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A Constantinescu

Publications and source records attributed to A Constantinescu.

At least 37 records · Page 2Linked to original sources
PubMed1997

Regional tumor oxygen dynamics: 19F PBSR EPI of hexafluorobenzene.

We demonstrate a novel approach to measuring regional tumor oxygen tension using 19F pulse burst saturation recovery echo planar imaging (EPI) relaxometry of hexafluorobenzene. Hexafluorobenzene offers exceptional sensitivity to changes in oxygen tension, and has a single resonance making it ideal for imaging studies. By combining a pulse burst saturation recovery preparation sequence with EPI, the relaxation experiments were performed in approximately 20 min facilitating measurements of dynamic changes in pO2 accompanying interventions. Direct intratumoral administration of hexafluorobenzene permitted labeling of specific regions of interest, and imaging provided maps of pO2, confirming distinct intra tumoral heterogeneity. For a group of three Dunning prostate adenocarcinoma R3327-AT1 tumors interrogation of the central tumor region showed skewed pO2 distributions with considerable radiobiological hypoxia (approximately 90% voxels had pO2 < 15 torr) when rats breathed 33% O2. Altering the inspired gas to pure oxygen caused distributions to shift towards increased pO2 with significant increases in mean oxygen tension (p < 0.05) in two cases. Interrogation of both central and peripheral regions in a fourth tumor showed bimodal distribution for tumor oxygenation including approximately 75% voxels with pO2 > 15 torr. EPI allows the fate of individual voxels to be traced: upon altering the inspired gas to pure oxygen those voxels with baseline pO2 > 30 torr showed significant changes (p < 0.05), whereas those with pO2 < 16 torr showed minimal response. The precision of the measurements, together with the ability to simultaneously examine dynamic changes in multiple regions should provide a useful technique for investigating tumor hypoxia with respect to therapy.

Adenocarcinoma↗
PubMed1997

H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct.

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.

Animals↗
PubMed1996

Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro.

A common V(D)J recombinase that recognizes a conserved recombination signal sequence (RSS) mediates the assembly of immunoglobulin (Ig) and T cell receptor (TCR) genes in B and T cell precursors. The rearrangement of particular Ig and TCR gene segments, however, is tightly regulated with respect to cell lineage and developmental stage. Using an in vitro system, we analyzed recombinase cleavage of RSSs flanking Ig and TCR gene segments in nuclei. We found that both the lineage-specificity and temporal ordering of gene rearrangement is reflected in the accessibility of RSSs within chromatin to in vitro cleavage.

Animals↗
PubMed1996

Activation of cyclic AMP-dependent protein kinase reverses tolerance of a nucleoside transporter to ethanol.

Adenosine mediates some of the acute and chronic effects of ethanol in neural cells. In cultured NG108-15 cells, ethanol inhibits adenosine uptake via a specific facilitative nucleoside transporter leading to an increase in extracellular adenosine, activation of adenosine A2 receptors and increases in intracellular cyclic AMP (cAMP). After chronic ethanol exposure, an adaptive decrease in receptor-stimulated cAMP levels occurs. Additionally, the transporter becomes insensitive to rechallenge with ethanol and adenosine uptake is not inhibited. cAMP levels are decreased in cells chronically exposed to ethanol and we show here that cAMP-dependent kinase (PKA) activity in cellular homogenates also is decreased. Therefore, decreased cAMP-dependent phosphorylation may be responsible for loss of ethanol sensitivity. To test this hypothesis, NG108-15 cells were treated with agents that alter PKA activity and the ethanol sensitivity of adenosine transport was measured. In naive cells, decreasing PKA activity with the cAMP antagonist, Rp-adenosine-3',5'-cyclic phosphorothioate, resulted in ethanol-insensitive adenosine uptake. This effect was blocked by the phosphatase inhibitor, okadaic acid. These results suggest that loss of ethanol sensitivity is correlated with decreased PKA activity. Therefore, stimulating PKA activity in chronically treated cells should restore sensitivity of adenosine uptake to inhibition by ethanol. Indeed, the cAMP agonist, Sp-adenosine-3',5'-cyclic phosphorothioate, restored ethanol sensitivity of transport in cells treated chronically with ethanol. Our results suggest that ethanol sensitivity of adenosine transport is regulated by PKA and protein phosphatase activities in NG108-15 cells. Moreover, the effects of chronic ethanol exposure on adenosine transport can be reversed by activating PKA.

Adenosine↗
PubMed1995

Reduction and transport of lipoic acid by human erythrocytes.

Reduction of exogenous lipoic acid to dihydrolipoate is known to occur in several mammalian cells and tissues. Dihydrolipoate is a potent radical scavenger, and may provide significant antioxidant protection. Because lipoic acid appears in the bloodstream after oral administration, we have examined the reduction of exogenous lipoate by human erythrocytes. Normal human erythrocytes reduced lipoate to dihydrolipoate only in the presence of glucose; deoxyglucose did not substitute for glucose, indicating that the reduction of lipoate requires glucose metabolism. Furthermore, the reduction was shown to be NADPH dependent. Erythrocytes isolated from a human subject with a genetic deficiency of glucose-6-phosphate dehydrogenase (and, therefore, deficient in the formation of NADPH) did not reduce lipoate. Dehydroepiandrosterone, a specific inhibitor of glucose-6-phosphate dehydrogenase, inhibited lipoate reduction. Our findings imply that some of the reduction of exogenous lipoic acid is catalysed by glutathione reductase, a flavoprotein dehydrogenase; mitomycin C, an inhibitor of FAD-dependent reductases, inhibited lipoate reduction by erythrocytes, and glutathione reductase purified from human erythrocytes was observed to reduce lipoic acid in a cell-free system. We further explored these findings with erythrocyte ghosts and liposomes. Our results indicate that a transport system exists for alpha-lipoic acid and dihydrolipoate; resealed erythrocyte ghosts, containing trapped lipoamide dehydrogenase and pyridine nucleotides, reduced externally added lipoate. By contrast, liposomes prepared with enzyme and pyridine nucleotides did not catalyze reduction of lipoate. This work indicates that uptake of exogenous lipoate and reduction to dihydrolipoate by normal human erythrocytes may contribute to oxidant protection in the human bloodstream.

Biological Transport↗
PubMed1995

Glutathione reductase and lipoamide dehydrogenase have opposite stereospecificities for alpha-lipoic acid enantiomers.

The reduction of exogenous alpha-lipoic acid to dihydrolipoate by mammalian cells and tissues confers additional antioxidant protection to the cell. Both (R+) and (S-) isomers of alpha-lipoic acid were analyzed as substrates with glutathione reductase from several sources and with mammalian lipoamide dehydrogenase. Mammalian glutathione reductase catalyzed faster reduction of (S)-lipoic acid (1.4-2.4-fold greater activity) than of (R)-lipoic acid, whereas lipoamide dehydrogenase had a very marked preference for (R)-lipoic acid (18-fold greater activity) over (S)-lipoic acid. Mammalian glutathione reductase showed better affinity for (S)-lipoic acid substrate; Km values were 3.5 mM for (S)-lipoic acid and and 7 mM for (R)-lipoic acid. Glutathione reductase from yeast reduced lipoic acid less efficiently than the mammalian enymes, had a Km for both stereoisomers of about 10 mM, and showed little stereospecificity. Although (S)-lipoic acid is not formed in nature, these findings indicate that exogenous (S)-lipoic acid may have a useful role as an antioxidant for mammalian systems.

Animals↗
PubMed1994

6-Fluoropyridoxol: a novel probe of cellular pH using 19F NMR spectroscopy.

6-Fluoropyridoxol was evaluated as an intracellular pH indicator. This molecule exhibits exceptional sensitivity to changes in pH (approximately 10 ppm acid/base shift) and a pKa approximately 8.2 appropriate for physiological investigations. Using 19F NMR spectroscopy we determined both intra- and extracellular pH in whole blood and confirmed the measurements using traditional techniques: ion-electrodes and 31P NMR spectroscopy.

Animals↗
PubMed1994

Non-invasive determination of tumor oxygen tension and local variation with growth.

PURPOSE: The objective was to develop and demonstrate a novel noninvasive technique of measuring regional pO2 in tumors. The method is based on measuring 19F nuclear magnetic resonance spin-lattice relaxation rate (R1 = 1/T1) of perfluorocarbon (PFC) emulsion discretely sequestered in a tumor. METHODS AND MATERIALS: We have examined pO2 in the Dunning prostate tumor R3327-AT1 implanted in a Copenhagen rat. Oxypherol blood substitute emulsion was administered intravenously and became sequestered in tissue. Proton magnetic resonance imaging (MRI) showed tumor anatomy and correlated 19F MRI indicated the distribution of perfluorocarbon. Fluorine-19 spectroscopic relaxometry was used to measure pO2 in the tumor and repeated measurements over a period of 3 weeks showed the variation in local pO2 during tumor growth. RESULTS: Perfluorocarbon initially resided in the vascularized peripheral region of the tumor: 19F nuclear magnetic resonance R1 indicated pO2 approximately 75 torr in a small tumor (approximately 1 cm) in an anesthetized rat. As the tumor grew, the sequestered PFC retained its original distribution. When the tumor had doubled in size the residual PFC was predominantly in the core of the tumor and the pO2 of this region was approximately 1 torr indicating central tumor hypoxia. CONCLUSION: We have demonstrated a novel noninvasive approach to monitoring regional tumor pO2. Given the critical role of oxygen tension in tumor response to therapy this may provide new insight into tumor physiology, the efficacy of various therapeutic approaches, and ultimately provide a clinical technique for assessing individual tumor oxygenation.

Adenocarcinoma↗
PubMed1994

Interactions between ubiquinones and vitamins in membranes and cells.

The interaction between ubiquinones and vitamin E was studied in the inner membranes of rat liver mitochondria, liposomes and human erythrocyte plasma membranes. Free radicals were produced by addition of exogenous oxidants, and their reaction with chromanols and ubiquinone was followed by ESR and HPLC. Membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added ubiquinone. With these membrane preparations it was shown that (i) in the inner mitochondrial membranes there is a requirements for ubiquinone in the enzymatic recycling of vitamin E; (ii) succinate-ubiquinone reductase incorporated in liposomes cannot protect vitamin E in the absence of ubiquinone and (iii) in human erythrocyte plasma membranes protection against the loss of vitamin E can be provided by NADH-cytochrome-b5-dependent enzymatic recycling. We conclude that ubiquinonols (ubisemiquinones) reduce vitamin E through electron transport.

Animals↗
PubMed1994

Synthesis and evaluation of a new class of technetium-99m monocationic radiotracers from dithienyldiazalkanes.

Monocationic 99mTc complexes, [99mTcO2 (THEN)]+ and [99mTcO2(THPN)]+ derived from a new class of N2S2 chelators diethienyldiazalkanes, namely 1,6-di(2-thienyl)-2,5-diazahexane (THEN) and 1,7-di- (2-thienyl)-4,4-dimethyl-2,6-diazaheptane (THPN), have been synthesized using Sn(II) tartrate reduction of [99mTc]pertechnetate at pH 9 and extraction of the complexes as ion pairs with trifluoromethane sulfonate anion (CF3SO3-) in high radiochemical yield (approximately 90%). Radiochemical analysis of the 99mTc complexes using chromatography (Whatman Cellulose CEL300 DEAE anion exchange strip, MEK) and paper chromatography (Whatman I, acetone) revealed high radiochemical purity (> 90%). High octanol-saline partition coefficients (> 37) of [99mTcO2(THEN)]+ and [99mTcO2(THPN)]+ indicated a lipophilic nature of the complexes. Relative stability of the 99mTc complex was high as measured by DTPA challenge studies. Biodistribution of [99mTcO2(THEN)]+ and [99mTcO2(THPN)]+ in rats at 15 min post-injection showed heart-to-blood activity ratios of 0.26 and 0.46, respectively, with rapid blood clearance. Further studies involving the variation of substituents on the basic core structure of this new class of 99mTc complexes may provide radiotracers suitable for myocardial imaging.

Animals↗
PubMed1994

alpha-Lipoic acid protects against hemolysis of human erythrocytes induced by peroxyl radicals.

The azo initiator of peroxyl radicals 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induces oxidative hemolysis in human erythrocytes and subsequent hemoglobin oxidation. Using the degree of hemolysis versus time as an indication of the oxidative damage it was found that i) both reduced and oxidized alpha-lipoic acid protected against oxidative damage; ii) simultaneous treatment of erythrocytes with ascorbate and dihydrolipoate or alpha-lipoate has a synergistic tendency to protect cells against hemolysis; iii) glutathione in combination with dihydrolipoic acid or alpha-lipoic acid has an additive effect on hemolysis protection. The spin trapping reagent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) formed an adduct with the peroxyl/alkoxyl radicals produced by thermal decomposition of AAPH in the presence of oxygen. The formation of this adduct was prevented by reduced or oxidized lipoic acid, reduced glutathione or ascorbate. It is concluded that AAPH-peroxyl radicals progressively damage the cells and the released hemoglobin is subsequently oxidized to methemoglobin which might further enhance the oxidative damage. The protective effect of antioxidants is exerted outside the cells by directly scavenging AAPH-alkoxyl radicals.

Amidines↗
PubMed1993

Vitamin E recycling in human erythrocyte membranes.

Vitamin E, the major lipid chain-breaking antioxidant in erythrocyte membranes, is present in low concentration, suggesting that mechanisms should exist to protect against its loss. Enzymatic pathways for the recycling of vitamin E from its tocopheroxyl radical have been observed previously in inner membranes of mitochondria and microsomes. These pathways use electron transport enzymes and their substrates to regenerate vitamin E. Erythrocyte membranes also contain significant NADH-cytochrome c reductase activity, as well as cytochrome b5, the function of which is not yet known. Using an enzymatic oxidation system composed of lipoxygenase and arachidonic acid, free radicals were produced in human erythrocyte membranes, and their reaction with chromanols was followed by ESR and high performance liquid chromatography (HPLC). Since the endogenous vitamin E content of the membranes is very low, we used a vitamin E homologue lacking the hydrocarbon chain (2,2,5,7,8-pentamethyl-6-hydroxychromane) as a probe molecule for ESR measurements. However, parallel HPLC determinations of lipid hydroperoxides and of endogenous vitamin E confirmed the results obtained by ESR. It was found that protection against the loss of vitamin E can be provided either by NADH-cytochrome b5-dependent enzymatic recycling or by a nonenzymatic pathway involving ascorbate and dihydrolipoic acid.

Arachidonic Acid↗
PubMed1993

Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated.

Immunoglobulin and T-cell receptor genes are assembled during lymphocyte development by a novel, highly regulated series of gene rearrangement reactions known as V(D)J recombination. All rearranging loci are flanked by conserved heptamer-nonamer recombination signal sequences. Gene rearrangement results in the imprecise fusion of coding sequences and the precise fusion of signal sequences. DNA molecules with double-stranded breaks near signal sequences have been detected in cells undergoing V(D)J recombination of the TCR delta locus. We have devised a ligation-mediated PCR assay that detects broken-ended molecules in purified genomic DNA. Using this assay we found that DNA breaks occurring precisely at the signal sequence-coding sequence junction are a general feature of V(D)J recombination, appearing in association with each type of rearranging immunoglobulin gene segment. We show that a significant fraction of these broken ends are blunt and 5'-phosphorylated. In addition, detection of these broken-ended signal sequences is dependent on the activity of RAG-1 and RAG-2, and is restricted to the G0/G1 phase of the cell cycle. The pattern of broken-ended molecules detected in cells at various stages of development reflects the activity of the V(D)J recombinase at different loci during B- and T-cell development.

Aging↗
PubMed1992

Recombinant antibody-metallothionein: design and evaluation for radioimmunoimaging.

We have produced a chimeric antibody (Ab) in which metallothionein, a well-characterized biological chelator of metals, was genetically fused to the F(ab') domain of the S107 Ab heavy chain. Coexpression with the Ab light chain that conveys specificity for the synthetic antigen phosphocholine was achieved in plasmacytoma cells. Metal- and antigen-binding domains of the Ab-metallothionein hybrid function with normal avidity and specificity. Ab-metallothionein can be efficiently loaded with 99mTc and used to specifically bind phosphocholine-haptenated cells in vitro or to localize plasma-cell ascites tumors in mice. The approach offers potential advantages for producing radiolabeled Ab for targeted radiotherapy and diagnostic imaging.

Animals↗
PubMed1992

Synthesis and evaluation of technetium-99m monocationic mixed ligand complexes of phenyl substituted/condensed tetradentate Schiff's bases and trimethylphosphine.

Tc-99m monocationic mixed ligand complexes of phenyl substituted/condensed Schiff's bases, N,N'-ethylene-bis-(benzoylacetone imine) (Lb) or N,N'-ethylene-bis-(salicylaldehyde imine) (Lc) or N,N'-ethylene-bis-(2-hydroxyacetophenone imine) (Ld) and trimethylphosphine were synthesized to determine the influence of the presence of a phenyl group in these tracers on their heart uptake in rats. A new formulation procedure using aq. beta-hydroxypropylcyclodextrin (HPB) solution was developed for intravenous administration of nonpolar 99mTc complexes. Comparison of biodistribution data for the reference 99mTc complex from N,N'-ethylene-bis-(acetylacetone imine) and trimethylphosphine using HPB formulation and alternate formulation (0.9% saline) showed the same results. Biodistribution of the title 99mTc complexes, [99mTc Lb (PMe3)2]+, [99mTc Lc (PMe3)2]+ and [99mTc Ld (PMe3)2]+ showed heart-to-blood activity ratios of 1.7, 2.1 and 1.7, respectively, at 15 min post-injection in rats.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1988

On the environment of zinc in beef heart cytochrome c oxidase: an x-ray absorption study.

The role of zinc in beef heart cytochrome c oxidase has been studied by using x-ray absorption spectroscopy, zinc depletion and secondary structure predictions of subunits of beef heart cytochrome c oxidase. The stoichiometry of zinc in cytochrome oxidase has been determined in 35 different preparations and found to be one-half of copper (Cu:Zu = 2:1). Zinc is tightly bound to this enzyme and cannot be removed by dialysis against EDTA. However, zinc could be partially (up to 50%) depleted by treating the enzyme with either dipicolinic acid or by trypsin digestion. This partial depletion of zinc does not change the O2 uptake rate. X-ray absorption spectroscopy shows that the atom is in a distorted tetrahedral environment with mostly sulfur ligands. Since subunit VIa removed by the digestion removes about one-half the zinc, a possible binding site involves the two S sites present in that subunit with an appropriate folding in a structural role.

Animals↗