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At least 1,243 records · Page 69Linked to original sources
PubMed1985

Mammotroph autoregulation: intracellular fate of internalized prolactin.

As part of a study concerning the mechanism of mammotroph autoregulation, the intracellular fate of [125I]iodo rat PRL, internalized by mammotrophs, was determined by quantitative electron microscope autoradiography. Simple grain density (GD) analysis of mammotroph autoradiograms showed that 5 cellular compartments (plasma membrane, Golgi apparatus, secretory granules, nucleus and mitochondria) were significantly labeled and that the labeling pattern in these compartments changed with time. Inclusion of a 25-fold excess of unlabeled PRL inhibited the binding of radiolabeled hormone to mammotrophs by approximately 85%. At no time were any grains found associated with lysosomes and there was no evidence of degradation of the radiolabeled PRL as judged by SDS-polyacrylamide gel electrophoresis or cold perchloric acid precipitation of cell extracts or media samples. Delivery of internalized PRL to a number of specific intracellular organelles suggests that these organelles may serve in the autoregulation of PRL secretion.

Animals↗
PubMed1990

31P nuclear magnetic resonance (NMR) identification of sugar phosphates in isolated rat ovarian follicular granulosa cells and the effects of follicle-stimulating hormone.

Immature female rats (23-30 days old) were implanted subcutaneously with diethylstilbestrol (DES) in silastic capsules. After 48 h their ovaries were removed and the granulosa cells isolated (Foreman et al. (1984) Life Sci. 35, 1273-1279). The cells were incubated in Hepes balanced saline buffer with substrates with or without follicle-stimulating hormone (FSH). At the end of incubation perchloric acid extracts were made for 31P NMR spectroscopy. The resonances of fructose 1-phosphate, fructose 6-phosphate, glucose 1-phosphate, and ribose 5-phosphate were identified in the granulosa cell extracts. The relative intensities of fructose 6-phosphate to ribose 5-phosphate decreased after incubation with FSH in vitro. This suggests that FSH increases the activity of the pentose pathway within 1 h. Thus, FSH can acutely activate those metabolic pathways which provide nicotinamide-adenine dinucleotide phosphate (NADPH) to be used in steroid synthesis and cholesterol mobilization.

Animals↗
PubMedDate not supplied

In vivo binding of N-nitrosopyrrolidine and N-nitrosohexamethyleneimine to non-purine sites on rat liver DNA.

Liver DNA and RNA were isolated from rats treated with the liver carcinogens N-nitrosopyrrolidine (NPYR) and N-nitrosohexamethyleneimine (NHX). After hydrolysis in 70% perchloric acid (100 degrees C, 1.0 h), 70% of the radioactivity in both the DNA and RNA hydrolysates chromatographed as a single peak. The material from both hydrolysates had comparable Rf values on cation exchange and Sephadex G-10 chromatography. Subsequent experiments indicated this material was volatile. After depurination (0.1 M HCl) of the DNA from NPYR- and NHX-treated rats, Sephadex G-10 chromatography separated only a single radioactive peak which co-eluted with the apurinic acid at the void volume. The material which comprised this peak was not volatile. After dialysis of the same 0.1 M HCl hydrolysate from NHX-treated rats, 98% of the radioactivity remained attached to the apurinic acid. These 2 cyclic nitrosamines appear to produce alkylating species that: (1) are capable of extensive, if not exclusive, phosphotriester formation; or (2) have 2 active sites that cross-link to keep purines attached to apurinic acid after 0.1 M HCl hydrolysis.

Animals↗
PubMed1990

31P nuclear magnetic resonance studies of growth inhibition and dexamethasone resistance in human leukemic cells.

31P Nuclear magnetic resonance spectra of perchloric acid extracts from wild-type human leukemic CEM-C7 cells and the dexamethasone-resistant CEM-C1 mutant reveal significant differences in concentrations of phospholipid precursors and ATP+, which indicate metabolic differences between these two cell lines. At high cell concentrations the CEM-C7 cells are growth inhibited, which is reflected by low phospholipid precursor levels, indicative of low phospholipid turnover. The CEM-C1 mutant does not exhibit this growth inhibition and has constant phospholipid precursor levels over the same cell concentration range. Dexamethasone causes phospholipid precursor and ATP levels in CEM-C7 to drop after 48 h, but spectra obtained for CEM-C1 cells continue to show high cell viability up to 72 h.

Adenosine Triphosphate↗
PubMed1982

Is noradrenaline a major catecholamine in the bovine retina?

Using high performance liquid chromatography (HPLC) and electrochemical detection, a major substance corresponding to noradrenaline was identified in crude perchloric acid extracts from bovine retina. Prior purification of the extract with aluminium oxide followed by HPLC confirmed this observation. By altering the oxidation potential of the detector, noradrenaline was confirmed by a further criterion. Bovine retina was also shown to metabolize [3H]dopamine to form a substance which was identified as noradrenaline by the same criteria used to establish its presence. The noradrenaline content was found to be 62 +/- 4 ng/g fresh weight of tissue. Its level is therefore of the same order as the dopamine content (86 +/- 3 ng/g fresh weight).

Animals↗
PubMed1983

Adrenaline: occurrence in the bovine retina.

Using high performance liquid chromatography (HPLC) and electrochemical detection, a substance corresponding to adrenaline was identified in crude perchloric acid extracts from bovine retina. The substance did not originate from exogenous contamination such as blood. Retinal tissues also contained phenylethanolamine-N-methyl-transferase (PNMT) activity and the retinal enzyme had a similar substrate specificity as purified bovine PNMT. The pigment epithelium/choroid complex also had PNMT activity and an analysis of extracts by HPLC revealed the presence of small amounts of adrenaline. The results suggest that adrenaline is of functional importance in the bovine retina.

Animals↗
PubMed1983

The occurrence of serotonergic nerves in the bovine cornea.

Using high performance liquid chromatography (HPLC) and electrochemical detection, serotonin and its precursor 5-hydroxytryptophan (5-HTP) were identified in crude perchloric acid extracts from bovine cornea. In addition, dopamine, noradrenaline and adrenaline were identified. Through the use of immunohistochemical procedures it was established that the serotonin is localized in nerve fibers in the cornea and they were always found subepithelially or in the stroma. The occurrence of serotonergic fibres in the cornea suggests that the amine has a specific functional role in this tissue.

Animals↗
PubMed1991

First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes.

Primary cultures of cerebral cortical neurons or astrocytes or the two cell types together (co-cultures) were incubated with [1-13C]glucose for 20 or 48 h. Subsequently, perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy in order to detect 13C-labeled amino acids (glutamine, glutamate, gamma-aminobutyrate (GABA)) and other metabolites (lactate, tricarboxylic acid cycle (TCA) constituents). NMR spectra of PCA extracts of neurons or co-cultures exhibited distinct peaks for glutamate and GABA whereas the PCA extracts of astrocytes and co-cultures showed peaks corresponding to glutamine and glutamate. This pattern is consistent with the neuronal location of the GABA synthesizing enzyme glutamate decarboxylase and the astrocytic localization of the glutamine synthesizing enzyme, glutamine synthetase. NMR spectra of the incubation media showed clearly that 13C-labeled citrate, alanine and glutamine were synthesized and released from astrocytes since only media from the astrocyte cultures or co-cultures or neurons and astrocytes contained these metabolites in detectable amounts. It may be concluded that astrocytes play an important role supplying neurons with precursors for biosynthesis of glutamate and GABA such as glutamine and TCA cycle constituents. Since among the latter only citrate could be found in significant amounts it may be hypothesized that this may be the quantitatively most important TCA constituent to be released from astrocytes and subsequently utilized by neurons.

Animals↗
PubMed1992

Regional and temporal glycerol changes induced by forebrain ischemia in gerbils.

Temporal ischemic changes in glycerol and energy metabolites were studied in the striatum, hippocampus and cortex of gerbils subjected to 15 min of bilateral carotid artery occlusion alone or with various periods of recirculation. The same tissue sample served for the determination of glycerol and energy reserve by a simple enzymatic fluoro- and spectrometric assay after perchloric acid extraction. Cerebral ischemia increased the levels of glycerol (8- to 10-fold) and depleted the energy stores. During the first hour of recirculation, the glycerol content decreased and thereafter (at 2 h), normalized in all structures. However, the glycerol content was still twice as high in the striatum and hippocampus as compared to their respective controls. At the same time, an incomplete restoration of energy reserves was observed in these structures. The findings indicate that glycerol is not a stable postischemic indicator of the ischemia-induced membrane damage.

Adenosine Triphosphate↗
PubMed1975

Glycoproteins from ascitic fluid of Ehrlich ascites tumor. Isolation and chemical characterization.

Three glycoprotein bands were identified by polyacrylamide disc gel electrophoresis in the perchloric acid soluble fraction of ascitic fluid of Ehrlich ascites tumor in mice. The three proteins were first separated by a new discontinuous preparative electrophoresis apparatus described previously [1]. They were further purified on Sephadex G-100 and then were subjected to chemical characterization. These glycoproteins were rich in glutamic and aspartic acids and contained the sugar moieties galactose, mannose, fucose, N-acetyl-D-glucosamine and sialic acid. The percent sugar composition ranged from 17.7-37.3% of the total weights of these glycoproteins.

Amino Acids↗
PubMed1984

Arginine uptake by isolated rat liver mitochondria.

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-14C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [14C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-14C]carboxylic acid or [14C]sucrose. There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH4Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0 degree C. Mitochondria did not concentrate citrulline.

Animals↗
PubMed1986

Occurrence of adenosine 2',5'-bisphosphate in rat liver.

Adenosine 2',5'-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 microM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5'-AMP than 2'-AMP was obtained as an intermediate of the reaction.

Adenosine Diphosphate↗
PubMed1989

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons.

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.

Adenine Nucleotides↗
PubMed1987

High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins.

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).

Amino Acids↗
PubMed1987

Proton magnetic resonance spectroscopy of leech muscle and nervous system.

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.

3-Hydroxybutyric Acid↗
PubMed1992

Immunochemical studies of histone H5 from Halobatrachus didactylus.

1. Histone H5 from Halobatrachus didactylus was isolated by using perchloric acid (PCA) extraction of fish liver nuclei and trichloroacetic acid (TCA) precipitation. 2. A polyclonal antiserum was generated by immunizing rabbits with the antigen purified from SDS-PAGE. 3. By immunofluorescence the serum stains erythrocyte nuclei from H. didactylus but it does not react with mammalian cells. 4. By Western blotting, the anti-H5 antibody reacts with the isolated antigen at high titers. 5. Digestion of histone H5 with pepsin and cyanogen bromide suggests that the epitopes are located in the globular and C-terminal region of the H5 molecule excluding the N-terminal.

Animals↗
PubMed1984

Measurement of glutathione-protein mixed disulfides.

We have undertaken the development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Treatment with 25 mM dithiothreitol (DTT) in 50 mM N-morpholinopropane sulfonic acid buffer, pH 8.0, reduces disulfides on the protein, and free thiols resulting from this procedure are analyzed by HPLC. We have found up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in our chromatography system with a retention volume similar to cysteine. Future experiments concerning this unidentified thiol are in progress.

Animals↗
PubMed1987

High-performance liquid chromatographic drug analysis by direct injection of whole blood samples. II. Determination of hydrophilic drugs.

The determination of hydrophilic drugs in whole blood by direct injection high-performance liquid chromatography was investigated. A pre-column equipped with an inlet filter of pore size 40 microns and an outlet filter of pore size 2 microns was packed with Butyl Toyopearl 650-M. A whole blood sample was injected directly into the pre-column to trap proteins, hydrophobic compounds and blood cytomembranes, and hydrophilic compounds emerged into an analytical column (Nucleosil 5SA, particle size 5 microns) and were determined after column switching. Proteins in 40 microliters of rabbit whole blood were adsorbed in the pre-column (0.63 ml of wet gel) in 0.4% perchloric acid solution. The recovery of procainamide and N-acetylprocainamide from whole blood was quantitative with good reproducibility (coefficient of variation less than 4%). It was shown that procainamide added to rabbit whole blood was subjected to N-acetylation by N-acetyltransferase in blood cells.

Acecainide↗