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PubMed1993

Synthesis and antitumor activity of ammine/amine platinum(II) and (IV) complexes.

Dimeric platinum complexes, [Pt(RNH2)I2]2 (where R = H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl), have been synthesized by reactions of diiodoplatinum compounds with perchloric acid in water/ethanol solutions. The dimerization varies from several hours to a few days depending upon the length of the carbon chain in the alkylamines and the process can be conveniently monitored by 195Pt NMR spectroscopy. All these dimers exhibit two closely separated resonances around -4000 ppm (vs K2PtCl4 at -1620 ppm) in dimethylformamide. Reactions of [Pt(NH3)I2]2 with alkylamines do not yield the desired mixed ammine/amine complexes, which are obtained subsequently by treatment of the alkylamine dimer [Pt(RNH2)I2]2 with ammonium hydroxide in water. By using this latter procedure, a novel class of ammine/amine platinum complexes of the type PtII(NH3)(RNH2)Cl2, PtIV(NH3)(RNH2)X2A2, and PtIV(NH3)(RNH2)(CBDCA)A2.H2O, where X2 = chloro or 1,1-cyclobutanedicarboxylato (CBDCA), A = OH, Cl, or OCOCH3, have been synthesized and characterized by elemental analysis, infrared, and 195Pt NMR spectroscopic techniques. The alicyclic ammine/amine Pt(II) complexes, where R is C3-C6 were selected as representative of the class to undergo antitumor evaluations. The compounds had excellent activity against murine leukemic L1210/0 cells with cyclobutylamine-, cyclopentylamine- and cyclohexylamine-containing complexes demonstrating cytotoxicity superior to that of the clinically established cisplatin.

Amines↗
PubMed1989

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups.

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.

Amines↗
PubMed1990

A rapid procedure for the analysis of phenylalanine in brain tissue utilizing electron-capture gas chromatography.

A rapid procedure has been developed for the analysis of phenylalanine in brain tissue. Perchloric acid extracts of brain tissue were made basic, and benzoyl chloride was added to derivatize the amine function. The aqueous layer was retained and made slightly acidic. To derivatize the carboxylic acid group, a solution of pentafluorobenzyl alcohol was added in the presence of the coupling agent dicyclohexylcarbodiimide and chloroform. After shaking for 15 min, the organic phase was retained and taken to dryness. The residue was taken up in toluene, washed, and an aliquot used for analysis on a gas chromatograph equipped with an automatic injector, a capillary column and an electron-capture detector. The procedure has been utilized for analysis of phenylalanine in brains of rats treated with vehicle or phenylalanine.

Animals↗
PubMed1982

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA).

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.

Antibodies, Monoclonal↗
PubMed1987

Comparisons of histones in retinal and brain nuclei from newborn and adult mice.

Histone proteins from purified nuclei of neonatal and adult mouse retinas were analyzed and compared utilizing SDS-polyacrylamide gel electrophoresis. Identical procedures were applied to examine the histones extracted from the brains of the same animals. In the newborn and mature retina and brain, 8 histone fractions have been separated, identified and quantified by scanning densitometry. These are the linker histone (H1), consisting of 3 subfractions (H1a, H1b, H1(0); the semi-histone uH2A (A24); and the 4 nucleosome core histones (H2A, H2B, H3, H4). Developmental differences are exhibited by the linker histone in both brain and retinal cells. The greatest differences between the histone patterns of retina and brain are also in the H1 group. Because the linker histone is subject to the greatest variability. H1 was selectively extracted with 5% perchloric acid from both neonatal and adult brain. This procedure established that the observed differences are a developmental phenomenon and are not due to interactions of the linker histones with other nuclear proteins. The ratio of the non-histone chromosomal proteins to total histone was found to be significantly greater in both adult and neonatal brain compared to retina at either age.

Aging↗
PubMed1996

Ontogenetic differences in energy metabolism and inhibition of protein synthesis in hippocampal slices during in vitro ischemia and 24 h of recovery.

The present study was designed to clarify whether ontogenetic differences in the vulnerability of the brain towards hypoxic-ischemic insults are only caused by the low cerebral energy demand of immature animals or whether there are additional mechanisms, such as protein synthesis (PSR), that may be involved in this phenomenon. We therefore measured tissue levels of adenylates and PSR in hippocampal slices from immature (E40) and mature (E60) guinea pigs fetuses and from adult guinea pigs during in vitro ischemia and 24 h of recovery using a recently modified method. Hippocampal slices were incubated in a temperature controlled flow-through chamber, gassed with 95% O2/5% CO2. In vitro ischemia was induced by transferring slices to a glucose-free artificial cerebrospinal fluid (aCSF) equilibrated with 95% N2/5% CO2. The duration of ischemia ranged from 10 to 40 min. Adenylates were measured by HPLC after extraction with perchloric acid. PSR was evaluated as the incorporation rate of [14C]leucine into proteins. Under control conditions, tissue levels in adenylates did not change, whereas PSR increased slightly in hippocampal slices from mature fetuses and adult animals during a 24-h control incubation period. In slices from immature fetuses ATP levels were only maintained for 2 h. During in vitro ischemia the decline in ATP, total adenylate pool, and adenylate energy charge was much slower in slices from immature fetuses than in slices from mature fetuses or adults. After in vitro ischemia, ATP and the total adenylate pool did not completely recover in mature fetuses and adults, whereas adenylate energy charge almost returned to control values independently of the developmental stage. Two hours after in vitro ischemia PSR was undisturbed in slices from immature fetuses, but severely inhibited in slices from mature fetuses and adults. With ongoing recovery, PSR in mature fetuses returned to control values, while in adults it was still inhibited even 24 h after in vitro ischemia. From these results we conclude that hippocampal slices prepared from mature guinea pig fetuses as well as from adult guinea pigs can be held metabolically stable during long-term incubation using a recently modified technique. However, in slices from immature fetuses a stable energy state could not be maintained for more than 2 h. We further conclude that postischemic disturbances in PSR closely reflect the ontogenetic changes in the vulnerability of the brain to ischemia and that low energy metabolism is certainly not the only cause of the increased vulnerability of the fetal brain to ischemia.

Adenosine Triphosphate↗
PubMed1983

Composition and synthesis during G1 and S phase of a high mobility group-E/G component from Chinese hamster ovary cells.

A perchloric acid soluble protein from the sedimented chromatin of blended Chinese hamster ovary (line CHO) cells has been isolated by guanidine hydrochloride gradient chromatography on Bio . Rex-70 ion exchange resin. The amino acid composition of the protein (designated as CHO HMG-E/G) is similar to that of mouse HMG-E, but it differs from that of bovine HMG-14 and HMG-17 or any possible mixture of the two. CHO HMG-E/G incorporates [32P]phosphate like HMG-14 and HMG-17 class proteins from other species, but all resolvable molecular species incorporate phosphate, and the more highly-phosphorylated band migrates faster, rather than slower, than the other in acid-urea gel systems. Incorporation of [3H]lysine into HMG-E/G following release from isoleucine deprivation G1 block indicates that the protein is extensively synthesized during both the G1 and S phases of the cell cycle.

Amino Acids↗
PubMed1988

Plasma proteins immunologically related to inter-alpha-trypsin inhibitor.

SDS-polyacrylamide gel electrophoresis and immunoblot were applied to analysis of plasma proteins immunologically related to inter-alpha-trypsin inhibitor (ITI). In this system, anti-ITI sera were able to identify ITI and other components with an Mr near 120 kDa which would be degradation products of ITI by limited proteolysis. An anti-UTI (urinary trypsin-inhibitor) serum could detect, beside these derivatives, two minor components (Mr values near 90 and 60 kDa). Analysis of perchloric acid supernatants of plasma samples, using the same technic, induced visualization of a new component, similar to urinary trypsin inhibitor which could not be detected by direct analysis. This one was also characterized in a higher content in pathological samples (renal failure and infectious diseases).

Alpha-Globulins↗
PubMed1988

Isolation and characterization of two new low-molecular-weight protein proteinase inhibitors from the granule-rich fraction of equine neutrophilic granulocytes.

A new species of protein proteinase inhibitors was detected in the granule-rich fraction of equine neutrophilic granulocytes. Five isoinhibitors were identified with a narrow enzyme specificity towards two microbial proteinases, e.g., proteinase K and subtilisin. Two isoinhibitors were purified and partially characterized. They had an Mr of 11,300 and 7400, respectively, and were resistant to perchloric acid and heat treatment at 100 degrees C for 20 min. The inhibitors retained their activity over a broad range of pH (1-9 and 1-12, respectively). The possible biological function of this species of protein proteinase inhibitors as defensins (= endogenous antibiotics) is tentatively discussed.

Animals↗
PubMed1986

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles.

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.

Animals↗
PubMed1988

Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlation with metabolic indices.

The 31P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1, 2 and 3 days post feeding showed a 40% decrease in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.

Cell Line↗
PubMed1989

The Na+/K+-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study.

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.

Calibration↗
PubMed1991

Determination of endogenous levels of cyclic ADP-ribose in rat tissues.

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10 degrees C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 +/- 0.64 pmol/mg, in heart is 1.04 +/- 0.08 pmol/mg and in brain is 2.75 +/- 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.

Adenosine Diphosphate Ribose↗
PubMed1992

Changes in phosphatidylethanolamine metabolism in regenerating rat liver as measured by 31P-NMR.

31P-NMR spectra of regenerating rat liver in vivo show increases in resonance intensities in the phosphomonoester (PME) region and decreases in the phosphodiester (PDE) region as early as 12 h post partial hepatectomy, which return to normal by 8 days. The compounds primarily responsible for these changes have been identified in perchloric acid extracts as the phosphomonoester phosphoethanolamine and the phosphodiester glycerophosphoethanolamine (GPE), indicating altered phosphatidylethanolamine metabolism. A corresponding increase in diacylglycerol (DAG) levels during regeneration indicates a possible role for a phosphatidylethanolamine-specific phospholipase C in cellular proliferation. These results suggest that changes in phospholipid metabolites previously associated with neoplastic tissue can also be induced by normal tissue undergoing rapid cellular proliferation. The spectral changes observed in the regenerating rat liver are similar to changes seen in spectra from the livers of human patients in several disease states, indicating that 31P-NMR may allow non-invasive study of cell turnover in liver disease.

Adenosine Triphosphate↗
PubMed1993

Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons.

Primary cultures of cerebral cortical astrocytes and neurons, as well as neurons growing on top of the astrocytes (sandwich co-cultures), were incubated with 1-[13C]glucose or 2-[13C]acetate and in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. [13C]NMR spectroscopy at 125 MHz was performed on perchloric acid extracts of the cells or on media collected from the cultures. In addition, the [13C/12C] ratios of the amino acids glutamine, glutamate and 4-aminobutyrate (GABA) were determined by gas chromatography/mass spectroscopy, showing a larger degree of labeling in GABA than in glutamate and glutamine from glucose. Glutamine and glutamate were predominantly labeled from acetate. A picture of cellular metabolism mainly regarding the tricarboxylic acid cycle and glycolysis was obtained. Due to the fact that acetate is not metabolized by neurons to any significant extent, it could be shown that precursors from astrocytes are incorporated into the GABA pool of neurons grown in co-culture with astrocytes. Spectra of media removed from these cultures revealed that likely precursor candidates for GABA were glutamine and citrate. The importance of glutamine is further substantiated by the finding that inhibition of glutamine synthetase, an enzyme present in astrocytes only, significantly decreased the labeling of GABA in co-cultures incubated with 2-[13C]acetate.

Acetates↗
PubMed1994

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: compartmentation of astrocyte metabolism.

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2-13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Adenosine Triphosphate↗
PubMedDate not supplied

Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR.

The sources of ammonia used by isolated, intact rat liver mitochondria in the production of citrulline have been investigated in situ using a novel methodology based on the analysis of 13C-15N heteronuclear couplings observed by 13C NMR. Isolated mitochondria from rat liver were incubated with ornithine, 13CO3H- and 15NH4Cl, using unlabeled glutamate or glutamine as alternative, intramitochondrial nitrogen donors. The production of (7-13C, 8-15N) or (7-13C, 8-14N) citrulline was determined in situ by 13C NMR and the relative proportions of 15N- and 14N-citrullines confirmed by high resolution 13C NMR analysis of the C-7 citrulline resonance observed in perchloric acid extracts prepared at the end of the incubations. The 15N fractional enrichment of the intramitochondrial NH3 pool was manipulated either by modifying the 15N enrichment of added 15NH4Cl, or by altering the concentration of the unlabeled nitrogen donors in the incubation medium. Fractional 15N enrichments measured in the N-8 nitrogen of the resulting (7-13C) citrulline closely paralleled those of the external 15NH4Cl with minor dilutions derived from the unlabeled nitrogen contribution from the alternative substrates. In the presence of 10 mM 15NH4Cl, 10 mM glutamate contributed 4% of the citrulline N-8 nitrogen. Under similar conditions, the contribution of nitrogen from 10 mM glutamine to N-8 citrulline was 6%. These results indicate that the primary source of ammonia used for citrulline synthesis by isolated, intact rat liver mitochondria is extramitochondrial, providing also an illustration of the use of 13C-15N spin coupling patterns observed by 13C NMR, as a new tool in the study of ammonia metabolism.

Ammonia↗
PubMed1994

Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells.

The level of diadenosine 5',5"-P1-P4-tetraphosphate (diadenosine tetraphosphate or Ap4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap4A, 0.019 +/- 0.004; ATP, 109 +/- 11; ADP, 23.8 +/- 5.8; AMP, 11.3 +/- 1.5; p4A, 0.18 +/- 0.08; catecholamines, 460 +/- 57. The level of Ap4A, catecholamines and ATP (nmol/mg protein) found in chromaffin granules and in chromaffin cells were, respectively: (0.15 +/- 0.07; 2175 +/- 99; 531 +/- 66) and (0.22 +/- 0.14; 1143 +/- 277; 222 +/- 53). In all the cases investigated, the ratio catecholamines/ATP and catecholamines/Ap4A were around 5 and in the order of 10(3), respectively. The amount of Ap4A found here, in bovine adrenal medulla, chromaffin granules and chromaffin cells, is two orders of magnitude lower than previously reported.

Adenosine Triphosphate↗