Biomedical subjects

I Constantinidis

Publications and source records attributed to I Constantinidis.

14 recordsLinked to original sources

PubMed1992

Energy metabolism, pH changes, and lactate production in RIF-1 tumors following intratumoral injection of glucose.

The metabolic consequences of increased glucose availability were examined in subcutaneous RIF-1 tumors in vivo, using 13C and 31P NMR spectroscopy. Significant increases in the levels of nucleotide triphosphates and phosphocreatine relative to low energy phosphates and in tumor pH were observed within 30 min following injection of 1 g/kg of glucose directly into the tumor. These changes did not occur following an equivalent intratumoral dose of the non-metabolizable sugar alcohol, mannitol. When [1-13C]-glucose was administered, [3-13C]-lactate and [3-13C]-alanine were the only labeled metabolites detected in the in vivo 13C NMR spectra during the period of bioenergetic improvement. Biochemical analysis revealed a substantial increase in tumor and plasma glucose concentration, but no increase in either tumor or plasma lactate, consistent with the absence of acidosis. Evaluation of the distribution of glucose in the tumor by quantitative autoradiography of [1-14C]-2-deoxyglucose administered with the glucose indicated that, on average, 7 mM of the added glucose distributed over the entire tumor within 10 min. The significant improvement in overall metabolic status of the tumors following glucose administration is attributed to the existence of substrate limited regions within the tumor.

Animals↗

PubMed1991

In vivo 14N nuclear magnetic resonance spectroscopy of tumors: detection of ammonium and trimethylamine metabolites in the murine radiation induced fibrosarcoma 1.

The in vivo 14N nuclear magnetic resonance spectra of s.c. implanted murine radiation induced fibrosarcomas (RIF-1) display narrow resonances assignable to betaine and other trimethylamines and broad resonances due to amino acids and peptides. In 19 of the 41 tumors studied a distinct resonance from the ammonium ion is detectable. The accumulation of ammonium in the tumor to nuclear magnetic resonance detectable levels may result from glutaminolysis (a possible pathway for energy production in the tumor), from the degradation of peptides and proteins, or from the deamination of adenine nucleotides. Estimates of the tissue ammonium concentration were obtained from the in vivo tumor spectrum and the spectrum of the nonlabile trimethylamines in the perchloric acid extract. In the extract, the 14N resonances of betaine, carnitine, choline, phosphorylcholine, and glycerophosphorylcholine were resolved, and a relatively high level of tissue urea was observed. Spin-lattice relaxation times were obtained for the 14N nucleus of each of these metabolites in phosphate buffer.

Ammonia↗

PubMed1991

In vivo 13CNMR spectroscopy of glucose metabolism of RIF-1 tumors.

An efficient method for measuring in vivo 13C NMR spectra of tumors has been developed and employed to monitor glucose metabolism in radiation-induced fibrosarcomas (RIF-1) subcutaneously implanted in C3H/HeN mice. [1-13C]Glucose was injected directly into the tumors at a dose of 1 g/kg body wt. Spectra were obtained with a Bruker AM 360-WB spectrometer (8.4 T/8.9 cm bore) employing a homebuilt probe equipped with a four-turn solenoidal coil (1.5 cm outer diameter) for detection of 13C signals and a Helmholtz coil (two 3-cm turns separated by a 3-cm gap, oriented orthogonally to the 13C coil) for 1H decoupling. In addition to the natural abundance 13C resonances of the tumors, signals were detected from the alpha- and beta-anomers of labeled glucose. Within 15 min following injection of labeled glucose [3-13C]lactate and [3-13C]alanine were detected. Lactate labeling approached steady state levels within about 50 min after glucose injection: in contrast, alanine labeling increased continuously over the duration of the experiment (70 min). Sixty minutes after glucose injection, the ratio of the intensity of [3-13C]lactate to the principal lipid methylene resonance (30 ppm from external tetramethylsilane), which served as an internal intensity reference, was correlated with tumor size, whereas the corresponding ratio of the [3-13C]alanine resonance was not. Labeling of glutamate was below the level of detection in the in vivo spectra; however, labeling of C4-glutamate at a level approximately 50-fold lower than the level of [3-13C]lactate was detected in perchloric acid extracts. Incorporation of 13C label into C2- and C3-glutamate and C2-lactate was also observed.

Animals↗

PubMed1990

Potentiation of interleukin 1 alpha mediated antitumor effects by ketoconazole.

In the present studies, the regulatory role of adrenal hormones on the antitumor activity of recombinant human interleukin 1 alpha (IL-1 alpha) was investigated. Ketoconazole, a potent but transient inhibitor of adrenal steroid hormone biosynthesis, inhibited IL-1 alpha induced increases in plasma corticosterone. In s.c. RIF-1 tumors (C3H/HeJ mice) ketoconazole potentiated IL-1 alpha induced hemorrhagic necrosis (59Fe labeled RBC uptake) and prolonged intervals of low tumor perfusion (86Rb+ uptake) and attendant depletion of tumor high energy phosphate reserves as determined by in vivo 31P nuclear magnetic resonance spectroscopy. In normal muscle and skin the ketoconazole-IL-1 alpha combination had no effect on RBC content and little or no effect on tissue perfusion. Ketoconazole potentiation of IL-1 alpha induced tumor pathophysiologies was accompanied by time and ketoconazole dose dependent potentiation of RIF-1 tumor clonogenic cell killing. Although ketoconazole at 40 mg/kg and IL-1 alpha at 25 micrograms/kg alone each produced approximately 50% clonogenic cell kill, a combined treatment (IL-1 alpha 1 h after ketoconazole) resulted in surviving fractions of approximately 1.5%. In vitro, ketoconazole and IL-1 alpha induced only additive clonogenic cell kill in primary RIF-1 explant cultures. The effect of elevated plasma corticosterone levels, induced by ketamine-acepromazine anesthesia, on IL-1 alpha responsiveness was also studied in the RIF-1 tumor model. In C3H/HeJ mice, anesthesia increased plasma corticosterone levels within 30 min, abrogated the IL-1 alpha effect on tumor perfusion, and prevented depletion of tumor high energy phosphate metabolite reserves. Our results are consistent with the hypothesis that IL-1 alpha mediated adrenal hormone responses exert a profound negative feedback on IL-1 alpha antitumor activities. Our data also indicate that adrenal steroid hormone biosynthetic pathways could provide a focus for modulation strategies to increase the efficacy of cytokine based therapeutic interventions.

Animals↗

PubMed1989

31P-nuclear magnetic resonance studies of the effect of recombinant human interleukin 1 alpha on the bioenergetics of RIF-1 tumors.

The effect of a single injection of human recombinant interleukin 1 alpha (IL-1 alpha) on s.c. RIF-1 tumors in mice was studied by in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were obtained before and up to 24 h after IL-1 alpha. At 2, 4, 6, and 8 h after IL-1 alpha injection, RIF-1 tumors exhibited a reduction in bioenergetic status compared to untreated controls. The Pi to beta-nucleoside triphosphate and the phosphomonoester to beta-nucleoside triphosphate ratios increased, while the phosphocreatine to Pi and phosphodiester to phosphomonoester ratios decreased. Tumor blood flow, estimated by 86RbCl uptake, decreased within 30 min after IL-1 alpha treatment. Minimum perfusion was detected at 4 h, with recovery between 6 and 12 h after IL-1 alpha treatment. Histological sections of the RIF-1 tumors revealed intravascular congestion by 2 h, extravascular hemorrhage by 4 h, and necrosis by 12 h after treatment with IL-1 alpha. The time course of bioenergetic changes in RIF-1 tumors determined by 31P-NMR spectroscopy was found to parallel the reduction and subsequent recovery of tumor blood flow.

Animals↗

PubMed1989

The effects of high pressure upon ligated and deoxyhemoglobins and myoglobin. An optical spectroscopic study.

The effects of high pressure (0.1-3.4 gigapascal (GPa)) on the ferrous heme active sites of human adult hemoglobin, sperm whale myoglobin, and Glycera dibranchiata hemoglobin (Fraction II) were probed using resonance Raman and absorption spectroscopies. High-to-low spin transitions of the heme iron occur for hemoglobin, myoglobin, and Glycera hemoglobin at 0.35, 0.75, and 0.50 GPa, respectively, for the deoxy species. These interspecies differences result from variations in the composition of the hemepockets and/or their rigidity to pressure-induced volume changes. Heme active sites initially bound to CO or O2 exhibit distinctive behavior at high pressures. For all proteins studied, O2 apparently dissociates from the heme at only moderately high pressure, while CO remains bound to the heme moiety even at extreme pressures. The Raman spectra demonstrate the differences in the ligated and deoxy species at 3.4 GPa in the high frequency region. Discrete changes (i.e. iron spin-state transitions and dissociation of O2) occur that are commensurate with the collapse of the distal pocket, while continuous shifts in the absorption and Raman spectra are observed at pressures above those required for pocket collapse.

Animals↗

PubMed1989

Purity of Glycera dibranchiata monomer hemoglobin components III and IV determined by isoelectric focusing.

1. The Glycera dibranchiata monomer hemoglobin components III and IV display behavior upon high voltage isoelectric focusing which is similar, but not identical to the behavior demonstrated by monomer hemoglobin component II (Constantinidis and Satterlee (1987). Biochemistry 26, 7779-7786). 2. Both components III and IV show multiple line behavior and formation of significant amounts of apoprotein when solutions of each holoprotein are focused on polyacrylamide gels. 3. The apoprotein of each component focuses as a single line, indicating that this is the most unambiguous estimate of purity for these proteins. 4. The purity of the component III and IV preparations can be estimated to be at least 95%.

Animals↗

PubMed1988

Assignment of selected hyperfine proton NMR resonances in the met forms of Glycera dibranchiata monomer hemoglobins and comparisons with sperm whale metmyoglobin.

This work indicates a high degree of purity for our preparations of all three of the primary Glycera dibranchiata monomer hemoglobins and details assignments of the heme methyl and vinyl protons in the hyperfine shift region of the ferric (aquo?) protein forms. The assignments were carried out by reconstituting the apoproteins of each component with selectively deuteriated hemes. The results indicate that even though the individual component preparations consist of essentially a single protein, the proton NMR spectra indicate spectroscopic heterogeneity. Evidence is presented for identification and classification of major and minor protein forms that are present in solutions of each component. Finally, in contrast to previous results, a detailed analysis of the proton hyperfine shift patterns of the major and minor forms of each component, in comparison to the major and minor forms of metmyoglobin, leads to the conclusion that the corresponding forms of the proteins from each species have strikingly similar heme-globin contacts and display nearly identical heme electronic structures and coordination numbers.

Animals↗

PubMed1988

CD studies on the reversed heme orientation in monomeric Glycera dibranchiata hemoglobins.

Circular dichroism spectra of three monomeric components of Glycera dibranchiata hemoglobins are reported. Contrary to what is found for most hemoglobins and myoglobins, G. dibranchiata hemoglobins display largely negative dichroic spectra in the Soret region. Independent NMR measurements have shown that the same monomeric hemoglobin components contain the heme moiety predominantly (greater than 85%) oriented in a reversed way with respect to the orientation which occurs in most hemoglobins and myoglobins. On the basis of these independent NMR studies, and also of previous data on other invertebrate hemoproteins, a correlation appears evident between reversed heme orientation in hemoglobins and negative ellipticity in the Soret CD spectrum. This represents a simple tool to evaluate this aspect of heme asymmetric environment.

Animals↗

PubMed1987

Isoelectric focusing purity criteria and 1H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata.

Three major monomeric hemoglobins have been isolated from the erythrocytes of Glycera dibranchiata. Their importance to structure-function studies of heme proteins lies in the fact that they have been shown to possess an exceptional amino acid substitution. In these proteins, the E-7 position is occupied by leucine rather than the more common distal histidine. This substitution alters the polarity of the heme ligand binding environment compared to myoglobin. Due to this, the G. dibranchiata monomer hemoglobins are attracting much attention. However, until now no purity criterion has been developed. Here we demonstrate that, for all of the Glycera monomer hemoglobins, multiple line patterns are shown on high-voltage isoelectric focusing (IEF) gels. Most of these lines are shown to be a consequence of heme-related phenomena and can be understood on the basis of changes in oxidation and ligation state of the heme iron. The multiple line pattern does not indicate significant impurities in the monomer hemoglobin preparations. Similar behavior is also demonstrated for horse heart myoglobin. The multiple line patterns on IEF gels disappear when gels of the apoproteins alone are focused. Single bands occur in this case for all of the monomer hemoglobins except component II, which displays two bands, one major and one minor. The minor band is found to be a modified apoprotein form. It is sensitive to apoprotein handling prior to focusing and depends upon whether the IEF gel is prefocused or not. From this analysis, IEF is shown to be a valuable purity criterion, and the purity of our monomer hemoglobin component II preparation is 97% one globin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

The structural bases for the unique ligand binding properties of Glycera dibranchiata hemoglobins. A resonance Raman study.

The hemoglobin of the marine annelid Glycera dibranchiata possesses several unique features: the hemoglobin consists of multiple monomeric and polymeric components, quaternary structure is lacking, the distal histidine is replaced by leucine in at least one monomeric constituent, and 4) the protein exhibits extremely rapid ligand binding kinetics. The effect of these structural modifications on the ligand binding process has been evaluated using resonance Raman spectroscopy to examine the vibrational modes of the porphyrin macrocycle in deoxy and carbonmonoxy equilibrium species of hemoglobin G. dibranchiata in both the unseparated monomeric and polymeric forms and in a single monomeric component designated Fraction II. Significant differences relative to hemoglobin were found in porphyrin pi electron density, vinyl environment, low frequency vibrational modes, and, in particular, the Fe-proximal histidine stretching mode. Spectra of the deoxy heme transients generated within 10 ns of ligand photolysis have also been examined. These clearly indicate large differences in the heme pocket dynamics subsequent to CO photolysis in G. dibranchiata hemoglobins relative to other hemoglobins. The significance of these results in terms of the kinetics and thermodynamics of ligand binding is discussed.

Animals↗

PubMed1985

A resonance Raman study of ligand binding geometry in Glycera dibranchiata carbonmonoxyhemoglobin.

Using 12CO and 13CO liganded protein and 406 nm laser excitation, multiple stretching (upsilon(Fe-CO), upsilon(C-O)) and bending (delta(Fe-C-O)) modes have been identified in the resonance Raman spectra of monomeric and polymeric Glycera hemoglobins. While the monomer fraction Glycera dibranchiata hemoglobin has upsilon(Fe-CO) = 496 cm-1, two distinct upsilon(Fe-CO) modes are found at 498 cm-1 and 520 cm-1 for the polymeric protein fraction. Data for upsilon(Fe-CO) and upsilon(C-O) were employed in an isolated three-body oscillator calculation to obtain approximate values for the Fe-C-O bond angle of 175 +/- 5 degrees for one polymeric component and 160 +/- 5 degrees for both the monomer and the second identifiable polymeric component. The differences in ligand binding geometry between the Glycera hemoglobins and other hemeproteins can be used to rationalize the relatively high values of on and off rates exhibited by the Glycera hemoglobins.

Animals↗

PubMed1985

Evaluation of the extent of heterogeneity in the Glycera dibranchiata monomer haemoglobin fraction by the use of n.m.r. and ion-exchange chromatography.

The coelomic haemoglobin of Glycera dibranchiata is known to be separable into monomeric and higher-Mr fractions. Although exhibiting homogeneity with respect to Mr, the extent of haemoglobin heterogeneity for the monomer fraction has never been adequately assayed. In the present paper we demonstrate that there exists in the monomer haemoglobin fraction reproducibly detectable heterogeneity regardless of the presence or absence of proteinase inhibitors during the isolations. These results show that, considered on the same time scale as previous preparations used for amino acid sequencing, crystallography and kinetics, the monomer haemoglobin fraction is highly heterogeneous. Application of ion-exchange chromatography and ion-filtration methods resulted in the isolation of four resolvable haem protein components from the Glycera monomer haemoglobin fraction. Three of these components were isolated in sufficient quantity to employ proton n.m.r. as a successful analytical tool for discriminating the individual haemoglobins. These results are not surprising. Several previous studies indicated less extensive heterogeneity in the monomer fraction. Moreover, the ability of the Glycera monomer haemoglobin to bind oxygen at even quite low partial pressures has been attributed to functional diversity originating in multiple haemoglobin components. The present work reveals the extent of the haemoglobin heterogeneity. The results show that it is more extensive than previously believed. Examination of this monomer fraction is particularly important, since crystallography indicates that one of the components of the monomer fraction lacks the E-7 (distal) histidine residue. As a consequence, the identification of such extensive heterogeneity is important to many previously published ligand-binding studies.

Animals↗

PubMed1985

Cherubism.

Cherubism is a benign, hereditary giant cell lesion of the jaws, that appears in children as a bilateral painless swelling, between the ages of 2 and 5 years and progresses until puberty, when it spontaneously regresses. It normally requires no treatment. We had the chance to operate on a 5-year-old boy with cherubism 8 years ago, and used homogenous bone grafts to replace the diseased tissue to avoid pathological fracture of the mandible. Since then we have been following the patient, witnessing the gradual involvement of other sites in the jaws and the displacement of teeth and tooth germs.

Anodontia↗