition to covalent binding with myoglobin, HNE may influence beef color stability by interacting with mitochondria.

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice

We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with other inhibitors of mitochondrial function, including 5-hydroxydecanoate (specific for mitochondrial potassium-ATP channels), benzylguanidine (complexes I and III), and the cell-permeable manganese tetrakis (4-benzoic acid) porphyrin (a mimic of mitochondrial superoxide dismutase). In parallel with the blood pressure-lowering effect of rotenone, the DOCA-salt-induced increases in urinary 8-isoprostane excretion and in reactive oxygen species production of isolated kidney mitochondria were both significantly attenuated. Conversely, the DOCA-salt-induced reduction of urinary nitrate/nitrite excretion was significantly elevated. Following DOCA-salt treatment, mice deficient in NADPH oxidase subunits gp91(phox) or p47(phox) exhibited a partial attenuation of the hypertensive response at early but not later time points. Thus, the mitochondrial respiratory chain is a major source of oxidative stress in DOCA-salt hypertension, whereas NADPH oxidase may have a relatively minor role during the early stage of hypertension.

Changes in content of coenzyme Q10 in beef muscle, beef liver and beef heart with cooking and in vitro digestion

The goals of this study were to determine the contents and digestibility of coenzyme Q10 (CoQ10) in beef muscle, beef liver and beef heart, which are rich in coenzyme Q10, as well as the effects of cooking on coenzyme Q10 content. Coenzyme Q10 contents were found as 109.97 ± 1.54 μg/g in beef heart, 33.34 ± 1.43 μg/g in beef liver and 23.47 ± 1.06 μg/g in beef M. longissimus dorsi muscle. The lowest retention in coenzyme Q10 content was found as 69.42 ± 0.56% ( p < 0.01) in beef heart after frying processing. Coenzyme Q10 retentions were also found as 76.38 ± 0.89% and 77.19 ± 1.09% after frying of beef liver and boiling of beef M. longissimus dorsi muscle, respectively. Digestibility of coenzyme Q10 in beef heart (65.84 ± 0.84%) and beef liver (68.17 ± 0.60%) was significantly higher than the digestibility of coenzyme Q10 in beef M. longissimus dorsi muscle (60.16 ± 0.53%) ( p < 0.01).

Community-Associated Methicillin-Resistant Staphylococcus aureus Survival on Artificial Turf Substrates

Artificial turf has been suggested as a risk factor for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). This is an experimental study looking at survival of CA-MRSA on artificial turf. MRSA strain USA-300-0114 was grown as either planktonic cells or biofilms in liquid cultures of beef heart infusion broth overnight at 37 °C. Beakers containing ProGrass (Pittsburgh, PA) turf were inoculated at the dirt interface with either ∼5 × 10 planktonic bacteria or with biofilms. The inoculum included varying nutrient conditions consisting of spent medium, saline, or 5% mucin. The beakers were incubated at 37 °C in ambient air. The main outcome measure was the number of surviving colony-forming units determined by plating on mannitol salt agar. Survival was biphasic with a colony-forming unit drop from ∼5 × 10 to ∼5 × 10 after the first week followed by survival of between 10 and 10 bacteria until termination of the experiment (20-50 d). Survival was dependent on nutrients, and washed cells survived less than 1 d. Mucin could serve as a nutrient source and slightly increased surviving numbers to 10-10 bacteria. Biofilm formation did not influence survival. CA-MRSA survivability on artificial turf surfaces is dependent on the availability of nutrients. These results suggest that CA-MRSA could survive on artificial turf in significant numbers for 1 wk, and lower numbers for at least 1 month, if supplied with appropriate nutrients. Outdoor environmental conditions may affect these findings.

5-Hydroxytryptamine is a substrate for both species of monoamine oxidase in beef heart mitochondria.

The activity of beef heart mitochondrial monoamine oxidase towards 5-hydroxytryptamine (5-HT) is inhibited by the selective inhibitors clorgyline, PCO [5-phenyl-3-(N-cyclopropyl)-ethylamine-1,2,4-oxidiazole] and Deprenyl with a biphasic dependence on the inhibitor concentration. The activities towards tyramine, dopamine and tryptamine were also inhibited in a biphasic manner, but the apparent proportions of the two enzyme species active on dopamine and tryptamine depended on the inhibitor used. Phenethylamine oxidation was inhibited in a monophasic manner suggesting that only a single enzyme species was responsible for the oxidation of this substrate. The biphasic response of 5-HT oxidation to inhibition by clorgyline persisted when functionally competent mitochondria were used and was unaffected by the soluble amine oxidase inhibitors semicarbazine and aminoguanidine. These results indicate that the behaviour of the beef heart enzyme towards selective inhibitors is considerably different from that of any preparations previously studied and suggest that the classification of monoamine oxidase activites into A and B types may be only of limited usefulness.

Relaxation of the guinea-pig tracheal chain preparation by N6,2'-O-dibutyryl 3',5'-cyclic adenosine monophosphate.

Abstract N 6,2′-O-Dibutyryl 3′,5′-cyclic adenosine monophosphate (dibutyryl 3′,5′-amp), isoprenaline and theophylline relax the guinea-pig tracheal chain preparation; whereas 3′,5′-cyclic adenosine monophosphate (3′,5′-amp) does not. The relaxant effect of isoprenaline, but not that of dibutyryl 3′,5′-amp, was blocked by propranolol. 3′,5′-amp is hydrolyzed rapidly by beef heart phosphodiesterase whereas dibutyryl 3′,5′-amp is not. The presence of equimolar concentrations of dibutyryl 3′,5′-amp does not alter the rate of phosphodiesterase mediated hydrolysis of 3′,5′-amp. These data are consistent with the theories that relaxation of the guinea-pig trachea may be mediated by 3′,5′-amp and that dibutyryl 3′,5′-amp acts by mimicking 3′,5′-amp at its site of action.

An Experimental Model of Tool Mark Striations in Soft Tissues Produced by Serrated Blades

Stab wounds produced by serrated blades are generally indistinguishable from stab wounds produced by non-serrated blades, except when visible tool mark striations are left on severed cartilage. We explored the possibility that similar striations may be produced on the soft tissues of internal organs. Loin of beef, bovine kidney, and pig heart, liver, and aorta were each stabbed 20 times with a coarsely serrated blade. The walls of the stab tracks were exposed and documented by photography, cast with dental impression material, and the casts photographed. Striations were identified in all of the tissues in every stabbing, but their consistency and quality varied between tissues. Striations were most easily seen in liver, heart, and aorta. Tool mark striations in soft tissues other than cartilage have not been described in homicidal stabbings, likely because they have not been sought. We suggest that the walls of stab wound tracks should be exposed, and tissue striations should be sought as a means of identifying the weapon as having a serrated blade.

HPLC Separation of Fluorescent Products of Lipid Peroxidation in Erythrocytes and Mitochondria

Polyunsaturated fatty acids found in membrane phospholipids are readily oxidized by free radicals producing highly reactive aldehydes such as malondialdehyde. These aldehydes react with biological material to form fluorescent lipid peroxidation end products known as lipofuscin-like pigments. We studied fluorescent pigments from beef heart mitochondria incubated with malondialdehyde in vitro and from erythrocytes of patients with Alzheimer’s disease to evaluate their potential as markers of oxidative stress and to develop HPLC methods for their qualitative analysis. We used tridimensional fluorescence spectral arrays and synchronous fluorescence spectra in connection with HPLC separation with fluorescence detection. Stable fluorophores were found in both models and were successfully resolved into several distinctive fractions. This creates the basis for further characterization of this relatively less studied group of products of oxidative damage.

Silicone Modeling of the Interior Spaces of Hollow Organs: Use in Dog and Manatee Respiratory Tract and in a Beef Heart

Problem statement: The mechanism, by which the Florida manatee (Trichechus manatus latirostris) vocalizes, remains unknown because the manatee larynx does not contain true vocal cords. Since sound can be generated when air passes through a narrow respiratory structure we needed to visualize the internal anatomy of manatee respiratory tract to locate any candidate regions for study. Approach: To visualize the internal anatomy of upper and lower manatee respiratory tract we have developed a rapid but accurate method of modeling these structures using liquid silicone. We first tested this technique on the respiratory structure of a cadaver dog and then applied it to two small manatees which had died through natural causes. Incisions were made in the trachea of both dog and manatees and commercially available liquid silicone was then forced into the upper and lower respiratory tracts used a slightly modified common automobile grease gun. The animals were then refrigerated overnight and the silicone was allowed to cure for a period of 24 h. Results: In dog, we removed cured silicone model by applying mild force to it after surgically opening the nasal cavity. In the manatees some dissection was necessary for release of mold from the upper nasal cavity, but only mild force was necessary with no dissection to release silicone model from the lower tract. Because the models created exhibited great accuracy and fine structure, including presence of tertiary bronchi in the manatee respiratory tract, we realized that the technique was applicable for use in other hollow organs. We applied this method to the visualization of internal structure of a fresh beef heart and were pleased with the accuracy and detail of model produced. Conclusion: We suggest that this technique can be adopted for three-dimensional visualization of the internal structure and volume estimation of many hollow organs in a wide variety of organisms with both minimal effort and cost.

Purification of the mitochondrial calcium uniporter from beef heart and characterization of its properties

A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.

Quantification of Methadone and its Metabolite 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in Third Instar Larvae of Lucilia sericata (Diptera: Calliphoridae) Using Liquid Chromatography-Tandem Mass Spectrometry

Entomotoxicology studies the application of toxicological analysis on necrophageous insects present on human remains. This paper describes the development and validation of a sensitive liquid chromatography tandem mass spectrometry method for quantification of methadone and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in developmental stages of Lucilia sericata. One single larva was pulverized in a disposable vial and then extracted with 1-chlorobutane. After evaporation of the organic layer, samples were reconstituted in the mobile phase. Chromatographic separation was achieved on a NUCLEODUR Sphinx RP column with a liquid chromatographic gradient (0.1% formic acid and methanol), ensuring the elution of methadone and EDDP within 15 min. The method was fully validated according to international guidelines. The use of liquid liquid extraction was demonstrated to be effective (matrix effect < 27% and recovery > 66%). The method was linear over the dynamic range (10-400 pg/mg larva) with excellent within- and between-run precision and bias (CV% < 5%). The lower limit of quantification was fixed at 10 pg/mg larva. No instability of the extracted samples was observed in the autosampler after three freeze/thaw cycles and after two months at -20 degrees C. The validated method was applied to third instar larvae of Lucilia sericata reared on beef heart spiked with 4 microg/g methadone and on a postmortem methadone overdose case. The validation and actual sample analysis showed that the method is sensitive, rugged, precise, accurate, and well-suited for routine analysis of methadone and EDDP in a single larva obtained from forensic cases.