Biomedical subjects

M L Chiu

Publications and source records attributed to M L Chiu.

At least 19 recordsLinked to original sources

PubMed1996

Characterization of the covalent binding of thiostrepton to a thiostrepton-induced protein from Streptomyces lividans.

Thiostrepton is a highly modified multicyclic peptide antibiotic synthesized by diverse bacteria. Although best known as an inhibitor of protein synthesis, thiostrepton is also a potent activator of gene expression in Streptomyces lividans. In these studies, we characterize the nature of the interaction between thiostrepton and two proteins that it induces, TipAL and TipAS. In the absence of added cofactors, thiostrepton formed a complex with either TipAL or TipAS in aqueous solution. The TipA-thiostrepton complex was not dissociated by denaturants such as SDS, urea, or disulfide reducing agents. The mass of the TipAS-thiostrepton complex as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) was equivalent to the sum of TipAS and thiostrepton. Thiostrepton also reacted spontaneously with free cysteine (but not with other amino acids tested) to generate stable compounds having masses equivalent to thiostrepton plus 3 to 4 cysteines. Blocking experiments indicated that complex formation required dehydroalanine residues on thiostrepton and cysteine residues on TipAS. When the TipAS-thiostrepton complex was digested with trypsin and analyzed by MS, the thiostrepton adduct was found bound only to the unique cysteine-containing TipAS peptide fragment. Amino acid analysis confirmed that the TipAS-thiostrepton complex contained lanthionine, the product of a reaction between dehydroalanine and cysteine. Together, these data document a covalent attachment of thiostrepton to TipA proteins mediated by bond formation between dehydroalanine of thiostrepton and cysteine of TipAS. Implications regarding the function of TipAS as a thiostrepton (electrophile)-sequestering protein and thiostrepton-mediated activation of TipAL as a model of irreversible transcriptional activation are discussed.

Alanine↗

PubMed1994

An adherens junction protein is a member of the family of lactose-binding lectins.

We previously described a pig junction protein of M(r) 37,000 found in oral epithelium but not in epidermis, limited to suprabasal cells, and colocalizing by immunofluorescence with adherens junction proteins. A 1.1-kilobase pair cDNA of the 37-kDa protein yielded an open reading frame encoding a 323-amino acid protein of 35,852 Da, and Northern analysis demonstrated a band of 1.2 kilobases in tongue RNA. Secondary structure predictions indicate that the 37% identical 16-17-kDa N- and C-terminal domains from beta-sheet-rich barrels linked by a compact proline-rich segment. The protein is 72% identical in amino acid sequence and shares symmetrical two-domain structure with L-36, a lectin of unknown function from rat intestine, indicating that the 37-kDa protein is the porcine form of L-36. Of the homologous lactose binding lectins known, two others, invertebrate lectins, share this symmetrical structure. Expression of the C-terminal domain of the pig lectin in bacteria yields a lectin which binds lactosyl-Sepharose, and binding is inhibited by lactose. The expressed protein binds a glycoprotein of 120 kDa from pig tongue epithelium on Western blots, and this is also inhibited by lactose. The findings suggest that the lectin function may be involved in the assembly of adherens junctions.

Amino Acid Sequence↗

PubMed1994

1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule.

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.

Animals↗

PubMed1994

The heme iron coordination complex in His64(E7)Tyr recombinant sperm whale myoglobin.

By using site-directed mutagenesis of recombinant sperm whale (SW) myoglobin, the native distal histidine residue, at position 64 (the helical position E7), has been replaced with a tyrosine. The mutation of His64Tyr SW myoglobin has an analogous heme iron electronic structure as that of native hemoglobins M Boston and M Saskatoon. Optical spectroscopy showed that the distal tyrosine bound to the heme iron had a pK value of 5.6. In the pH range of 4.7-11.0, electron spin resonance spectroscopy suggested the presence of two heme iron ligation schemes: the heme iron bound to a distal water molecule or to a distal tyrosine residue. The heme iron coordination in the wild-type myoglobin and in the His64Tyr SW Mb mutant was studied by X-ray absorption near-edge structure (XANES) spectroscopy. Indeed, the heme iron K-edge reflects the electronic organization of the metal inside the six-coordinated complex. Comparative analysis of X-ray absorption heme iron K-edge shapes showed that the heme iron of His64Tyr SW myoglobin is bound to the oxygen atom from the phenol group of the distal tyrosine residue (Fe-OH phi). When the pH value decreased from pH 7 to 5.6, the Fe-OH phi bond strength decreased, resulting in an increase of the heme iron high-spin population of His64Tyr SW myoglobin. At low pH values, the Fe-OH phi bond can be disrupted with the possibility of heme iron binding of another ligand having a higher affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Correlation between the steric bulk of the distal E7 and E11 residues and the tilt of the FeCN unit in cyanometmyoglobin as determined by NMR from the orientation of the magnetic axes in single and double point mutants.

The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)-->Gly (H[E7]G), Val68(E11)-->Ile (V[E11]I), and Val68(E11)-->Ala (V[E11]A) and the double mutants His64-(E7)-->Gly:Val68(E11)-->Ile (H,V[E7,E11]G,I) and His64(E7)-->Gly:Val68(E11)-->Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (approximately 8 degrees) from that in the wild type (WT) (approximately 16 degrees), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C delta H3 directed away from the iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin.

Sequence-specific backbone 1H and 15N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D 1H-15N-correlated spectra obtained from uniformly 15N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.

Amino Acid Sequence↗

PubMed1994

Activity of fluconazole: postantifungal effect, effects of low concentrations and of pretreatment on the susceptibility of Candida albicans to leucocytes.

Exposure times and serum concentrations in humans after treatment with multiple doses of fluconazole have been studied to measure: (a) the postantifungal effect (PAFE) on Candida albicans, at two concentrations of the drug in the presence or absence of 10% human serum; (b) the activity of low concentrations of the drug on yeasts previously exposed to fluconazole with or without 10% human serum; and (c) the effect of fluconazole pretreatment on the fungicidal activity of leucocytes and serum against C. albicans. Fluconazole showed no PAFE against C. albicans between -0.1 and -0.7 h, but when the assays were performed in the presence of serum, concentration dependent PAFEs were obtained (1.1 h-3.6 h). Pretreated yeasts were more susceptible than untreated yeasts to low concentrations of the drug. The decrease in growth was dependent on the concentration used in pretreatment. Growth delay was also more marked when the yeast cells were tested in the presence of serum. Pretreatment of the growing C. albicans cells with fluconazole increased their vulnerability to killing by leucocytes mainly in the first hour (P < 0.05). These results could partially explain why fluconazole has more activity in vivo than in vitro.

Blood Physiological Phenomena↗

PubMed1994

Expression of recombinant human multidrug resistance P-glycoprotein in insect cells confers decreased accumulation of technetium-99m-sestamibi.

UNLABELLED: The multidrug-resistant P-glycoprotein is a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR) which appears to function as an efflux transporter of a variety of potent chemotherapeutic agents. METHODS: To directly demonstrate that 99mTc-sestamibi is recognized by the human P-glycoprotein, we overexpressed recombinant human MDR1 P-glycoprotein in host Sf9 insect cells using a baculoviral vector and correlated expression of the gene product with 99mTc-sestamibi accumulation. RESULTS: In parental Sf9 cells and in wild-type baculoviral infected (control) cells, 99mTc-sestamibi accumulation asymptotically approached a plateau of 650 fmoles (mg protein)-1 (nMo)-1 and 337 fmoles (mg protein)-1 (nMo)-1, respectively. In MDR1 baculoviral infected cells, P-glycoprotein expression was maximal at 72 hr postinfection, while 99mTc-sestamibi accumulation was reduced to 12 fmole (mg protein)-1 (nMo)-1. Verapamil (500 microM), the classical MDR modulator, produced an approximately 300% enhancement of 99mTc-sestamibi accumulation in Sf9 cells expressing MDR1 P-glycoprotein, but only a 50% enhancement in parental Sf9 cells, consistent with verapamil-induced inhibition of P-glycoprotein-mediated 99mTc-sestamibi efflux. CONCLUSIONS: These data demonstrate that the recombinant protein is transiently expressed in a functional state capable of drug transport in Sf9 cell membranes and that 99mTc-sestamibi is a transport substrate recognized by the human MDR1 P-glycoprotein. Technetium-99m-sestamibi may prove useful for functionally characterizing P-glycoprotein expression in human tumors in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Solution structure determination of the heme cavity in the E7 His-->Val cyano-met myoglobin point mutant based on the 1H NMR detected dipolar field of the iron: evidence for contraction of the heme pocket.

The 1H NMR spectrum of the cyanomet complex of the sperm whale His[E7]Val myoglobin (Mb) point mutant has been analyzed by 2D methods to yield the assignments for the active site residues, including the substituted Val E7. The dipolar shifted proximal residues are used to quantitatively locate the magnetic axes for the paramagnetic susceptibility tensor in the molecular framework. The orientation of the major axis, which correlates with the ligand tilt, is approximately 15 degrees from the heme normal, as found in wild-type (WT) Mb, but is tilted in a direction rotated approximately 40 degrees toward the heme gamma-meso position with respect to WT and similar to that in the His[E7]Gly mutant [Rajarathnam, K., La Mar, G. N., Chiu, M., & Sligar, S. G. (1992) J. Am. Chem. Soc. 114, 9048-9058]. The altered direction of an unchanged tilt angle for the Fe+3-CN unit is shown to be qualitatively consistent with earlier computations of the potential energy surface for MbCO [Kuriyan, J., Wilz, S., Karplus, M., & Petsko, G. A. (1986) J. Mol. Biol. 192, 133-154]. It is concluded that His E7 does not significantly contribute to the ligand tilt but strongly influences the direction of tilt. Deviations between observed and predicted dipolar shifts for the E-helix backbone protons and perturbed patterns of their respective nuclear Overhauser effect between the E-helix and the heme 1,8-methyls are separately analyzed for movement of the E-helix and agree on a translation of the E-helix of the order of 0.8 A in a direction toward the iron. The discrepancy between observed and predicted dipolar shifts for Phe CD1 indicates a approximately 0.5-A movement by the ring parallel to the heme and towards the E-helix. The E-helix and Phe CD1 movements are consistent with a contraction of the pocket to fill the space created by the His-->Val substitution. The correlation between the observed dipolar shifts of the substituted Val E7 side chain and those calculated as a function of rotation of the residue with and without movement of the E-helix confirm the movement of the E-helix and allow a quantitative description of the Val orientation. It is concluded that the dipolar field of the paramagnetic susceptibility tensor provides an important quantitative constraint for defining the heme cavity structure in cyanomet complexes of distal point mutants of myoglobin and hemoglobin.

Amino Acid Sequence↗

PubMed1993

Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex.

The multidrug-resistant P-glycoprotein (Pgp), a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR1), appears to function as an energy-dependent efflux pump. Many of the drugs that interact with Pgp are lipophilic and cationic at physiological pH. We tested the hypothesis that the synthetic gamma-emitting organotechnetium complex, hexakis(2-methoxyisobutylisonitrile)technetium(I) ([99mTc]SESTAMIBI), a lipophilic cationic radiopharmaceutical, could be a suitable Pgp transport substrate capable of functional imaging of the MDR phenotype. The cellular pharmacological profile of [99mTc]SESTAMIBI transport was examined in Chinese hamster V79 lung fibroblasts and the 77A and LZ derivative cell lines which express modestly low, intermediate, and very high levels of Pgp, respectively. Steady-state contents of [99mTc]SESTAMIBI in V79, 77A, and LZ cells were 10.0 +/- 0.5 (SEM) (n = 9), 3.6 +/- 0.5 (n = 8), and 0.4 +/- 0.02 (n = 9) fmol.(mg protein)-1 (nMo)-1, respectively, consistent with enhanced extrusion of the imaging agent by Pgp-enriched cells. Maximal doses (> 100 microM) of the multidrug-resistant reversal agents verapamil and cyclosporin A enhanced [99mTc]SESTAMIBI accumulation in V79, 77A, and LZ cells by approximately 10-, 25-, and 200-fold, respectively. The median effective concentration values for tracer accumulation in the presence of verapamil in V79, 77A, and LZ cells were 4, 100, and 200 microM, and those for cyclosporin A were 0.9, 3, and > 25 microM, respectively. Pgp-mediated [99mTc]SESTAMIBI transport occurred against its electrochemical gradient and was found to be ATP dependent displaying an apparent Km of 50 microM. Carrier-added [99Tc]SESTAMIBI was 11- to 13-fold less toxic in multidrug-resistant cells, and inhibited photolabeling of Pgp by [125I]iodoaryl azidoprazosin in a concentration-dependent manner; half-maximal displacement was observed at approximately 100- to 1000-fold molar excess [99Tc]SESTAMIBI. Exploiting the favorable gamma emission properties of 99mTc, functional expression of Pgp was successfully imaged in human tumor xenographs in nude mice with pharmacologically inert tracer quantities of [99mTc]SESTAMIBI. Functional imaging with these organotechnetium complexes may provide a novel mechanism to rapidly characterize Pgp expression in human tumors in vivo, target reversal agents in vivo, and ultimately provide a means to direct patients to specific cancer therapies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Detection of adriamycin-induced cardiotoxicity in cultured heart cells with technetium 99m-SESTAMIBI.

Adriamycin, a broad-spectrum cytotoxic agent useful in cancer chemotherapy, is limited by a dose-dependent cardiomyopathy mediated in part by disruption of mitochondrial energetics. Hexakis(2-methoxyisobutyl isonitrile)technetium(I) (99mTc-SESTAMIBI) is a gamma-emitting radiopharmaceutical with myocellular accumulation properties dependent on mitochondrial membrane potential. To test the hypothesis that 99mTc-SESTAMIBI could monitor Adriamycin-induced alterations in cardiac energetics, cultured chick heart cells were treated with Adriamycin and 99mTc-SESTAMIBI tracer kinetics were determined. Concentration- and time-dependent depression of 99mTc-SESTAMIBI accumulation was evident within 60 min of treatment. The apparent Ki for acute Adriamycin inhibition of tracer accumulation was 82 microM. After 24 h of treatment, Adriamycin concentrations as low as 0.1 microM demonstrated detectable inhibitory effects. The apparent Ki for this subchronic Adriamycin inhibition of 99mTc-SESTAMIBI accumulation was 18 microM. Subchronic concentration-dependent increases in adriamycin-induced myocellular injury as reflected by lactate dehydrogenase (LDH) release correlated inversely with decreases in 99mTc-SESTAMIBI accumulation. These data further support a contribution from altered mitochondrial energetics to Adriamycin-induced injury and establish a pharmacological foundation for pursuing the possibility of noninvasive imaging of chronic Adriamycin cardiotoxicity in cancer patients using 99mTc-SESTAMIBI.

Analysis of Variance↗

PubMed1992

Comparison of neutral and cationic myocardial perfusion agents: characteristics of accumulation in cultured cells.

Uptake and washout kinetics of two new neutral lipophilic technetium-99m-labeled boronic acid adducts of technetium tris(dioxime) (BATO complexes) were studied in monolayers of contractile chick heart cells and compared to the cationic myocardial perfusion agents, 99mTc(CNCH2C(CH3)2OCH3)6+ (Tc-MIBI) and 201Tl+. 99mTcCl(CDOH)2(CDO)(BCH3), where CDO = cyclohexanedione dioxime (CDO-MeB), had a 7-fold greater net accumulation than Tc-MIBI and the most rapid unidirectional washout with a fast initial phase and a slower secondary component. Incubation with cationic membrane transport inhibitors or metabolic inhibitors had little or modest influence, respectively, on uptake of these BATO complexes. Studies with NIH 3T3 fibroblasts indicated that the neutral complexes did not show myocyte specific accumulation.

3T3 Cells↗

PubMed1992

Restricted tissue distribution of a 37-kD possible adherens junction protein.

A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.

Actins↗

PubMed1992

Comparative effects of neutral dipolar compounds and lipophilic anions on technetium 99m-hexakis (2-methoxyisobutyl isonitrile) accumulation in cultured chick ventricular myocytes.

RATIONALE AND OBJECTIVES: Non-flow-dependent myocellular accumulation and uptake kinetics of the myocardial perfusion and viability imaging agent, hexakis (2-methoxyisobutyl isonitrile) technetium 99m(I) (Tc-SESTAMIBI), are thermodynamically driven by large negative sarcolemmal and mitochondrial membrane potentials, and can be enhanced by addition of the lipophilic anion, tetraphenylborate (TPB). To further understand the general properties required of a co-administered compound for increasing the kinetic response of Tc-SESTAMIBI to membrane potential, a systematic appraisal of additional candidate lipid-soluble anions and neutral dipolar compounds was undertaken. METHODS: Each compound was biologically tested for its ability to enhance Tc-SESTAMIBI accumulation in a cultured heart cell model, and electronic dipole moments were evaluated using semi-empirical molecular orbital calculations. RESULTS: Of this series, phloretin (100 microM), TPB (10 microM), and to a lesser degree, 8-anilino-1-naphthalene sulfonate (100 microM) enhanced myocellular accumulation of Tc-SESTAMIBI. Phloretin enhancement was pH-dependent, showing maximal effect at pH 7.4, and was not additive to the augmentation induced by TPB. A series of additional lipid soluble anions and structural analogues of phloretin were without effect. CONCLUSION: Although selected compounds enhanced Tc-SESTAMIBI accumulation, overall, no direct relationship of dipole moment to biologic enhancement was demonstrated.

Anilino Naphthalenesulfonates↗

PubMed1992

Severe gastric mucosal changes following vagotomy with duodenogastric reflux.

We conducted an experimental study of the morphological changes in gastric mucosa following vagotomy, in particular of the chronic effects of duodenogastric reflux. Male Wistar rats were divided into four groups: sham operation, duodenogastric reflux (DGR), truncal vagotomy with duodenogastric reflux (TV + DGR), and truncal vagotomy with bypass (TV + bypass). The last operation was performed to observe the effects of vagotomy without reflux. The duodenogastric reflux procedure was established by cutting the duodenum just distal to the orifice of bile and pancreatic ducts following gastrojejunostomy. After 12 and 30 weeks, animals were killed for morphological studies of gastric mucosa. The TV + DGR group showed formation of multiple elevated lesions macroscopically, and microscopically showed marked atrophy of gastric glands and proliferation of cystically dilated adenomatous lesions, which were regarded as precancerous or paracancerous lesions. The DGR group showed chronic gastric ulcer and hyperplasia of the gastric glands. However, the TV + bypass group showed no remarkable changes in the gastric mucosa. These results indicate that chronic effects of duodenogastric reflux following vagotomy induce severe inflammatory changes as well as structural alteration of the gastric mucosa. Therefore, a regular follow-up observation is necessary after vagotomy when combined with a drainage procedure because the latter could promote duodenogastric reflux and produces pathological changes to the stomach.

Animals↗

PubMed1992

Divergent kinetics of 201Tl and 99mTc-SESTAMIBI in cultured chick ventricular myocytes during ATP depletion.

BACKGROUND: Thallous chloride (201Tl) and hexakis(2-methoxyisobutyl isonitrile) technetium (I) (99mTc-SESTAMIBI) are myocardial perfusion imaging agents with biological properties that also reflect tissue viability. Initial myocellular uptake rates of 201Tl reflect activity of Na,K-ATPase, whereas those of 99mTc-SESTAMIBI reflect mean plasma membrane potential. METHODS AND RESULTS: To better understand the mechanistic responses of these tracers to myocellular injury, cultured chick embryo cardiac myocytes were metabolically inhibited in iodoacetate (1 mM) and rotenone (10 microM) for up to 2 hours, and initial uptake rates of each agent were determined at successive intervals along with correlative cellular contents of ATP, sodium, and potassium and lactate dehydrogenase release. ATP content fell from 30.5 +/- 1.4 to 2.7 +/- 0.9 nmol.(mg protein)-1 within 2 minutes, whereas sodium and potassium contents ran down their thermodynamic gradients more slowly (t 1/2 approximately 60 minutes). Modestly severe cell injury was produced at 2 hours as estimated by lactate dehydrogenase release (18% of total). Initial uptake rates of 201Tl declined from 6.9 +/- 0.8 to 4.0 +/- 0.4 fmol.(mg protein)-1.(nMo)-1.(min)-1 by 20 minutes and remained depressed and ouabain (100 microM)-insensitive at 30 +/- 13% of control. Conversely, initial uptake rates of 99mTc-SESTAMIBI increased from 10.6 +/- 0.8 to 15.0 +/- 0.6 fmol.(mg protein)-1.(nMo)-1.(min)-1 within 10 minutes, remained elevated for 40-60 minutes, and later declined to low values. Injury-induced enhancement of initial uptake rates of 99mTc-SESTAMIBI were insensitive to ouabain (100 microM), carbonyl cyanide-m-chlorophenyl hydrazone (5 microM), and valinomycin (1 microgram/ml) but were significantly inhibited by 130 mM Ko buffer, Ba2+ (1 mM), glybenclamide (100 microM), and quinacrine (10 microM). CONCLUSIONS: Uptake rates of 201Tl monotonically decline, correlating with Na-K pump inhibition from ATP depletion. Conversely, uptake rates of 99mTc-SESTAMIBI at first increase above control for 40-60 minutes, indicating a mean plasma membrane hyperpolarization possibly resulting from opening of ATP-sensitive and arachidonic acid-activated potassium channels, before declining to low values with more severe cell injury. Correlative non-flow-dependent relations between 201Tl and 99mTc-SESTAMIBI contain information regarding the degree of myocellular injury.

Adenosine Triphosphate↗

PubMed1992

Subcellular distribution and analysis of technetium-99m-MIBI in isolated perfused rat hearts.

To address the apparent discrepancy between cultured cells and whole heart preparations, Langendorff-perfused rat hearts loaded with hexakis (2-methoxyisobutyl isonitrile) technetium (I) (99mTc-MIBI) were fractionated by a standard differential centrifugation method and fractional contents of 99mTc-MIBI were correlated with the mitochondrial marker, malate dehydrogenase (MDH), and mitochondrial substrates. The "cytosolic" fraction nominally contained 89% +/- 3% of total 99mTc-MIBI, but also contained 91% +/- 1% of total MDH activity by this method. Chromatographic analysis of activity in the "cytosolic" fraction demonstrated greater than 95% of the agent was present as the original free cationic complex; binding to a small molecular weight cytosolic protein was not involved in localization. Addition of the mitochondrial uncoupler CCCP (5 microM) to both "mitochondrial" and "cell fragment" pellets released up to 84% +/- 8% of 99mTc-MIBI content and addition of the mitochondrial substrate succinate (10 microM) in the presence of rotenone (1 microM) enhanced 99mTc-MIBI content by up to 139% +/- 52% over the control. These correlative data from rat hearts indicate that approximately 90% of 99mTc-MIBI activity in vivo is associated with mitochondria in an energy-dependent manner as a free cationic complex, but migrates during fractionation/centrifugation.

Animals↗

PubMed1991

Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species.

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.

Carbon Isotopes↗