An improved chemically defined culture medium for strain L mouse cells based on growth responses to graded levels of nutrients including iron and zinc ions

Higuchi, Kiyoshi

doi:10.1002/jcp.1040750108

Cited by 143 publications

(48 citation statements)

References 8 publications

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“…Murine L cells were cultured to confluency at 37 Њ C and 5% CO 2 in a chemically defined medium (37) to which 10% fetal bovine serum (Hyclone, Logan, UT) was added. Mock-transfected cells (designated as control) and cells transfected with cDNA corresponding to the pro-SCP-2 and SCP-x were obtained as described earlier (19,38).…”

Section: Cell Culturementioning

confidence: 99%

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Atshaves

Storey

Schroeder

2003

Journal of Lipid Research

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Sterol carrier protein-2 (SCP-2) and SCP-x are ubiquitous proteins found in all mammalian tissues. Although both proteins interact with fatty acids, their relative contributions to the uptake, oxidation, and esterification of straight-chain (palmitic) and branched-chain (phytanic) fatty acids in living cells has not been resolved. Therefore, the effects of each gene product on fatty acid metabolism was individually examined. Based on the following, SCP-2 and SCP-x did not enhance the uptake/translocation of fatty acids across the plasma membrane into the cell: i ) a 2-fold increase in phytanic and palmitic acid uptake was observed at long incubation times in SCP-2-and SCP-x-expressing cells, but no differences were observed at initial time points; ii ) uptake of 2-bromo-palmitate, a nonoxidizable, poorly metabolizable fatty acid analog, was unaffected by SCP-2 or SCP-x overexpression; and iii ) SCP-2 and SCP-x expression did not increase targeting of radiolabeled phytanic and palmitic acid to the unesterified fatty acid pool. Moreover, SCP-2 and SCP-x expression enhanced fatty acid uptake by stimulating the intracellular metabolism via fatty acid oxidation and esterification. In summary, these data showed for the first time that SCP-2 and SCP-x stimulate oxidation and esterification of branched-chain as well as straight-chain fatty acids in intact cells. 1)]. More recent studies indicate that SCP-2 also functions in the uptake and esterification of straight-chain (e.g., oleic acid) fatty acids (FAs) (2), while SCP-x participates in the oxidation of branchedchain (e.g., phytanic acid) FAs (3, 4). However, almost nothing is known about the function of SCP-x in FA uptake or esterification. Likewise, the relative roles of SCP-2 versus SCP-x in straight-chain or branched-chain FA oxidation remain unresolved. Despite the lack of conclusive evidence, several observations point to an important role for SCP-2 and SCP-x in functioning in these capacities, including FA binding affinities where SCP-2 binds straightchain FAs (5-11) and their metabolically active fatty acyl CoA derivatives (9, 12) to a high degree, as exhibited by K d s in the submicromolar and nM range, respectively. In addition, SCP-2 also binds branched-chain FAs such as phytanic or pristanic acid (9), as well as their respective acyl CoAs with high affinity (7, 9).The intracellular localization of SCP-x and SCP-2 also indicate a role in FA metabolism. The highest concentration of SCP-2 and SCP-x is in peroxisomes (13), where ␣ -oxidation of branched-chain FAs as well as the ␤ -oxidation of some straight chain FAs occurs (14-16). Additionally, it has been shown that half or more of total SCP-2 is extraperoxisomal (13, 17), where part of the SCP-2 may arise from the partial posttranslational cleavage of the SCP-x protein (18,19). Extraperoxisomal SCP-2 locations include: i ) plasma membrane in caveolae, wherein several FA translocase/transporter proteins reside (20); ii ) cytoplasm, where SCP-2 enhances diffusion of fluorescent fatty acid (21); ii...

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Section: Cell Culturementioning

confidence: 99%

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Atshaves

Storey

Schroeder

2003

Journal of Lipid Research

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“…[5,6]), it has been suggested that inhibition of oligosaccharide-lipid synthesis in cells arrested in protein synthesis results because of a deficiency in dolichyl phosphate [3,4]. Such deficiency might occur in the absence of synthesis of acceptor polypeptides, since the lack of transfer of the oligosaccharide chain from the oligosaccharide-lipid to the acceptor polypeptide would preclude concomitant regeneration of dolichyl (di)phosphate.We have studied the regulation of glycoprotein synthesis in LM cells, a mouse fibroblast line that grows in suspension in chemically defined medium [7], thus allowing one to control conditions for exogenous polyisoprenoid supplementation. Labeling experiments in these cells in vivo indicate that the synthesis of mannose-labeled oligosaccharide-lipid was markedly inhibited in the presence of cycloheximide or puromycin.…”

mentioning

confidence: 99%

“…We have studied the regulation of glycoprotein synthesis in LM cells, a mouse fibroblast line that grows in suspension in chemically defined medium [7], thus allowing one to control conditions for exogenous polyisoprenoid supplementation. Labeling experiments in these cells in vivo indicate that the synthesis of mannose-labeled oligosaccharide-lipid was markedly inhibited in the presence of cycloheximide or puromycin.…”

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confidence: 99%

Relationship between Oligosaccharide‐Lipid Synthesis and Protein Synthesis in Mouse LM Cells

Grant

Lennarz

1983

European Journal of Biochemistry

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Previous studies from several laboratories have reported that inhibition of protein synthesis results in a concomitant reduction in synthesis of oligosaccharide-diphosphoryldolichol. We have investigated this phenomenon in LM cells grown in defined culture medium. The results of this study indicate that incubation of LM cells with cycloheximide at a concentration sufficient to totally arrest polypeptide synthesis, results in a rapid reduction in the synthesis of [3H]mannose-labeled or [3H]glucosamine-labeled oligosaccharide-lipid within 2 min. Cycloheximide treatment had only a slight inhibitory effect on synthesis of GDP-Man. Furthermore, during the first 5 min after the addition of cycloheximide to L a cells, when [3H]mannose incorporation into oligosaccharidelipid was maximally reduced, the specific activity of the GDP-Man pool was identical to that observed in control cells. Labeling in vivo of the lipid-linked saccharide precursors of oligosaccharide-lipid revealed that cycloheximide had virtually no effect on the synthesis of Man-P-Dol, GlcNAc-PP-Dol, GlcNAcz-PP-Dol, and fLMan-(GlcNAc)z-PP-Dol (Do1 = dolichol). In agreement with this finding, results of experiments in vitro using microsomes prepared from LM cells indicate that cycloheximide did not directly inhibit the enzymes responsible for the synthesis of these lipid-linked saccharide precursors.Supplementation of mouse LM cells by preincubation for 1 h with dolichyl phosphate (5 pg/ml) resulted in a 300 % stimulation of oligosaccharide-lipid synthesis when compared to non-supplemented cells. However, dolichyl phosphate supplementation of cycloheximide-treated cells failed to restore oligosaccharide-lipid synthesis to the level observed in control cells. In control cells pre-labeled oligosaccharide-lipid turned over rapidly and, as expected, cycloheximide addition to the chase medium significantly retarded this turnover process. However, preincubation of cells with exogenous dolichyl phosphate had little or no effect on the turnover of oligosaccharide-lipid in control or cycloheximide-treated cells. These findings argue against dolichyl phosphate deficiency as the primary cause of reduced oligosaccharide-lipid synthesis when protein synthesis is blocked. The implications of these results, and an alternative hypothesis to explain the effect of inhibition of protein synthesis on oligosaccharide-lipid synthesis based on elevation of intracellular GTP levels, are discussed.Recently, several laboratories [l -41 have reported that inhibition of protein synthesis by a variety of drugs leads to inhibition of the incorporation of t3H] Man into oligosaccharide-PP-Dol. It seems likely that the mechanism of this inhibitory effect is related to regulation for N-linked glycosylation, since the various drugs employed [l -41 inhibit protein synthesis by different modes of action. Since it is well established in a variety of systems in vitro that supplementation with dolichyl phosphate leads to enhanced glycosylation (e.g. [5,6]), it has been suggested that inhibition ...

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“…Y. pestis strain KIM D27 (wild type) (29) and the ⌬lcrV mutant (KLD29) (27) were grown in heart infusion broth (HIB), M9-Casamino Acids (M9-Ca) minimal medium, or thoroughly modified Higuchi's (TMH) medium (27,29,30). Y. enterocolitica strain W22703 (wild type) (31) and the ⌬lcrV mutant (CT1) (17) were grown in tryptic soy broth (TSB) or M9-Ca medium, as indicated.…”

Section: Methodsmentioning

confidence: 99%

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

Ligtenberg

Miller

Mitchell

et al. 2012

Journal of Bacteriology

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c LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly.

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An improved chemically defined culture medium for strain L mouse cells based on growth responses to graded levels of nutrients including iron and zinc ions

Cited by 143 publications

References 8 publications

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Relationship between Oligosaccharide‐Lipid Synthesis and Protein Synthesis in Mouse LM Cells

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

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