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1.
J Biol Chem ; 292(12): 5110-5122, 2017 03 24.
Article in English | MEDLINE | ID: mdl-28087695

ABSTRACT

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.


Subject(s)
Lipid Metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Transferases (Other Substituted Phosphate Groups)/metabolism , Ceramides/metabolism , Diglycerides/metabolism , Fatty Acids/metabolism , Hep G2 Cells , Hepatocytes/metabolism , Humans , Membrane Proteins/analysis , Membrane Proteins/genetics , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/genetics , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/genetics , Triglycerides/metabolism , Up-Regulation
2.
Leuk Res ; 47: 32-40, 2016 08.
Article in English | MEDLINE | ID: mdl-27244255

ABSTRACT

Despite BCR-ABL tyrosine kinase inhibitors (TKIs) improved outcome of patients with chronic myeloid leukemia (CML), resistance still develops when progresses to blast phase (BP). The mechanisms underlying resistance to TKIs are not well understood. In this study, we analyzed ceramide levels in CD34 cells derived from BP-CML patients and healthy donor bone marrow (BM) using liquid chromatography mass spectrometry. We found that ceramide level was significantly lower in BP-CML CD34 compared with normal BM counterparts. BP-CML CD34 ceramide(low) were more resistant to BCR-ABL TKIs compared to BP-CML CD34 ceramide(normal). Both mRNA and proteins levels of sphingomyelin synthase 1 and 2 are lower in BP-CML CD34 ceramide(low) compared to normal BM CD34 cells, suggesting that these two ceramide synthesis enzymes maybe the mechanism of how ceramide level is suppressed. Importantly, up-regulation of cellular ceramide level induces apoptosis of multiple CML cell lines and BP-CML CD34 progenitors. Combination of BCR-ABL TKIs with ceramide analog is synergistic in targeting BP-CML 34 progenitors. Collectively, our work provides evidence that down-regulation of ceramide level is involved in the resistance of BP-CML CD34 progenitors to TKIs treatment. Targeting ceramide metabolism together with BCR-ABL inhibition makes it an attractive addition to the armamentarium in BP-CML treatment.


Subject(s)
Ceramides/metabolism , Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Protein Kinase Inhibitors/pharmacology , Adult , Aged , Antigens, CD34 , Bone Marrow Cells/chemistry , Bone Marrow Cells/enzymology , Cell Line , Ceramides/analysis , Ceramides/biosynthesis , Female , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Male , Middle Aged , Protein Kinase Inhibitors/therapeutic use , Transferases (Other Substituted Phosphate Groups)/analysis , Tumor Cells, Cultured
3.
Res Microbiol ; 167(3): 202-14, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26708983

ABSTRACT

The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.


Subject(s)
Bacillus subtilis/enzymology , Cell Membrane/enzymology , Conserved Sequence , Membrane Proteins/analysis , Protein Sorting Signals , Transferases (Other Substituted Phosphate Groups)/analysis , DNA Mutational Analysis , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Membrane Proteins/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Sequence Deletion , Transferases (Other Substituted Phosphate Groups)/genetics
4.
Mol Gen Mikrobiol Virusol ; 33(2): 38-41, 2015.
Article in Russian | MEDLINE | ID: mdl-26182666

ABSTRACT

Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.


Subject(s)
Biochemistry/methods , Membrane Proteins/analysis , Membrane Proteins/chemistry , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/chemistry , Tissue Extracts/chemistry , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/chemistry , Buffers , Chemical Fractionation , Humans , Immunoblotting/methods , Kidney Cortex/chemistry , Quillaja/chemistry , Saponins/chemistry
5.
Assay Drug Dev Technol ; 12(9-10): 506-13, 2014.
Article in English | MEDLINE | ID: mdl-25415593

ABSTRACT

With the public availability of biochemical assays and screening data constantly increasing, new applications for data mining and method analysis are evolving in parallel. One example is BioAssay Ontology (BAO) for systematic classification of assays based on screening setup and metadata annotations. In this article we report a high-throughput screening (HTS) against phospho-N-acetylmuramoyl-pentapeptide translocase (MraY), an attractive antibacterial drug target involved in peptidoglycan synthesis. The screen resulted in novel chemistry identification using a fluorescence resonance energy transfer assay. To address a subset of the false positive hits, a frequent hitter analysis was performed using an approach in which MraY hits were compared with hits from similar assays, previously used for HTS. The MraY assay was annotated according to BAO and three internal reference assays, using a similar assay design and detection technology, were identified. Analyzing the assays retrospectively, it was clear that both MraY and the three reference assays all showed a high false positive rate in the primary HTS assays. In the case of MraY, false positives were efficiently identified by applying a method to correct for compound interference at the hit-confirmation stage. Frequent hitter analysis based on the three reference assays with similar assay method identified additional false actives in the primary MraY assay as frequent hitters. This article demonstrates how assays annotated using BAO terms can be used to identify closely related reference assays, and that analysis based on these assays clearly can provide useful data to influence assay design, technology, and screening strategy.


Subject(s)
Biological Assay/methods , Escherichia coli Proteins/analysis , High-Throughput Screening Assays/methods , Transferases (Other Substituted Phosphate Groups)/analysis , Biological Assay/standards , Fluorescence Resonance Energy Transfer/methods , Fluorescence Resonance Energy Transfer/standards , High-Throughput Screening Assays/standards , Retrospective Studies
6.
Anal Bioanal Chem ; 406(9-10): 2467-75, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24493334

ABSTRACT

DNA adductome analysis using liquid chromatography-tandem mass spectrometry is a promising tool to exhaustively search DNA modifications. Given that the molecular weight of chemical-specific adducts is determined by the total molecular weights of the active form and nucleotide bases, we developed a new method of comprehensive analysis for chemical-specific DNA adducts based on the principle of adductome analysis. The actual analytical mass range was 50 mass units up or down from the average molecular weight of the four DNA bases plus the molecular weight of the expected active form of the chemical. Using lucidin-3-O-primeveroside (LuP), lucidin-modified bases formed by its active form were exhaustively searched using this new method. Various DNA adducts, including Luc-N (2)-dG and Luc-N (6)-dA, were identified in the kidneys of rats given LuP. Together with measurement of 8-hydroxydeoxyguanosine (8-OHdG) levels, the combined application of this new method with a reporter gene mutation assay was performed to clarify renal carcinogenesis induced by madder color (MC) that includes LuP and alizarin (Alz) as constituent agents. A DNA adductome map derived from MC-treated rats was almost identical to that of LuP-treated rats, but not Alz-treated rats. Although 8-OHdG levels were elevated in MC- and Alz-treated rats, significant increases in gpt and Spi(-) mutant frequencies were observed only in MC- and LuP-treated rats. In addition, the spectrum of gpt mutants in MC-treated rats showed almost the same pattern as those in LuP-treated rats. The overall data suggest that LuP may be responsible for MC-induced carcinogenicity and that the proposed methodology is appropriate for exploring and understanding mechanisms of chemical carcinogenesis.


Subject(s)
DNA Adducts/analysis , Kidney/enzymology , Plant Extracts/chemistry , Rubia/chemistry , Transferases (Other Substituted Phosphate Groups)/genetics , Animals , Chromatography, High Pressure Liquid , DNA Adducts/genetics , Genes, Reporter , Male , Mass Spectrometry , Mutation , Rats , Rats, Inbred F344 , Transferases (Other Substituted Phosphate Groups)/analysis
7.
Plant Cell ; 26(1): 391-409, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24443516

ABSTRACT

Mitochondria are essential and dynamic organelles in eukaryotes. Cardiolipin (CL) is a key phospholipid in mitochondrial membranes, playing important roles in maintaining the functional integrity and dynamics of mitochondria in animals and yeasts. However, CL's role in plants is just beginning to be elucidated. In this study, we used Arabidopsis thaliana to examine the subcellular distribution of CL and CARDIOLIPIN SYNTHASE (CLS) and analyzed loss-of-function cls mutants for defects in mitochondrial morphogenesis and stress response. We show that CL localizes to mitochondria and is enriched at specific domains, and CLS targets to the inner membrane of mitochondria with its C terminus in the intermembrane space. Furthermore, cls mutants exhibit significantly impaired growth as well as altered structural integrity and morphogenesis of mitochondria. In contrast to animals and yeasts, in which CL's effect on mitochondrial fusion is more profound, Arabidopsis CL plays a dominant role in mitochondrial fission and exerts this function, at least in part, through stabilizing the protein complex of the major mitochondrial fission factor, DYNAMIN-RELATED PROTEIN3. CL also plays a role in plant responses to heat and extended darkness, stresses that induce programmed cell death. Our study has uncovered conserved and plant-specific aspects of CL biology in mitochondrial dynamics and the organism response to environmental stresses.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/physiology , Cardiolipins/physiology , Mitochondria/metabolism , Stress, Physiological , Arabidopsis/metabolism , Arabidopsis/ultrastructure , Arabidopsis Proteins/analysis , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Base Sequence , Cardiolipins/metabolism , Membrane Proteins/analysis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/physiology , Molecular Sequence Data , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/chemistry , Transferases (Other Substituted Phosphate Groups)/genetics , Transferases (Other Substituted Phosphate Groups)/physiology
8.
Mol Microbiol ; 87(3): 569-79, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23190171

ABSTRACT

Maintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate-limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knock-down results in loss of PG and a reduction of another mitochondria-specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin-isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co-migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.


Subject(s)
Genes, Essential , Mitochondrial Membranes/enzymology , Transferases (Other Substituted Phosphate Groups)/analysis , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/growth & development , Cardiolipins/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Knockdown Techniques , Immunoblotting , Immunohistochemistry , Mitochondrial Membranes/chemistry , Molecular Weight , Multiprotein Complexes/chemistry , Phosphatidylglycerols/metabolism , Sequence Homology, Amino Acid , Transferases (Other Substituted Phosphate Groups)/genetics , Trypanosoma brucei brucei/chemistry , Trypanosoma brucei brucei/genetics
9.
Exp Parasitol ; 120(3): 215-20, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18691575

ABSTRACT

Giardia lamblia is a protozoan parasite with many characteristics common among eukaryotic cells, but lacking other features found in most eukaryotes. Cardiolipin is a phospholipid located exclusively in energy transducing membranes and it was identified in mitochondria, bacteria, hydrogenosomes and chloroplasts. In eukaryotes, cardiolipin is the only lipid that is synthesized in the mitochondria. Biochemical procedures (TLC, HPLC) and fluorescent tools (NAO) were applied in order to search for cardiolipin in G. lamblia. In addition, BLAST searches were used to find homologs of enzymes that participate in the cardiolipin synthesis. Cardiolipin synthase was searched in the Giardia genome, using Saccharomyces cerevisiae and Mycoplasma penetrans sequences as bait. However, a good match to G. lamblia related proteins was not found. Here we show that mitosomes of G. lamblia apparently do not contain cardiolipin, which raises the discussion for its endosymbiotic origin and for the previous proposal that Giardia mitosomes are modified mitochondria.


Subject(s)
Cardiolipins/analysis , Giardia lamblia/chemistry , Mitochondria/chemistry , Amino Acid Sequence , Animals , Cardiolipins/isolation & purification , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Conserved Sequence , Giardia lamblia/enzymology , Iodine , Lipids/isolation & purification , Membrane Proteins/analysis , Membrane Proteins/chemistry , Mycoplasma penetrans/enzymology , Organelles/chemistry , Organelles/enzymology , Saccharomyces cerevisiae/enzymology , Sequence Alignment , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/chemistry
10.
World J Gastroenterol ; 14(16): 2566-71, 2008 Apr 28.
Article in English | MEDLINE | ID: mdl-18442207

ABSTRACT

AIM: To study the hepatoprotective capacity of Sapindus mukorossi (S. mukorossi) and Rheum emodi (R. emodi) extracts in CCl(4) treated male rats. METHODS: The dried powder of S. mukorossi and R. emodi was extracted successively with petroleum ether, benzene, chloroform, and ethanol and concentrated in vacuum. Primary rat hepatocyte monolayer cultures were used for in vitro studies. In vivo, the hepatoprotective capacity of the extract of the fruit pericarp of S. mukorossi and the rhizomes of R. emodi was analyzed in liver injured CCl(4)-treated male rats. RESULTS: In vitro: primary hepatocytes monolayer cultures were treated with CCl(4) and extracts of S. mukorossi & R. emodi. A protective activity could be demonstrated in the CCl(4) damaged primary monolayer culture. In vivo: extracts of the fruit pericarp of S. mukorossi (2.5 mg/mL) and rhizomes of R. emodi (3.0 mg/mL) were found to have protective properties in rats with CCl(4) induced liver damage as judged from serum marker enzyme activities. CONCLUSION: The extracts of S. mukorossi and R. emodi do have a protective capacity both in vitro on primary hepatocytes cultures and in in vivo in a rat model of CCl(4) mediated liver injury.


Subject(s)
Hepatocytes/physiology , Liver/physiology , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Protective Agents/pharmacology , Rheum , Sapindus , Animals , Carbon Tetrachloride/toxicity , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/pathology , L-Lactate Dehydrogenase/analysis , Liver/drug effects , Male , Protective Agents/therapeutic use , Rats , Rats, Wistar , Transferases (Other Substituted Phosphate Groups)/analysis
11.
J Endocrinol ; 192(1): 17-32, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17210739

ABSTRACT

Sphingomyelin synthase 2 (SMS2) is an enzyme that catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol, and it is crucial to cellular lipid metabolism. Using the technique of subtraction hybridization, we have isolated a full-length cDNA encoding SMS2 from rat testes, which shared 93 and 87% identity at the nucleotide level with SMS2 in mice and humans respectively. A specific polyclonal antibody was prepared against a 20 amino acid peptide of NH(2)-FSWPLSWPPGCFKSSCKKYS-COOH near the C-terminus of SMS2. Studies by RT-PCR and immunoblotting have shown that the expression of SMS2 was limited to late round spermatids and elongating spermatids, but it was not detected in late elongate spermatids and Sertoli cells. Furthermore, SMS2 was shown to associate with the developing acrosome beginning in late round spermatid through elongating spermatids (but not late elongate spermatids) and the cell membrane in studies using fluorescent microscopy and immunohistochemistry. These data were further confirmed by studies using immunogold electron microscopy. The expression of SMS2 in the seminiferous epithelium is stage-specific with its highest expression detected in the acrosome region in late round spermatids from stages VIII-IX, and also in the acrosome in elongating spermatids with diminished intensity in stages X-V; however, it was not found in the acrosome in elongate spermatids in stages VI-VIII. Collectively, these results suggest that SMS2 may play a crucial role in the lipid metabolism in acrosome formation and the plasma membrane restructuring from late round spermatids to early elongating spermatids.


Subject(s)
Acrosome/enzymology , Cell Membrane/enzymology , Seminiferous Epithelium , Spermatids/enzymology , Transferases (Other Substituted Phosphate Groups)/analysis , Adherens Junctions/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/pharmacology , Base Sequence , Cells, Cultured , DNA, Complementary/analysis , Hydrazines/pharmacology , In Situ Hybridization/methods , Indazoles/pharmacology , Male , Microscopy, Fluorescence , Microscopy, Immunoelectron , Molecular Sequence Data , Open Reading Frames , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sertoli Cells/chemistry , Spermatogenesis , Transferases (Other Substituted Phosphate Groups)/genetics
12.
Hum Mutat ; 27(8): 830-1, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16835905

ABSTRACT

Mucolipidosis type II (ML II; I-cell disease) and mucolipidosis III (ML III; pseudo Hurler polydystrophy) are autosomal recessively inherited disorders caused by a defective N-acetylglucosamine 1-phosphotransferase (phosphotransferase). The formation of mannose 6-phosphate markers in soluble lysosomal enzymes is impeded leading to their increased excretion into the serum, to cellular deficiency of multiple hydrolases, and lysosomal storage of non-digested material. Phosphotransferase deficiency is caused by mutations in GNPTA and GNPTG encoding phosphotransferase subunits. Here we report on an adolescent with progressive joint contractions and other signs of mucolipidosis II who survived to the age of 14 years. Impaired trafficking of lysosomal enzymes cathepsin D and -hexosaminidase in metabolically labeled fibroblasts was documented. Mutations in the GNPTG gene and alterations in the GNPTG mRNA level were not detected. A different electrophoretic mobility of the 97 kDa GNPTG dimer suggested posttranslational modification abrogating the compartmentalization of GNPTG in the Golgi apparatus. A nucleotide substitution in the GNPTA gene (c.3707A>T) was identified altering the predicted C-terminal transmembrane anchor of the phosphotransferase subunit. The data demonstrate that defective GNPTA not only impairs lysosomal enzyme targeting but also the availability of intact GNPTG required for phosphotransferase activity and assembly of subunits.


Subject(s)
Mucolipidoses/diagnosis , Mucolipidoses/genetics , Mutation, Missense , Transferases (Other Substituted Phosphate Groups)/genetics , Transferases (Other Substituted Phosphate Groups)/metabolism , Adolescent , Cathepsin D/metabolism , Cells, Cultured , Child , Fibroblasts/metabolism , Humans , Infant , Male , Mucolipidoses/diagnostic imaging , Protein Subunits/metabolism , Radiography , Transferases (Other Substituted Phosphate Groups)/analysis , beta-N-Acetylhexosaminidases/metabolism
13.
Biochem J ; 386(Pt 1): 137-43, 2005 Feb 15.
Article in English | MEDLINE | ID: mdl-15458384

ABSTRACT

CL (cardiolipin) is a major mitochondrial membrane phospholipid important for the regulation of mitochondrial function. We examined CL de novo biosynthesis and its resynthesis in isolated rat liver hepatocytes prepared 48 h subsequent to two-thirds PHx (partial hepatectomy). The pool size of CL and its de novo biosynthesis from [1,3-(3)H]glycerol were increased 3.3-fold (P<0.05) and 3.1-fold (P<0.05) respectively in hepatocytes prepared from PHx rats compared with sham-operated controls. The reason for the increased CL biosynthesis was a 65% increase (P<0.05) in enzymic activity in PGP-S (phosphatidylglycerolphosphate synthase), a key enzyme in de novo CL biosynthesis. The increase in PGP-S activity was due to a 3-fold increase (P<0.05) of hepatic PGP-S mRNA expression. The increase in de novo CL biosynthesis and pool size corresponded to a 2.3-fold increase (P<0.05) in the amount of [1-14C]linoleic acid incorporated into CL of hepatocytes prepared from PHx rats compared with sham-operated controls, indicating an increase in CL resynthesis. The activity of MLCL-AT (monolysocardiolipin acyltransferase), a rate-limiting enzyme of CL resynthesis, was increased by 43% (P<0.05) in hepatocytes prepared from PHx rats compared with sham-operated controls; this result would explain the increase in [1-14C]linoleic acid incorporation into CL. The increase in MLCL-AT activity was due to an increase in hepatic MLCL-AT protein expression. The results show that CL de novo biosynthesis and its resynthesis are increased during liver regeneration.


Subject(s)
Cardiolipins/biosynthesis , Hepatocytes/metabolism , Liver Regeneration/physiology , Acyltransferases/analysis , Animals , Hepatectomy , Mitochondria, Liver/enzymology , Phospholipids/analysis , Proliferating Cell Nuclear Antigen/analysis , Proteins/analysis , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Transferases (Other Substituted Phosphate Groups)/analysis
14.
Neuroscience ; 130(3): 657-66, 2005.
Article in English | MEDLINE | ID: mdl-15590150

ABSTRACT

Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimer's disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimer's disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.


Subject(s)
Alzheimer Disease/metabolism , Apoptosis/physiology , Astrocytes/metabolism , Ceramides/biosynthesis , Neurons/pathology , Aged , Alzheimer Disease/cerebrospinal fluid , Alzheimer Disease/pathology , Amyotrophic Lateral Sclerosis/cerebrospinal fluid , Amyotrophic Lateral Sclerosis/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Ceramides/cerebrospinal fluid , Extracellular Space/metabolism , Glucosyltransferases/analysis , Glucosyltransferases/biosynthesis , Humans , Immunohistochemistry , Indicators and Reagents , Lipid Metabolism , Mice , Serine/metabolism , Solvents , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/biosynthesis , Tretinoin/metabolism , Tretinoin/pharmacology
15.
Am J Chin Med ; 32(4): 531-9, 2004.
Article in English | MEDLINE | ID: mdl-15481643

ABSTRACT

Analgesic, antipyretic activity and toxicity study of the leaves of Bryonia laciniosa Linn. (Family: Cucurbitaceae) was evaluated in the standard animal models. The methanol extract of Bryonia laciniosa (MEBL) was evaluated by hot plate and acetic acid-induced writhing methods to assess analgesic activity. The antipyretic activity of the extract was also evaluated by normal body temperature and yeast-induced hyperpyrexia. The extract showed significant analgesic and antipyretic activity. The MEBL was further evaluated for toxicity at the doses of 125 and 250 mg/kg administered orally for 14 days in rats. At the end of experiments, the blood, liver function and kidney metabolism were observed. The hematological profile and different biochemical parameters such as SGOT, SGPT and ALP were estimated. The present study revealed that MEBL exhibited significant analgesic and antipyretic activity in the tested experimental animal models. The toxicity study indicates that the extract is not toxic at the tested doses.


Subject(s)
Bryonia , Plant Extracts/pharmacology , Alanine Transaminase/analysis , Alkyl and Aryl Transferases/analysis , Analgesics/adverse effects , Analgesics/pharmacology , Animals , Body Temperature/drug effects , Dose-Response Relationship, Drug , Female , Kidney Function Tests , Liver Function Tests , Male , Malignant Hyperthermia/prevention & control , Mice , Phytotherapy , Plant Leaves/chemistry , Rats , Rats, Wistar , Time Factors , Transferases (Other Substituted Phosphate Groups)/analysis
16.
Cell Biochem Funct ; 21(3): 211-5, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12910472

ABSTRACT

After the first histochemical demonstration by Chayen and Gahan of the presence of phospholipids and especially of sphingomyelin in chromatin, this became the object of long debate and of contradictory results. The general conclusion was that the presence of phospholipids may due to contamination during the isolation of chromatin. More recently the existence of a phospholipid chromatin fraction was confirmed by demonstrating that isolated hepatocyte nuclei, labelled by saturated and unsaturated radioiodination method, showed the presence of radioactivity only in the membrane and not in the isolated chromatin. The phospholipid composition showed an enrichment in sphingomyelin which increased during hepatocyte maturation or erythroleukemic cell differentiation induced by DMSO. A decrease in sphingomyelin was observed at the beginning of the S-phase in regenerating liver or in cultured proliferating cells. These changes were due to the presence of sphingomyelinase and sphingomyelin synthase in the chromatin, the activity of which paralleled the variation in sphingomyelin content. The sphingomyelin was co-localized with RNA as shown by biochemical and electron microscopy methods. Using bromo-uridine it was demonstrated that labelled RNA and sphingomyelin were present in actively transcribing nuclear regions. Isolated nuclear complexes after DNase and RNase digestion contained not only protein, but also RNA and sphingomyelin. After hydrolysis of sphingomyelin the RNAse-resistant RNA becomes RNAse sensitive. It can therefore be concluded that sphingomyelin and the related enzymes are present in the chromatin; sphingomyelin may have a role in RNA transcription protecting RNA by RNAse digestion before its transfer to the cytoplasm.


Subject(s)
Chromatin/metabolism , Sphingomyelins/metabolism , Animals , Apoptosis/physiology , Cell Differentiation/physiology , Cell Division/physiology , Phospholipids/analysis , RNA/metabolism , Rats , Ribonucleases/metabolism , Sphingomyelin Phosphodiesterase/analysis , Sphingomyelin Phosphodiesterase/metabolism , Sphingomyelins/analysis , Transferases (Other Substituted Phosphate Groups)/analysis , Transferases (Other Substituted Phosphate Groups)/metabolism
17.
Folia Parasitol (Praha) ; 49(2): 103-8, 2002.
Article in English | MEDLINE | ID: mdl-12194482

ABSTRACT

Physiological parameters were measured after experimental infection of roach (Rutilus rutilus L.) with Rhipidocotyle fennica Gibson, Valtonen et Taskinen, 1992 (Digenea) cercariae. The fish were caught from two lakes: a eutrophic bleached kraft mill effluent (BKME)-contaminated lake and an oligotrophic unpolluted lake. The intensity of infection was followed up to 10 days post infection (p.i.) and physiological parameters indicating non-specific stress responses and the condition of fish were examined simultaneously. The mean abundance, the number of parasites per fish, of R. fennica was significantly higher in the fish from the contaminated water during the first two days p.i., probably reflecting the decreased resistance of these fish to infection. The decrease of leukocrit, as well as the increase of the activity of transaminases (GOT and GPT) in infected fish of both groups are suggestive of pathological processes caused by cercariae penetrating the fish. A significantly lower leukocrit value, as well as higher alkaline phosphatase activity and plasma chloride levels were noted in fish originating from the contaminated lake compared to those from the unpolluted lake. No significant differences were noted in haematocrit, plasma protein and calcium values between the fish from the uncontaminated and contaminated lakes, or between the infected and uninfected control fish.


Subject(s)
Cyprinidae/physiology , Fish Diseases/parasitology , Industrial Waste/adverse effects , Trematode Infections/veterinary , Water Pollutants, Chemical/toxicity , Alkaline Phosphatase/analysis , Alkyl and Aryl Transferases/analysis , Animals , Calcium/analysis , Chlorides/analysis , Cyprinidae/parasitology , Fish Diseases/epidemiology , Fish Diseases/physiopathology , Hematocrit , Host-Parasite Interactions , Paper , Transferases (Other Substituted Phosphate Groups)/analysis , Trematoda/growth & development , Trematoda/isolation & purification , Trematode Infections/parasitology , Trematode Infections/physiopathology
18.
J Biol Chem ; 276(9): 6093-7, 2001 Mar 02.
Article in English | MEDLINE | ID: mdl-11078734

ABSTRACT

Coliphage phi X174 encodes a single lysis protein, E, a 91-amino acid membrane protein. Dominant mutations have been isolated in the host gene mraY that confer E resistance. mraY encodes translocase I, which catalyzes the formation of the first lipid intermediate in bacterial cell wall synthesis, suggesting a model in which E inhibits MraY and promotes cell lysis in a manner analogous to cell wall synthesis inhibitors like penicillin. To test this model biochemically, we monitored the effect of E on cell wall synthesis in vivo and in vitro. We find that expression of Emyc, encoding an epitope-tagged E protein, from a multicopy plasmid inhibits the incorporation of [(3)H]diaminopimelic acid into cell wall and leads to a profile of labeled precursors consistent with MraY inhibition. Moreover, we find that membranes isolated after Emyc expression are drastically reduced in MraY activity, whereas the activity of Rfe, an enzyme in the same superfamily, was unaffected. We therefore conclude that E is indeed a cell wall synthesis inhibitor and that this inhibition results from a specific block at the MraY-catalyzed step in the pathway.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Coliphages/chemistry , Escherichia coli Proteins , Peptidoglycan/biosynthesis , Transferases , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Viral Proteins/physiology , Bacterial Proteins/physiology , Catalysis , Cell Wall/metabolism , Nucleosides/pharmacology , Transferases (Other Substituted Phosphate Groups)/analysis , Uridine Diphosphate N-Acetylmuramic Acid/metabolism , Viral Proteins/genetics
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