[Biosynthesis and intracellular transport of sphingolipids in mammalian cells].
Explore the source record for details and available documents.
Acyltransferases↗
Biomedical subjects
K Hanada
Publications and source records attributed to K Hanada.
Ethnicity and skeletal Class III morphology: a pubertal growth analysis using thin-plate spline analysis.
A longitudinal retrospective study using thin-plate spline analysis was used to investigate skeletal Class III etiology in Japanese female adolescents. Headfilms of 40 subjects were chosen from the archives of the Orthodontic department at Niigata University Dental Hospital, and were traced at IIIB and IVA Hellman dental ages. Twenty-eight homologous landmarks, representing hard and soft tissue, were digitized. These were used to reproduce a consensus for the profilogram, craniomaxillary complex, mandible, and soft tissue for each age and skeletal group. Generalized least-square analysis revealed a significant shape difference between age-matched groups (P < .001), except for the craniomaxillary complex at stage IVA. T test for size analysis showed unequivocally increased mandibular size in skeletal Class III, which directly increased the craniofacial size collectively (P < .05). A deviant profilogram showed anisotropy displaying as maxillary deficiency, acute cranial base, and obtuse gonial angle in addition to increased facial height at stage IIIB. Maxillary retrusion decreased while the mandible showed excessive incremental growth and a forward position caused by deficient orthocephalization at stage IVA. Craniomaxillary complex total spline and partial warps (PW)3 and 2 showed a maxillary retrusion at stage IIIB opposite an acute cranial base at stage IVA. Mandibular total spline and PW4, 5 showed changes affecting most landmarks and their spatial interrelationship, especially a stretch along the articulare-pogonion axis. In soft tissue analysis, PW8 showed large and local changes which paralleled the underlying hard tissue components. Allometry of the mandible and anisotropy of the cranial base, the maxilla, and the mandible asserted the complexity of craniofacial growth and the difficulty of predicting its outcome.
Adolescent↗
[The clinical application and modification of the Quad Helix appliance].
OBJECTIVE: The aims of this study are to investigate the working mechanism, the characteristics, the clinical application and the suggested modification of the Quad Helix appliance. METHODS: A 7-year-old, female patient with Pierre-Rokin syndrome, who was preformed with palatorrhaphy at 21-month-old treated by using a Quad Helix appliance for one year. RESULTS: After one-year treatment, the wide between the maxillary first molars increased 9.65 mm, and the wide between maxillary canines increased 5.20 mm. The wide between the mandibular first molars also increased 3.60 mm, however the wide between mandibular canines decreased 5.20 mm. CONCLUSION: The Quad-helix appears to be a successful Orthodontic appliance to expand the narrow maxillary or mandibular arches.
Child↗
Reduction of sphingomyelin level without accumulation of ceramide in Chinese hamster ovary cells affects detergent-resistant membrane domains and enhances cellular cholesterol efflux to methyl-beta -cyclodextrin.
We examined the effects of reduction of sphingomyelin level on cholesterol behavior in cells using 2 types of Chinese hamster ovary cell mutants deficient in sphingomyelin synthesis: LY-A strain defective in intracellular trafficking of ceramide for sphingomyelin synthesis, and LY-B strain defective in the enzyme catalyzing the initial step of sphingolipid biosynthesis. Although the sphingomyelin content in LY-A and LY-B cells was approximately 40 and approximately 15%, respectively, of the wild-type level without accumulation of ceramide, these mutant cells were almost identical in cholesterol content and also in plasma membrane cholesterol level to the wild-type cells. However, density gradient fractionation analysis of Triton X-100-treated lysates of cells prelabeled with [(3)H]cholesterol showed that the [(3)H]cholesterol level in the low-density floating fraction was lower in sphingomyelin-deficient cells than in wild-type cells. When cells were exposed to methyl-beta-cyclodextrin, cholesterol was more efficiently fluxed from sphingomyelin-deficient cells than wild-type cells. These results suggest that the steady state level of cholesterol at the plasma membrane is little affected by the sphingomyelin levels in Chinese hamster ovary cells, but that sphingomyelin levels play an important role in the retention of cholesterol in the plasma membrane against efflux to extracellular cholesterol-acceptors, due to interaction between sphingomyelin and cholesterol in detergent-resistant membrane domains.
Amphotericin B↗
Reconstitution of ATP- and cytosol-dependent transport of de novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells.
Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment, where sphingomyelin (SM) synthase exists, was reconstituted within semi-intact Chinese hamster ovary cells. When [(3)H]ceramide that had been produced from [(3)H]sphingosine at 15 degrees C in perforated cells was chased at 37 degrees C, [(3)H]ceramide-to-[(3)H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [(3)H]ceramide-to-[(3)H]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a Chinese hamster ovary cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [(3)H]ceramide-to-[(3)H]SM conversion in perforated wild-type cells, whereas the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgi apparatus were merged, no longer required cytosol for conversion of [(3)H]ceramide to [(3)H]SM. These results indicated that the assay of [(3)H]ceramide-to-[(3)H]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. In addition, conversion of [(3)H]ceramide to [(3)H]glucosylceramide in semi-intact cells was little dependent on cytosol, suggesting that ceramide reached the site of glucosylceramide synthesis by a cytosol-independent (or less dependent) pathway.
Adenosine Triphosphate↗
Isolation and characterization of a novel liver-specific gene, hepassocin, upregulated during liver regeneration.
By differential cDNA cloning coupled with Xenopus oocyte expression screening, we isolated a cDNA encoding a novel protein, termed 'hepassocin', the expression of which is upregulated in the regenerating rat liver. The cDNA contained a single open reading frame encoding a protein of 314 amino acids (ca. 34 kDa), including 24 amino acids of signal sequence. The protein expressed from the cDNA in Verots cells had activity to stimulate DNA synthesis in primary rat hepatocytes and was of 66 kDa or 34 kDa, under non-reducing or reducing conditions, respectively. Using an affinity column conjugated with the antibody raised against a peptide in a hydrophilic region, we purified hepassocin from the rat liver: it had a DNA synthesis-stimulating activity in hepatocytes. The hepassocin obtained here was 66 kDa, and the 34 kDa protein obtained under reducing conditions contained five cysteine residues, indicating that hepassocin is active as a homodimer. Northern blot analysis revealed that hepassocin mRNA (1.4 kb in length) occurred only in the liver, and in situ hybridization studies revealed its presence in parenchymal hepatocytes but not in endothelial cells. Furthermore, the expression of hepassocin mRNA was upregulated during compensatory hyperplasia after partial hepatectomy and regeneration after galactosamine treatment in the rat liver. These results suggest that hepassocin plays an important role in stimulating liver cell growth, through an autocrine mechanism.
Amino Acid Sequence↗
D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis.
Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [(3)H]KDS from L-[(3)H]serine was performed using purified SPT. D-Serine inhibited [(3)H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1-(14)C]palmitoyl coenzyme A and D-serine did not produce [(14)C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.
Acyltransferases↗
UvrA and UvrB suppress illegitimate recombination: synergistic action with RecQ helicase.
Illegitimate recombination is a major cause of genetic instability in prokaryotes as well as in eukaryotes. This recombination usually occurs at a low frequency, but it is greatly enhanced by UV irradiation or other environmental stresses. DNA damages produced by these environmental stresses are thought to induce DNA double-strand breaks, leading to illegitimate recombination. In this paper we show that UV-induced illegitimate recombination is enhanced by mutations of nucleotide excision repair genes, uvrA or uvrB, and partially by uvrC mutation, but not by uvrD mutation. Unexpectedly, the recombination was enhanced by the uvrA uvrB double mutation even without UV irradiation, but the uvrB uvrC double mutation has not shown this effect, suggesting that illegitimate recombination is mostly suppressed by UvrA and UvrB. Moreover, illegitimate recombination was synergistically enhanced by the recQ uvrA double mutation. In addition, overproduction of the UvrA protein suppressed the hyperrecombination phenotype of the recQ or uvrB mutant, but it did not affect the UV-sensitive phenotype of the uvrB mutant. We concluded that the UvrAB complex suppresses illegitimate recombination in a pathway shared with RecQ helicase. In addition, UvrA protein alone can suppress illegitimate recombination in the pathway, in which RecQ helicase and UvrAB complex work. Possible functions of the proteins involved in these pathways are also discussed.
Adenosine Triphosphatases↗
Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant.
In the present study, we demonstrate a model cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) cell mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the mutant. Although LY-B cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 cells under sphingosine-supplied conditions, indicating that, in CHO cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. Cultivation of LY-B/cLCB1 cells in sphingolipid-deficient medium in the presence of 10 microM sphingofungin B and ISP-1 (myriocin, thermozymocidin), potent inhibitors of SPT activity, caused severe growth inhibition with approximately 95% inhibition of de novo sphingolipid synthesis. The growth inhibition by sphingofungin B and ISP-1 was rescued substantially by exogenous sphingosine, whereas the cytotoxicity of two other types of SPT inhibitor, L-cycloserine and beta-chloro-L-alanine, was hardly rescued. Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [(3)H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not L-cycloserine or beta-chloro-L-alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity.
Acyltransferases↗
Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts.
In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose- and time-dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk-1 and Flt-1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up-regulated by a common mediator such as VEGF.
Animals↗
Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques.
Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.
Acyl Coenzyme A↗
Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum.
Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg(2+) and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn(2+) could not substitute for Mg(2+) to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn(2+) and Mg(2+) activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo.
Amides↗
Enantioselective binding of propranolol, disopyramide, and verapamil to human alpha(1)-acid glycoprotein.
We investigated the binding of propranolol (PL), disopyramide (DP), and verapamil (VP) enantiomers by human alpha(1)-acid glycoprotein (AGP; also called orosomucoid) and the relationships between the extent of drug binding and lipophilicity, desialylation, and genetic variants of AGP. Desialylation had little effect on the affinity of AGP for the drugs tested. The percentage binding correlated significantly with the partition coefficients for the drugs tested. Each enantiomer was competitively displaced from AGP by another enantiomer of the same drug, suggesting that they bind to the same site. However, the enantiomers bound to AGP with stereospecific affinities; the (-)-isomers of DP and VP had higher Kd values (4.27 and 4.97 microM, respectively) than the (+)-isomers (1.51 and 2.48 microM, respectively). When enantiomers of the different drugs were used in competitive binding experiments, VP binding was only partially inhibited by DP. This result suggested that drug binding is specific to different variants of AGP (A, F1, S). DP was found to specifically bind to variant A, whereas PL and VP bind to both A and F1/S variants.
Disopyramide↗
Selection of mammalian cell mutants in sphingolipid biosynthesis.
Explore the source record for details and available documents.
Animals↗
Photoprotective role of metallothionein in UV-injury - metallothionein-null mouse exhibits reduced tolerance against ultraviolet-B.
We have shown previously that injection of cadmium chloride (Cd(2+)) depletes the number of ultraviolet B (UVB)-induced sunburn cells (SBC) in the mouse skin in vivo and that Cd(2+) treatment enhances UVB resistance in cultured keratinocytes in vitro, indicating the photoprotective role of Cd(2+)-induced metallothioneins (MTs) with antioxidant property against UVB injury. However, there has been no direct evidence for the role of MTs in UV protection. To improve our understanding of MTs in photoprotection, MT-null mouse deficient in MT-1 and MT-2 genes was studied. Skin explants were preliminarily exposed to medium alone, Cd(2+) and Cd(2+) plus buthionine S, R-sulfoximine (BSO), an inhibitor of glutathione synthesis. We then compared the number of UVB-induced SBC and apoptotic cells (AC) in the epidermis of MT-null mice with that of control mice using organ culture systems. The skin of MT-null mice developed a greater number of SBC and AC than did that of normal mice in all experimental conditions. These findings indicate that the skin of MT-null mouse is readily injured by UVB irradiation. MT-null mouse provided direct evidence of the photoprotective effect of cellular MTs in the skin.
Animals↗
Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum.
Malaria is clinically manifested only when the human malaria parasites in the genus Plasmodium enter the obligatory intraerythrocytic life cycle. Elucidation for the roles of the serum, the key nutrient, and its components is then deemed essential for thorough understanding of the proliferation of Plasmodium cells at the erythrocytic stage. Fractionation and analysis of serum and its components was performed by chromatography, solvent extraction, and subsequent reconstitution experiments. Only fractions containing serum albumin (SA) from the serum and purified intact bovine serum albumin (BSA) showed comparable growth promoting activity with human serum (HS). Delipidated BSA can only effect parasite growth after reconstitution with lipids extracted from intact BSA. Fatty acid (FA) species in the neutral lipid fraction from intact BSA proved likewise when reconstituted with delipidated BSA. Furthermore, the involved FA species have to come in a pair of one saturated and one unsaturated, with palmitic and oleic acids as the best combination. The results were further substantiated by morphological analysis as well as biochemical analysis of the DNA synthesis during the intraerythrocytic development. This study can be a basis to explore the molecular mechanism of lipid traffic within the parasitized red blood cell (RBC), which can be an important adjunct to the development of drugs for malaria therapy.
Animals↗
Control of genetic stability in Escherichia coli: the SbcB 3'-5' exonuclease suppresses illegitimate recombination promoted by the RecE 5'-3' exonuclease.
BACKGROUND: The Escherichia coli sbcB gene, which codes for a 3'-5' exonuclease, ExoI, is known to suppress illegitimate recombination. In contrast, the recE gene, which codes for a 5'-3' exonuclease, Exo VIII promotes joining between DNA ends having short stretches of homology. Therefore, it seems likely that the 3'-5' and 5'-3' exonucleases regulate genetic instability that is mediated by illegitimate recombination. However, there has been little evidence to substantiate the involvement of exonuclease activity in the promotion and suppression of illegitimate recombination. RESULTS: Using a plasmid system for the analysis of deletion formation, we first demonstrated that deletion formation is increased by the sbcA mutation, which activates the expression of RecE 5'-3' exonuclease. It is thought that DNA ends having 3'-single stranded overhangs are important for illegitimate recombination. Next, we found that a large supply of SbcB 3'-5' exonuclease suppresses the deletion formation enhanced by the RecE exonuclease. Moreover, the SbcB exonuclease even suppressed deletion formation in cells not expressing RecE exonuclease. CONCLUSION: We conclude that DNA ends with 3'-overhangs produced by 5'-3' dsDNA exonuclease activity are proficient for illegitimate recombination, while blunt DNA ends produced by 3'-5' ssDNA exonuclease activity are deficient for illegitimate recombination. Therefore, both exonucleases may play important roles in genetic stability by controlling end-joining between DNA molecules.
Base Sequence↗
The relationship between frontal facial morphology and occlusal force in orthodontic patients with temporomandibular disorder.
The aim of this study was to investigate the relation between frontal craniofacial morphology and the distribution of occlusal force in orthodontic patients with and without TMD. Experimental data were obtained from 45 female orthodontic patients (TMD-free group: 22 subjects; TMD group: 23 subjects) using the pressure-sensitive sheet. The following results were obtained: (1) There were significant differences in the gonial angle width and the mandibular deviation of the postero-anterior cephalogram between the two groups. (2) There were significant differences in occlusal force, occlusal contact area and average occlusal pressure between the two groups. (3) On the relation between the frontal craniofacial morphology and the occlusal data, the asymmetry indices of occlusal force and occlusal contact area showed significant positive correlations to the mandibular deviation and the ratios of maxillary and gonial angle widths in the TMD group. The TMD-free group showed the smaller mandibular deviation and the smaller asymmetry indices of occlusal force and occlusal contact area than those in the TMD group. It could be concluded that orthodontic patients with TMD may have not only the morphological disharmony but also the imbalance of occlusal force.
Adolescent↗