Bovine Heart Muscle Research Articles

Post‐embryonic development of Chrysomya putoria (Diptera: Calliphoridae) in swine and bovine tissues in South America: implications for forensic entomology

AbstractNecrophagous diptera are used in forensic entomology to measure the post‐mortem interval (PMI) of cadavers. PMI estimates based on fly larvae, however, can be inaccurate when larval development is retarded, or accelerated, by the nutritional composition of the cadaveric tissue. We compared the post‐embryonic development of Chrysomya putoria (Wiedemann, 1830) on different tissues of pigs and cows. Forty newly hatched fly larvae per treatment were placed on 60 g of the following swine tissues: liver (T1), swine lung (T2), swine heart (T3), swine muscle (T4), bovine heart (T5), bovine lung (T6) and bovine muscle (T7). There were four replicates/treatment. Experiments were conducted in controlled conditions (28°C day/26°C night, RH 70% ± 10% and 12 h of photophase). Fly larvae reared on swine liver took longer to develop (5.2 days) than flies reared on other substrates (from 3.5 to 4.5 days), and this difference was statistically significant. The total duration of development on swine liver (T1) was significantly slower (8.7 days) when compared to the other treatments. Larvae reared on swine lungs (T2) were significantly heavier in the third instar ( = 61.2 mg) than larvae reared on swine liver (T1), swine muscle (T4), bovine lung (T6) and bovine muscle (T7). Total larval survival was high (above 76%) in all treatments except for T1 (66%). There was no deviation from the expected adult sex ratio, and the percentage of abnormalities remained within the expected range. These results reinforce the idea that, to achieve greater accuracy in PMI estimates, the type of tissue on which the larvae develops needs to be taken into account.

Imaging myocardial fiber orientation using polarization sensitive optical coherence tomography

Knowledge of myocardial fiber architecture is essential towards understanding heart functions. We demonstrated in this study a method to map cardiac muscle structure using the local optical axis obtained from polarization-sensitive optical coherence tomography (PSOCT). An algorithm was developed to extract the true local depth-resolved optical axis, retardance, and diattenuation from conventional round-trip results obtained in a Jones matrix-based PSOCT system. This method was applied to image the myocardial fiber orientation in a bovine heart muscle sample.

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.

3B1112 Preparation and structural analysis of the membrane embedded rotor ring of the FoF1 ATPsynthase from bovine heart muscle(3B Membrane proteins 2,The 49th Annual Meeting of the Biophysical Society of Japan)

3B1112 Preparation and structural analysis of the membrane embedded rotor ring of the FoF1 ATPsynthase from bovine heart muscle(3B Membrane proteins 2,The 49th Annual Meeting of the Biophysical Society of Japan)

Immunohistochemistry of the monkey retina with a monoclonal antibody against subunit V of cytochrome c oxidase.

A monoclonal antibody against subunit V of cytochrome c oxidase was newly prepared from purified bovine heart muscle enzyme. This antibody showed cross-reactivity against the bovine and the human cytochrome c oxidase on Western blot. Cytochrome c oxidase in paraffin embedded sections of monkey retinas and optic nerves was examined immunohistochemically using the monoclonal antibody. The inner segment of cone and rod photoreceptors, the inner nuclear layer and the inner plexiform layer showed marked reactivities. The optic nerve fiber axons also showed high reactivities, with more in the unmyelinated fibers peripheral to the lamina cribrosa. These immunohistochemical results using the antibody are mostly agreeable with previously reported histochemical localization of cytochrome c oxidase activity in the retina, but there were some noticeable differences in the plexiform layers.

Structurally Unstable Regions in the Tropomyosin-Troponin Complex from Bovine Heart Muscle

Regulation of contraction in striated muscles requires a semi-independent movement of various domains of the tropomyosin-troponin (TmTn) complex on the actin filament. Such movement is facilitated by the presence of flexible or structurally unstable regions in these proteins. In particular, it is well documented that the central part of tropomyosin comprises a region that unfolds readily at physiological temperatures. To identify flexible regions in the native TmTn complex isolated from bovine heart muscle, we have used limited proteolysis with Staphylococcus aureus V8 protease that cleaves specifically the peptide bond on the C-terminal side of Glu. At 4°C only troponin T and troponin I are digested at positions 59 and 166, respectively. At 35°C Tm is also digested, initially at position 145, followed by a rapid digestion at positions 142,131 and 150. The resulting two fragments of Tm spanning residues 1-131 (2x14.9 kDa) and 151-284 (2x15.5 kDa) are resistant to further digestion in the presence of troponin, which indicates that they retain their coiled-coil structure. However, in the absence of Tn the C-terminal fragment of Tm undergoes further proteolysis. The troponin C component of the complex is resistant to proteolysis under the conditions used. The two Tm fragments, together with TnC, the N-terminal fragment of TnI (residues 1-166) and the C-terminal fragment of TnT (residues 60-284) form a 125 kDa complex that is stable at low salt concentrations, binds to F-actin irrespective of calcium, and imparts significant calcium-dependent regulation to actomyosin ATPase. These results demonstrate that the central segment of Tm spanning residues 131-151 is structurally unstable irrespective of the presence of troponin and it structure stabilizing effect on the C-terminal part of Tm.

Expression Profiling of Galectin-3-Depleted Melanoma Cells Reveals its Major Role in Melanoma Cell Plasticity and Vasculogenic Mimicry

Galectin-3 (Gal-3) is a beta-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells. In vitro, Gal-3 silencing resulted in loss of tumor cell invasiveness and capacity to form tube-like structures on collagen ("vasculogenic mimicry"). cDNA microarray analysis after Gal-3 silencing revealed that Gal-3 regulates the expression of multiple genes, including endothelial cell markers that appear to be aberrantly expressed in highly aggressive melanoma cells, causing melanoma cell plasticity. These genes included vascular endothelial-cadherin, which plays a pivotal role in vasculogenic mimicry, as well as interleukin-8, fibronectin-1, endothelial differentiation sphingolipid G-protein receptor-1, and matrix metalloproteinase-2. Chromatin immunoprecipitation assays and promoter analyses revealed that Gal-3 silencing resulted in a decrease of vascular endothelial-cadherin and interleukin-8 promoter activities due to enhanced recruitment of transcription factor early growth response-1. Moreover, transient overexpression of early growth response-1 in C8161-c9 cells resulted in a loss of vascular endothelial-cadherin and interleukin-8 promoter activities and protein expression. Thus, Gal-3 plays an essential role during the acquisition of vasculogenic mimicry and angiogenic properties associated with melanoma progression.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of cardiolipin extracted from detergent-solubilized mitochondrial electron transfer complexes

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of cardiolipin extracted from detergent-solubilized mitochondrial electron transfer complexes

Fluorescence studies on glyceraldehyde-3-phosphate dehydrogenase from bovine heart muscle.

Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that catalyses conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. ATP has been found to have an inhibitory effect on this enzyme. To establish the interaction between the enzyme and ATP, a fluorescence technique was used. Fluorescence quenching in the presence of ATP suggests cooperative binding of ATP to the enzyme (the Hill obtained coefficient equals 2.78). The interaction between glyceraldehyde-3-phosphate dehydrogenase and ATP may control not only glycolysis but other activities of this enzyme, such as binding to the cytoskeleton.

Synthesis of an unsaturated fatty acid analogue (18-(4′-azido-2′-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine: acyl-CoA-O-acyltransferase

Acylation/deacylation reactions represent a basic requirement of triglyceride as well as phospholipid metabolism, and maintenance of membrane lipid composition. In order to examine enzymes participating in these pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acids or acyl-CoAs. For the synthesis of omega-amino-oleic acid, coupling two bifunctional Cg-components was used. The described synthesis scheme is also suited for the specific generation of other fatty acid analogues with distinct positions of the double bond. The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsaturated fatty acyl-CoAs. It could be shown that the photolabel, esterified with coenzyme A, acts in the dark as a reversible inhibitor of the enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis. Several proteins could be specifically visualized using the iodinated analogue. The data indicate that this new photoaffinity label may have application to identify and characterize lipid biosynthetic enzymes using unsaturated fatty acids as well as acyl-CoA binding proteins and the active site of these proteins.

A novel UDP-sugar, UDP-3-ketoglucosamine or UDP-4-ketoglucosamine, from bovine heart muscle reduces metmyoglobin with NAD(P)H.

3-Ketoglucose and similar ketosugars have been identified in microorganisms only and little is known about their functions. UDP-sugars are widely found as an intermediate in sugar metabolism in living organisms. Yet what role UDP-sugars play, or whether they play a direct role in metabolism, is still unknown. UDP-sugars were isolated and purified from bovine heart muscle, and a UDP-sugar fraction capable of NAD(P)H-dependent catalytic reduction of metmyoglobin was detected. Subsequent identification revealed that the active UDP-sugar was UDP-3- or UDP-4-ketoglucosamine. These compounds were purified from bovine cardiac muscle by ultrafiltration, anion-exchange column chromatography and reverse-phase chromatography. They were further characterized by determination of their chemical reducing activity, by comparison with synthetic UDP-3- or UDP-4-ketoglucosamine standards using high-performance liquid chromatography, by estimation of molecular mass using fast atom bombardment mass spectrometry, and by Fourier transform infrared microspectroscopy and electron probe microanalysis. The results suggest that UDP-3- or UDP-4-ketoglucosamine reduces metmyoglobin in bovine cardiac muscle. It is important that the reducing activity displayed by this ketosugar is not the effect of UDP-3- or UDP-4-ketoglucosamine alone but depends on NAD(P)H. In other words, this action of UDP-3- or UDP-4-ketoglucosamine is catalytic.

Molecular Cloning and Biochemical Characterization of Bovine Spleen Myristoyl CoA:ProteinN-Myristoyltransferase

Myristoyl-CoA:proteinN-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. We have isolated full-length cDNA encoding bovine spleen NMT (sNMT). The single long open reading frame of 1248 bp of sNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The protein coding sequence was expressed inEscherichia coliresulting in the production of functionally active 50-kDa NMT. Deletion mutagenesis showed that the C-terminus is essential for activity whereas up to 52 amino acids can be deleted from the N-terminus without affecting the function. One of the N-terminal deletions resulted in threefold higher NMT activity. Genomic Southern analysis indicated the presence of two strong hybridizing bands with three different restriction enzyme digests suggesting the possibility of two copies of the NMT gene in the bovine genome. RNA blot hybridization analysis of total cellular RNA prepared from bovine brain, heart, spleen, lung, liver, kidney, and skeletal muscle probed with bovine sNMT cDNA revealed a single 1.7-kb mRNA. Western blot analysis of various bovine tissues with human NMT peptide antibody indicated a common prominent immunoreactive band with an apparent molecular mass of 48.5–50 kDa in all tissues. Additional immunoreactive bands were observed in brain (84 and 50 kDa), lung (58 kDa), and skeletal muscle (58 kDa). Activity measurements demonstrated that brain contained the highest NMT activity followed by spleen, lung, kidney, heart, skeletal muscle, pancreas, and liver. It appears therefore that mRNA and protein expression do not correlate with NMT activity, suggesting the presence of regulators of the enzyme activity.

HPLC of phenylthiocarbamyol labelled uridine-diphosphate-hexosamine

The biochemical functions of uridine-diphosphate-amino-sugar conjugates are largely unknown. As a first step to investigate their biological roles in glycolysis of higher organisms, bovine heart muscle, which metabolizes large quantities of glycogen, was assayed for uridine-diphosphate-hexosamine. A low molecular mass ultrafiltrate was prepared from the methanolsoluble fraction of a beef heart homogenate, and the fraction was further purified using anion-exchange and reverse-phase HPLC. Samples were analyzed by two HPLC methods; anion-exchange in borate buffer and reverse-phase ion-pair chromatography. One newly established technique employing phenylthiocarbamoyl labelled uridine-diphosphate-hexosamine also was used selectively to separate uridine-diphosphate-hexosamine from other uridine-diphosphate-sugars. Hydrolysate of purified sample was classified further according to molecular structure. As a result, phenylthiocarbamoyl labelled HPLC is suitable and easy for the detection of uridine-diphosphate-hexosamine and its derivatives.

Internal flexibility of cardiac myosins

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and segmental flexibility of cardiac myosins. Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns. DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.

Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels

Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.

Open-channel block by internally applied amines inhibits activation gate closure in batrachotoxin-activated sodium channels

We have studied the action of several pore-blocking amines on voltage-dependent activation gating of batrachotoxin(BTX)-activated sodium channels, from bovine heart and rat skeletal muscle, incorporated into planar lipid bilayers. Although structurally simpler, the compounds studied show general structural features and channel-inhibiting actions that resemble those of lidocaine. When applied to the cytoplasmic end of the channel, these compounds cause a rapid, voltage-dependent, open-channel block seen as a reduction in apparent single-channel amplitude (companion paper). Internal application of phenylpropanolamine, phenylethylamine, phenylmethylamine, and diethylamine, as well as causing open-channel block, reduces the probability of channel closure, producing a shift of the steady-state activation curve toward more hyperpolarizing potentials. These gating effects were observed for both cardiac and skeletal muscle channels and were not evoked by addition of equimolar N-Methyl-D-Glucamine, suggesting a specific interaction of the blockers with the channel rather than a surface charge effect. Kinetic analysis of phenylpropanolamine action on skeletal muscle channels indicated that phenylpropanolamine reduced the closed probability via two separate mechanisms. First, mean closed durations were slightly abbreviated in its presence. Second, and more important, the frequency of the gating closures was reduced. This action was correlated with the degree, and the voltage dependence, of open-channel block, suggesting that the activation gate cannot close while the pore is occluded by the blocker. Such a mechanism might underlie the previously reported immobilization of gating charge associated with local anesthetic block of unmodified sodium channels.

The Characterization of a Mitochondrial Acyl Carrier Protein Isoform Isolated from Arabidopsis thaliana

A cDNA clone was isolated from an Arabidopsis leaf cDNA library that shared a high degree of protein sequence identity with mitochondrial acyl carrier proteins (mtACPs) isolated from Neurospora crassa and bovine heart muscle. The cDNA encoded an 88-amino acid mature protein that was preceded by a putative 35-amino acid presequence. In vitro protein import studies have confirmed that the presequence specifically targets this protein into pea mitochondria but not into chloroplasts. These studies indicated that pea mitochondria were not only able to import and process the precursor protein but also possessed the ability to acylate the mature protein. The mitochondrial localization of this protein, mtACP-1, was confirmed by western blot analysis. Arabidopsis mitochondrial protein extracts contained two cross-reacting bands that comigrated with the mature mtACP-1 and acylated mtACP-1 proteins. The acylated form of mtACP-1 was approximately 4 times more abundant than the unacylated form and appeared to be localized predominantly in the mitochondrial membrane where the unacylated mtACP-1 was present mostly in the matrix fraction. A chloroplast fatty acid synthase system was used, and mtACP-1 was able to function as a cofactor for fatty acid synthesis. However, predominantly short- and medium-chain fatty acids were produced in fatty acid synthase reactions supplemented with mtACP-1, suggesting that mtACP-1 may be causing premature fatty acid chain termination.

Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation

We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels.

Cloning of the Cα catalytic subunit of the bovine cAMP-dependent protein kinase

The bovine Cα type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3′ untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3′ oligonucleotide was taken from the partial cDNA clone whereas the 5′ oligonucleotide was chosen by comparison of sequences of published Cα subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine Cα protein sequence, aspartic acid 286 is exchanged by an asparagine. The Cα mRNA was found to be expressed differentially in various bovine tissues.

Interaction of Zn 2+, and Cu 2+, Ions with glyceraldehyde-3-phosphate dehydrogenase from bovine heart and rabbit muscle

1. 1. Binding of Zn 2+, and Cu 2+, ions to GAPDHs from bovine heart and rabbit muscle resulted in a partial loss of enzymatic activity of both enzymes, in a time and metal ion concentration dependent manner. Cu 2+, ions caused a much larger decrease of the activity than Zn 2+, ions. 2. 2. Addition of NAD 2+, or EDTA to either enzyme resulted in a protective effect on GAPDH activity. A similar protective effect was observed following addition of 2-mercaptoethanol to the enzyme solution. 3. 3. The association constant for GAPDH-Zn 2+, complex, calculated from equilibrium dialysis data, was 0.9 × 10 4M −1 for the bovine heart GAPDH and 1.3 × 10 4M −1, for the rabbit muscle enzyme. The association constant for GAPDH-Cu 2+, complex was the same for both enzymes, 11.3 × 10 4M −1. 4. 4. Equilibrium dialysis data also revealed that in either enzyme the specific sites, binding the metal ions, are identical or very similar, and independent from each other. They are situated in the most conserved part of the enzyme molecule. 5. 5. Some zinc was found in GAPDH preparations from bovine heart. It is discussed if Zn 2+, ions could have a kind of modulation effect on GAPDH activity.