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1.
Biosci Biotechnol Biochem ; 81(11): 2164-2167, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28934905

ABSTRACT

In this study, we investigated the physiological function of glycerophosphodiesterase 5 (GDE5) in the proliferation of NIH3T3 fibroblasts. We used transcription activator-like effector nuclease (TALEN) in NIH3T3 cells with an intron targeting-mediated GDE5 gene knockout. The heterozygously GDE5-targeted NIH3T3 fibroblasts were isolated and showed decreased cell proliferation and up-regulation of EGFR mRNA expression, indicating that GDE5 modulates fibroblastic cell proliferation.


Subject(s)
Fibroblasts/cytology , Gene Editing/methods , Phospholipases/genetics , Transcription Activator-Like Effector Nucleases/metabolism , Animals , Base Sequence , Cell Proliferation , Fibroblasts/metabolism , Gene Knockout Techniques , Heterozygote , Mice , NIH 3T3 Cells , Phospholipases/deficiency , Phospholipases/metabolism
2.
EMBO J ; 36(6): 707-717, 2017 03 15.
Article in English | MEDLINE | ID: mdl-28228439

ABSTRACT

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.


Subject(s)
Ovule/growth & development , Phospholipases/metabolism , Plant Proteins/metabolism , Pollen/enzymology , Reproduction , Zea mays/physiology , Gene Expression Regulation, Plant , Phospholipases/deficiency , Zea mays/enzymology
3.
Mol Plant ; 7(8): 1288-1302, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24719469

ABSTRACT

Chlorophyll (Chl) degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 (SGR1) interacts with Chl catabolic enzymes (CCEs) and light-harvesting complex II (LHCII) at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 (At4g22920), the Arabidopsis thaliana genome contains an additional homolog, SGR2 (At4g11910), whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulating Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Cellular Senescence , Chlorophyll/metabolism , Phospholipases/metabolism , Pigmentation , Plant Leaves/cytology , Arabidopsis/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Darkness , Gene Expression Regulation, Plant/radiation effects , Gene Knockout Techniques , Light-Harvesting Protein Complexes/metabolism , Mutation , Phenotype , Phospholipases/deficiency , Phospholipases/genetics , Pigmentation/radiation effects , Stress, Physiological/radiation effects , Thylakoids/metabolism , Thylakoids/radiation effects
4.
Biochim Biophys Acta ; 1436(3): 363-9, 1999 Jan 04.
Article in English | MEDLINE | ID: mdl-9989267

ABSTRACT

Mucolipidosis (ML) II and III are rare autosomal recessively inherited diseases characterized by deficiency of multiple lysosomal enzymes and, as a result, a generalized storage of macromolecules in lysosomes of cells of mesenchymal origin. In ML II and ML III fibroblasts, most, but not all, newly synthesized lysosomal enzymes are secreted into the medium instead of being targeted correctly to lysosomes. Defects in the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase underlie this effect. It is unknown how lysosomal phospholipases are targeted to the lysosomes of fibroblasts. In the present study lysosomal phospholipase activity was determined in delipidated fibroblast homogenates and plasma from ML II and ML III patients and controls using a [3H]choline-labeled phosphatidylcholine. After incubation, residual phosphatidylcholine and its labeled degradation products (lysophosphatidylcholine, glycerophosphorylcholine and choline phosphate) were quantified. We found that ML II and ML III fibroblasts are deficient in lysosomal phospholipase A and C activity. These enzymes were present in elevated amounts in plasma of ML II and ML III patients. These data indicate that phospholipases, like most other lysosomal enzymes in these diseases, are secreted into the blood instead of being targeted specifically to lysosomes. Thus, the mannose-6-phosphate receptor pathway is needed for proper delivery of lysosomal phospholipases to lysosomes. We also found that production of labeled choline phosphate was mainly due to the activity of acid sphingomyelinase instead of phospholipase C under the assay conditions used. Other active lipolytic enzymes were phospholipase A and lysophospholipase. No evidence for phospholipase D activity was found.


Subject(s)
Lysosomes/enzymology , Mucolipidoses/enzymology , Phospholipases/deficiency , Biological Transport, Active , Case-Control Studies , Cells, Cultured , Fibroblasts/enzymology , Humans , Lysophospholipase/metabolism , Mucolipidoses/classification , Phosphatidylcholines/metabolism , Phospholipases/blood , Phospholipases A/blood , Phospholipases A/deficiency , Receptor, IGF Type 2/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Type C Phospholipases/blood , Type C Phospholipases/deficiency
6.
J Clin Invest ; 97(3): 799-805, 1996 Feb 01.
Article in English | MEDLINE | ID: mdl-8609237

ABSTRACT

Hepatic lipase (HL) is an endothelial-bound lipolytic enzyme which functions as a phospholipase as well as a triacylglycerol hydrolase and is necessary for the metabolism of IDL and HDL. To evaluate the feasibility of replacing an enzyme whose in vivo physiologic function depends on its localization on the vascular endothelium, we have infused recombinant replication-deficient adenovirus vectors expressing either human HL (HL-rAdV; n = 7) or luciferase cDNA (Lucif-rAdV; n = 4) into HL-deficient mice with pretreatment plasma cholesterol, phospholipid, and HDL cholesterol values of 176 +/- 9, 314 +/- 12, and 129 +/- 9, respectively. After infusion of HL-rAdV, HL could be detected in the postheparin plasma of HL-deficient mice by immunoblotting and postheparin plasma HL activities were 25,700 +/- 4,810 and 1,510 +/- 688 nmol/min/ml on days 5 and 15, respectively. Unlike the mouse HL, 97% of the newly synthesized human HL was heparin releasable, indicating that the human enzyme was virtually totally bound to the mouse vascular endothelium. Infusion of HL-rAdV in HL-deficient mice was associated with a 50-80% decrease in total cholesterol, triglyceride, phospholipids, cholesteryl ester, and HDL cholesterol (P < 0.001) as well as normalization of the plasma fast protein liquid chromatography lipoprotein profile by day 8. These studies demonstrate successful expression and delivery of a lipolytic enzyme to the vascular endothelium for ultimate correction of the HL gene defect in HL-deficient mice and indicate that recombinant adenovirus vectors may be useful in the replacement of endothelial-bound lipolytic enzymes in human lipolytic deficiency states.


Subject(s)
Endothelium, Vascular/enzymology , Genetic Therapy/methods , Hyperlipidemias/therapy , Lipase/therapeutic use , Phospholipases/therapeutic use , Adenoviridae/genetics , Animals , Cholesterol/blood , Humans , Lipase/blood , Lipase/deficiency , Lipase/genetics , Lipids/blood , Lipoproteins/blood , Male , Mice , Mice, Mutant Strains , Phospholipases/blood , Phospholipases/deficiency , Phospholipases/genetics , Recombinant Proteins/therapeutic use
7.
Infect Immun ; 60(3): 916-21, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1311708

ABSTRACT

A phospholipase-deficient mutant, termed JL762, was obtained from a virulent strain of Listeria monocytogenes by screening a bank of 5,000 Tn1545 transposon-induced mutants on 2.5% egg yolk brain heart infusion agar. As previously shown (J. Mengaud, C. Geoffroy, and P. Cossart, Infect. Immun. 59:1043-1049, 1991), the transposon insertion took place inside the gene mpl, which encodes a zinc metalloprotease. By Western blot (immunoblot) analysis, we showed that loss of phospholipase activity was associated with loss of a 29-kDa zinc-dependent phosphatidylcholine-phospholipase C (PC-PLC) in culture supernatant of JL762 and of EGD-SmR incubated with ion chelator. As the parental strain, JL762 still produced in supernatants approximately 33-kDa proteins antigenically closely related to the 29-kDa PC-PLC. These results strongly suggest that the zinc metalloprotease of L. monocytogenes might play a role in the maturation of the 29-kDa PC-PLC. Although the uptake and the intracellular growth of bacteria were not affected in vitro, we found that the virulence of mutant JL762 was strongly impaired in the mouse.


Subject(s)
DNA Transposable Elements , Listeria monocytogenes/pathogenicity , Metalloendopeptidases/genetics , Phospholipases/deficiency , Animals , Blotting, Western , Female , Listeria monocytogenes/enzymology , Listeria monocytogenes/genetics , Mice , Mice, Inbred ICR , Mutation , Rabbits , Virulence , Zinc
9.
Acta Med Scand ; 222(1): 23-6, 1987.
Article in English | MEDLINE | ID: mdl-3630775

ABSTRACT

The phospholipase A2 and acyltransferase activities characteristic of human plasma lecithin: cholesterol acyltransferase have been evaluated in incubation mixtures of lipoprotein depleted plasma of fish eye disease patients and autologous HDL or homologous normal HDL3. Both enzyme activities were strongly reduced as compared to those of normal controls. These findings further support the claim that fish eye disease plasma has a specific lack of high density lipoprotein lecithin:cholesterol acyltransferase (alpha-LCAT deficiency), although the cholesterol esterification of combined VLDL and LDL in such plasma proceeds at a normal rate.


Subject(s)
Acyltransferases/deficiency , Corneal Opacity/blood , Hypolipoproteinemias/blood , Lecithin Cholesterol Acyltransferase Deficiency/blood , Lipoproteins, HDL/blood , Phospholipases A/deficiency , Phospholipases/deficiency , Acyltransferases/blood , Corneal Opacity/enzymology , Humans , Hypolipoproteinemias/enzymology , Lipoproteins, HDL3 , Phospholipases A/blood , Phospholipases A2
10.
Neurology ; 33(1): 67-73, 1983 Jan.
Article in English | MEDLINE | ID: mdl-6681561

ABSTRACT

We examined the degradation of a labeled phosphatidylglycerol (PG) by fibroblasts from a normal control and a patient with Niemann-Pick (NP) disease. The control homogenate had both phospholipase A and phospholipase C activities toward PG, but NP cells had only phospholipase A. The PG phospholipase C of control fibroblasts was solubilized by sonication and freezing and thawing, was most active at pH 5.0, and was inhibited by Ca2-, detergents, sphingomyelin, and 5' AMP. Assay of PG phospholipase C in fibroblast cultures from NP patients with sphingomyelinase deficiency (three designated type A and four type B) confirmed absence of activity, whereas cultures from NP patients without sphingomyelinase deficiency (three designated type C and one with neurovisceral lipidoses and vertical supranuclear ophthalmoplegia) had activities close to those of normal controls. These findings substantiate previous observations of low phosphodiesterase activities in NP disease and suggest that the enzymatic function affected by the NP genes includes specificity toward PG and sphingomyelin. Deficiency of PG phospholipase C may explain the accumulation of bis(monoacylglycero)phosphate in NP disease.


Subject(s)
Lysophospholipids , Niemann-Pick Diseases/metabolism , Phosphatidylglycerols/metabolism , Phospholipases/deficiency , Type C Phospholipases/deficiency , Adolescent , Adult , Child , Child, Preschool , Female , Fibroblasts/metabolism , Humans , Infant , Male , Monoglycerides , Phosphatidic Acids/metabolism , Phosphatidylglycerols/analysis , Sphingomyelin Phosphodiesterase/metabolism
11.
Neurochem Res ; 8(1): 89-98, 1983 Jan.
Article in English | MEDLINE | ID: mdl-6856019

ABSTRACT

Examination of release of labeled glyceride from 2-[1-14C]oleoyl phosphatidylcholine by a soluble extract of human fibroblasts confirmed the presence of phosphodiesterase which is stimulated strongly by sodium taurocholate. This activity was maximal at pH 4.5 and was inhibited by sphingomyelin and 5' AMP. Assay of the phosphatidylcholine phosphodiesterase activity in fibroblast cultures from patients with Niemann-Pick disease revealed a severe deficiency in those cultures also deficient in sphingomyelinase (3 type A and 4 type B) whereas assay of cultures from Niemann-Pick patients without sphingomyelinase deficiency (3 type C and 1 with neurovisceral lipidosis and vertical supranuclear ophthalmoplegia) gave activities similar to controls. The distribution of label in the products of the reactions catalyzed by both control and Niemann-Pick extracts indicates that the phosphodiesterase activity observed was phospholipase C and that phospholipase D was not involved. The close correlation of phosphatidylcholine phospholipase C and sphingomyelinase activities in the control and mutant fibroblasts strongly suggests that both activities are catalyzed by one enzyme. Various alterations in the regulation of the specificity of a multifunctional phospholipase C may underlie phenotypic variation in Niemann-Pick disease.


Subject(s)
Niemann-Pick Diseases/enzymology , Phosphatidylcholines/metabolism , Phospholipases/deficiency , Taurocholic Acid/pharmacology , Type C Phospholipases/deficiency , Cell Line , Fibroblasts/enzymology , Glycerides/metabolism , Humans , Sphingomyelin Phosphodiesterase/deficiency
12.
Clin Chim Acta ; 108(3): 403-14, 1980 Dec 22.
Article in English | MEDLINE | ID: mdl-6781796

ABSTRACT

Radioactive phosphatidyl choline substrates specifically labeled in the one position or two position fatty acid were used to establish conditions for the detection of acidic phospholipase A1, A2 and C activities in extracts of cultured human fibroblasts. Maximal activity was detected at a pH of 3.0, 4.0 and 5.0 respectively, suggesting that the enzymes are of lysosomal origin. None of the activities were stimulated or inhibited markedly by Ca2+ or EDTA. The A1 and A2 activities, but not the C activity, were inactivated by the sulfhydryl reactive Ellman reagent. All three enzyme activities were in the normal range for cultured fibroblasts which were deficient in acid lipase, indicating that these activities are not attributable to the acid lipase gene product. Phospholipase A activity was deficient in fibroblast extracts from patients with Niemann-Pick disease, types A, B and C. These data suggest either identity or a genetic relationship between sphingomyelinase and phospholipase C. The activities examined were within the normal range in fibroblasts from patients with neuronal ceroid lipofuscinosis, sea blue histiocyte disease and selected uncharacterized degenerative diseases.


Subject(s)
Niemann-Pick Diseases/enzymology , Phospholipases A/analysis , Phospholipases/analysis , Phospholipases/deficiency , Skin/enzymology , Type C Phospholipases/deficiency , Calcium/pharmacology , Cells, Cultured , Edetic Acid/pharmacology , Fibroblasts/enzymology , Humans , Hydrogen-Ion Concentration , Phospholipases A1
13.
Am J Hematol ; 4(4): 387-99, 1978.
Article in English | MEDLINE | ID: mdl-717398

ABSTRACT

The structure and functions of platelets from a patient in whom albinism and hemorrhagic diathesis were associated have been investigated. Electron microscope studies showed a large reduction in the number of dense bodies and this was confirmed by an examination of fluorescent platelets loaded with mepacrine. The rare dense bodies were much bigger than normally observed; their density was diminished and was localized in a peripheral ring. Other platelet constituents were found to be normal. Platelet peroxidase activity was normal in the canaliculi of the dense tubular system; catalase-positive granules were also present. Serotonin uptake by the patient's platelets was much decreased and reserpine, a potent inhibitor of serotonin accumulation by normal human platelets, did not further decrease this incorporation. The uptake of free 14 C-arachidonic acid by the platelets was greatly diminished, as was its thrombin-induced liberation from phosphatidyl-choline and phosphatidyl inositol. Moreover, platelet phospholipase A1 activity was much reduced and phospholipase A2 activity was undetectable.


Subject(s)
Blood Platelet Disorders/blood , Blood Platelets/ultrastructure , Genetic Variation , Phospholipases/deficiency , Adult , Arachidonic Acids/metabolism , Female , Humans , Megakaryocytes/ultrastructure , Reserpine/pharmacology , Serotonin/metabolism , Serotonin Antagonists , Syndrome
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