Phosphatase inhibitor-1 (I-1) is a distal amplifier element of b-adrenergic signaling that functions by preventing dephosphorylation of downstream targets. I-1 is downregulated in human failing hearts, while overexpression of a constitutively active mutant form (I-1c) reverses contractile dysfunction in mouse failing hearts, suggesting that I-1c may be a candidate for gene therapy. We generated mice with conditional cardiomyocyte-restricted expression of I-1c (referred to herein as dTG I-1c mice) on an I-1–deficient background. Young adult dTG I-1c mice exhibited enhanced cardiac contractility but exaggerated contractile dysfunction and ventricular dilation upon catecholamine infusion. Telemetric ECG recordings revealed typical catecholamine-induced ventricular tachycardia and
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ABSTRACT The polyps of Cassiopea andromeda produce spindle shaped, freely swimming buds which do not develop a head (a mouth opening surrounded by tentacles) and a foot (a sticky plate at the opposite end) until settlement to a suited substrate. The buds, therewith, look very similar to the planula larvae produced in sexual reproduction. With respect to both, buds and planulae, several peptides and the phorbolester TPA have been found to induce the transformation into a polyp. Here it is shown that cantharidin, a serine/threonine protein phosphatase inhibitor, induces head and foot formation in buds very efficiently in a 30 min treatment, the shortest yet known efficient treatment. Some resultant polyps show malformations which indicate that a bud is ordinary polyp tissue in which preparatory steps of head and foot formation mutually block each other from proceeding. Various compounds related to the transfer of methyl groups have been shown to affect head and foot formation in larvae of the hydrozoon Hydractinia echinata. These compounds including methionine, homocysteine, trigonelline, nicotinic acid and cycloleucine are shown to also interfere with the initiation of the processes which finally lead to head and foot formation in buds of Cassiopea andromeda.
The metalion vanadate has insulin-like effects and has been advocated for use in humans as a therapeutic modality for diabetes mellitus. However, since vanadate is a tyrosine phosphatase inhibitor, it may result in undesirable activation of target cells. We studied the effect of vanadate on human mesangial cells, an important target in diabetic nephropathy. Vanadate stimulated DNA synthesis and PDGF B chain gene expression. Vanadate also inhibited total tyrosine phosphatase activity and stimulated tyrosine phosphorylation of a set of cellular proteins. Two chemically and mechanistically dissimilar tyrosine kinase
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The ALP activity in parental cancer cells, SR cells, and differentiated SR cells was assayed using an Alkaline Phosphatase Detection Kit (Millipore). The stem-like phenotypes of the parental CP70 and SR cells were assessed by detecting specific stem cell gene markers using flow cytometry (BD Biosciences) and immunocytochemistry. Cells were stained intracellularly with antibodies against human NANOG (GeneTex), OCT4 (Millipore), stagespecific embryonic antigen 4 (SSEA4; BioLegend), and SOX2 (Millipore), according to the manufacturer’s instructions as described previously [15, 21]. The cells were stained with CD44 (BioLegend), CD105 (BioLegend), CD117 (BioLegend), CD133 (Abcam), E-cadherin (BioLegend), or N-cadherin (BioLegend) fluorescence-conjugated monoclonal antibodies and analyzed by flow cytometry, fluorescence microscopy, or the DeltaVision Imaging System (DV Elite System; Applied Precision).
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Our prior studies had identified sodium stibogluconate (SSG), a drug used for nearly 60 years to treat visceral leishmaniasis in humans , as a potent inhibitor of multi-PTPs that include SHP-1 and other PTPs critical in negative regulation cytokine signaling and immunity [12- 14]. Targeting intracellular PTPs by SSG was suggested by the reduced PTP activity of SHP-1 and SHP-2 from cells cultured in the presence of SSG (10 mcg/ml) [13, 14]. At clinically achievable level of the drug when administered at half the currently recommended dose (10 mg/kg body weight), SSG inhibited recombinant SHP-1 (100%), SHP-2 (80%) and PTP1B (70%) [12, 15]. Selectivity was indicated by its limited impact on recombinant MKP1 PTP under comparable conditions . It is worth noting that all cancer therapeutic kinase inhibitors target multi-kinases. This may provide corresponding multiple impacts against the redundant pro-cancer mechanisms in vivo and could be critical for clinical efficacy . Multi- PTPs inhibitors may have clinical potential via a similar mode of operation and warrant investigation.
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Effect of Pdc phosphorylation on its interaction with G ␤␥ The effect of the phosphorylation level of ciliate Pdc on its interaction with G ␤␥ in vivo was examined in dark-adapted and illuminated Blepharisma cells. Cell lysates were immunoprecipitated with antibody selectively recognizing Pdc protein (28·kDa) (Fabczak et al., 2004) and the phosphorylation level of Pdc was estimated by immunoblotting using antibody against phosphoserine residues (Fig.·1A). In both dark-adapted and illuminated cells, immunoblot analysis demonstrated that Pdc antibody exclusively precipitated one phosphorylated protein of molecular mass 28·kDa (Fig.·1A, lanes 1 and 2). Illumination of cells caused a significant decrease in the phosphorylation level of Pdc (Fig.·1A, lane 2), while phosphorylation of this protein was markedly higher in cells not treated with light (Fig.·1A, lane 1). These experiments also showed that light-induced dephosphorylated Pdc was strongly coimmunoprecipitated with G ␤ (Fig.·1B, lane 2), whereas cell adaptation to darkness resulted in a marked decrease in the amount of G ␤ in the immunoprecipitate (Fig.·1B, lane 1). A result similar to that seen with dark-adapted cells was observed in cells incubated with 1· mol·l –1 of okadaic acid (Fig.·1B, lane 3). Treatment with this serine or threonine phosphatase inhibitor resulted in a significant decrease in the amount of G ␤ bound to Pdc, despite cell stimulation by light. These data are consistent with the observed effect of okadaic acid on the levels of Pdc phosphorylation in cells (Fig.·1A, lane 3). The phosphorylation level of Pdc in cells incubated with this phosphatase inhibitor and subsequently exposed to light was similar to that found in cells adapted to darkness (Fig.·1A, lane 3), and in both cases binding of G ␤ by Pdc was lower (Fig.·1B, lanes 1 and 3). These results showed that the decrease in phosphorylation of ciliate Pdc in response to cell illumination correlates well with the significant increase in G ␤ binding by this protein.
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People are also working to manage arsenic toxicity in plant through various fertilization and nutrient strategies like macronutrients microelements and plant hormones. Phytohormones have profound role in counteracting the effects of toxic metals and metalloids. Phytohormones were found capable of reducing lead toxicity in rice seedlings  and alleviating salinity stress (used as pretreatment chemical) in mungbean plants  Phosphorus is a necessary constituent of many important metabolites. Phosphorus (P) is a critical element required for optimum plant growth, and is essential for sustainable production of food across the globe. There is a growing need to improve the efficiency of Phosphorus use at the plant and whole farm scale . Phosphorus has a vital functional role in energy transfer, and acts as modulator of enzyme activity and gene transcription. Hydrolytic breakdown of phosphate esters is brought about by phosphatases which occur I both acidic and alkaline medium. Acid phosphatases (EC 188.8.131.52) catalyze non-specific hydrolysis of Pi from phosphate monoesters and play a major role in the supply and metabolism of phosphate in plants . Similarly alkaline phosphatases (EC 184.108.40.206) have a potential role in utilization of phosphomonoesters as the source of Pi required for maintenance of cellular metabolism . Again, Adenosine triphosphatases (ATPases) (EC 220.127.116.11) are enzymes that produce inorganic phosphate (Pi) by cleavage of the γ-phosphate of ATP. Geiger et al.,  reported that soil acid phosphatases generally are strongly inhibited by heavy metals. Therefore, the analysis of the potential inhibitory effect of metalloids on phosphatases is of importance, due to the putative role of their activities in mobilizing phosphate. However, little information is available on the effect of arsenic on Phosphorus metabolism and activity of phosphatase enzymes in pulse plants. Phosphorus has another important role in the present context, as it inhibits arsenate uptake in plant system . Arsenic is analogous to phosphate (in the periodic table, both are placed in the same group, VA); both ____________________
Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in many human malignant tumors including cervical cancer. Human papillomavirus (HPV) oncoprotein E7 is the key transformation factor in cervical cancer. Our previous data showed a positive association of CIP2A and HPV-16E7 protein levels; however, how CIP2A is regulated by HPV-E7 and the roles of CIP2A in HPV-E7-mediated cell proliferation are unknown. In this study, we demonstrated that HPV-16E7 protein significantly upregulating CIP2A mRNA and protein expression depended on retinoblastoma protein pRb rather than p130. CIP2A siRNA knockdown in HPV-E7-expressing cells inhibited cell proliferation, DNA synthesis and G1/S cell cycle progression. CIP2A siRNA decreased the protein levels of cyclin-dependent kinase 1 (Cdk1), Cdk2 and their partner cyclin A2, with no change in levels of Cdk4, Cdk6 and their partner cyclin D1. The downregulation of Cdk1 and Cdk2 was independent of c-Myc; instead, E2F1 was the main target of CIP2A in this process, as overexpression of E2F1 rescued the inhibitory effects of CIP2A siRNA knockdown on cell proliferation and G1 arrest of HPV-E7-expressing cells. Our studies reveal a novel function of CIP2A in HPV-16E7-mediated cell proliferation.
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Among the three binding sites of I2, the best-identified and most widely found in PP1 partners is the [R/K]X0-1 [V/I]X0-1[F/W] consensus motif, which corresponds to KTISW in PfI2. The presence of RVxF in about 25-30% of eukaryotic proteins is not a sufficient indicator in it- self to classify a protein as a PP1c regulator . These observations, together with the fact that PfI2 is the shortest I2 protein identified so far (144 amino acids for PfI2 versus 164–205 for other I2), the absence of one binding site (KGILK) and the fundamental difference in the RVxF motif (KTISW) raised the question of the cap- acity of PfI2 to bind and to regulate PfPP1. Using wild- type recombinant proteins, we showed that labeled PfPP1 was able to bind to PfI2 and vice versa. This was further validated by the use of a yeast two-hybrid system that confirmed the interaction of wild-type PfI2 with PfPP1c and suggested that it was strong since the mated PfI2 and PfPP1 yeast strains were able to grow under stringent conditions (SD-LWHA medium). In order to explore the contribution of PfI2 RVxF and HYNE motifs for the interaction with PfPP1, two types of construc- tions were used, one deleted for the Nt moiety of PfI2 and the other with a single mutation in the RVxF motif. Binding was unaffected on SD-LWH medium, whatever the construction tested and only one strain, carrying the PfI2 Y103A, mutant was unable to grow under the most stringent conditions (SD-LWHA medium). These obser- vations show that there is no one, major site of inter- action in PfI2 unlike Pf Inhibitor-3 (PfI3), for which we showed that the mutation of 16 W (localized within the RVxF domain of PfI3) completely abolished its binding/ function . PfI3 exhibits a totally disorganized struc- ture and seems to bind first to PfPP1 via the RVxF groove and folds afterwards to accomplish its function . Regarding I2, previous studies suggested a major role for the RVxF motif along with secondary binding sites which should be intrinsically structured for efficient binding to PP1c [33-35]. PfI2 secondary structure ana- lysis predicted that the RVxF motif is a part of an un- structured region, while the HYNE is within an α-helix. The role of this structure in PfI2-PfPP1c interaction was substantiated by the lack of binding of PfI2 deleted for the region containing the α-helix (PfI2 (1–94)). In the case of mutated PfI2, the yeast two-hybrid method sup- ported a role for 103 Tyr (localized within the HYNE do- main of PfI2) in the stabilization of PfI2-PfPP1 binding under stringent culture conditions.
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phatase when phytin was added to the soil, com- pared to the addition of KH 2 PO 4 or Ca(H 2 PO 4 ) 2. Marschner et al. (2007) showed that the P uptake by plants (Brassicae and Poaceae) significantly correlated with the activity of acidic phosphatase only at low P levels in soils. Interestingly, the activity of the acidic phosphatase and P uptake by plants correlated well at early stages of plant growth (phase of 6 leaves, blooming phase), but not at later stages of mature plants. Therefore, the authors assume that the mobilization of organic P is more emphasized at earlier growth stages. Similarly, the alkaline phosphatase hydrolyzes P from organic compounds. It is produced by soil fungi and bacteria (Dick and Kandeler 2005). Bhadraray et al. (2002) found a high correlation between the activity of the alkaline and acidic phosphatases in the rhizosphere of plants and the degree of evolution of the rice panicle.
activity of alkaline phosphatase. Kidney lesions due to toxic damage were largely confined to tubular epithe- lium, resulting in the suppression of tubular reabsorbing function as evidenced by structural alterations in tu- bules. These observations are similar to the reports pub- lished on the effect of proprietary herbal formulation [ 43]. Propolis treatment recovered ALPase activity after subchronic toxicity substantiating regenerative property [ 19]. ACPase is a lysosomal enzyme and its liberation include ionic imbalance followed by mitochondrial damage and stimulation of lysosomes leading to enzyme release. It is evident that lysosomal enzymes play im- portant role in pathogenesis of CCl 4 -induced hepatic
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Hydrolytic enzymes play an important role dur- ing germination in the mobilisation of endosperm reserves. They are rapidly activated during imbi- bition; however, some of them exist in the active form already in the aleurone layer and scutellum of dry mature grains (Fincher 1989). Glucosidase, phosphatase and protease activity was found mainly in the protein bodies and spherosomes of ungerminated seeds (Adams and Novellie 1975), however phosphatase and esterase activity was reported also from the testa of ungerminated wheat grains (Price and Ey 1970). In our previ- ous work we reported the significant increase in acid phosphatase in barley roots during Al treat- ment (Huttová et al. 2002). In contrast Al at 8mM concentration markedly inhibited the release of acid phosphatase from imbibiting barley grains. Our results demonstrated that inhibition of acid phosphatase did not occur in in vitro conditions. Bailey et al. (1976) reported that during barley grain imbibition new acid phosphatase isoen- zymes occurred, which are probably inhibited by Al. The role of secreted phosphatases is the hydrolysis of phosphate-esters from inorganic and organic phosphates, which can be disturbed at the presence of Al. Our results indicated that Al-inhibition of hydrolytic enzymes might cause phosphate deficiency during the first period of the germination. Similarly to phosphatases Al-induced inhibition of glucosidase and esterase may induce several defects in seed reserve mobilization. In ad- dition secreted forms of hydrolytic enzymes are generally involved in plant defence mechanism in plant microbe interaction (Boller 1987). Inhibition of hydrolytic enzymes and activation of antioxida- tive enzymes suggest that Al-induced stress had already started in the early stage of the grain im- bibition which was supported by the incapability of grains to change the pH of imbibition solution at higher Al concentration.
In the present study the alkaline phosphatase was found to be inhibited in the gill and liver exposed to the sublethal concentrations of effluent for 30 days. The inhibition may be due to altered membrane permeability which is brought about by the binding of the heavy metal ions present in the effluent to the enzyme configuration. Furthermore, the inhibition of alkaline phosphatase activity may have hampered glycogen and lipid metabolisms and disrupted the transfer of these catabolites of the hepatic cells and it falls in line with the report of Jignasa Dalela et al ., (1980). In the liver, the inhibition of enzyme may be due to disruption in the membrane permeability of the hepatic cells which ultimately affects other functions of the liver (Dalela et al., 1980). In the case of intestine, the activity of acid phosphatase showed massive inhibition in all the concentrations. The reduction in Table 1. Activity of acid phosphatase (µ mole PNP/mg) in the tissues of Arius macculatus exposed to sublethal concentrations (% 96
It is well known that the monomeric C subunit is unstable and requires binding to the A subunit or other non-canonical B subunits to preserve its activity . Amounting evidence shows that PP2A phosphatase activation is regulated by PTMs of PP2A-C subunit protein, B subunit diversity, the association of regulatory subunits with substrate proteins or binding with a 4 [49, 50], an important regulator of PP2A activity. The current investigations have shown that the C-terminus of PP2A-Cα undergoes methylation at L309 by the regulation of two enzymes, methyltransferase LCMT-1 that is expressed in the cytoplasm and a lipase-like methylesterase PME-1 that is expressed in the nucleus . On the other hand, the phosphorylation of multiple residues on the C terminus of PP2A-C is also critical for modulating PP2A activity via B subunit interactions. A recent report showed that ubiquitin E3 ligase NOSIP induces the ubiquitination of PP2A-C subunit and the loss of NOSIP enhances PP2A activity, resulting in perinatal lethality . Also, progestin-inducible EDD E3 ubiquitin ligase could polyubiquitinate PP2A-C by binding to the C-terminal of α4 . Here we also demonstrated that FAM122A potentiates poly- ubiquitination of anti-PP2A-Cα precipitates, which suggests that FAM122A enhances ubiquitination of PP2A- Cα and/or its partner(s) such as CIP2A, SET and TIPRL. To address this, in vitro ubiquitination assay by using recombinant PP2A-C protein deserves to be performed. Also, it remains to be further investigations how
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Diabetes is a metabolic disorder wherein blood glucose level is increased along with some other abnormal conditions like polyuria, polydipsia and polyphagia. As per WHO estimation, 380 million people will become diabetic by 2025. Tyrosine residues are selectively dephosphorylated by Protein tyrosine phosphatases (PTPs) and thus a wide variety of cellular processes are regulated by their action. Protein tyrosine phosphatase 1B (PTP1 B) has shown to be a negative regulator in the insulin signaling pathways. Recent gene knockout studies carried out on mice portrays PTP1B as an effective target for drug discovery process related to anti-diabetic and anti-obese agents. PTPs are also involved in several other disorders like cancer. The structure of compounds synthesized by the present method were confirmed by TLC, IR, NMR and Mass spectroscopy. The anti-diabetic activity of the synthesized compounds were tested against PTP1B enzyme by using Calbiochem® PTP1B colorimetric assay kit. Among all synthesized compounds 4c, 4d, 4e, 4f had shown promising anti-diabetic activity, while other compounds have shown lesser potency as anti-diabetic agent.
AKT: serine/threonine kinase 1; Bcl-2: B-cell CLL/lymphoma 2; CIP2A: cancer- ous inhibitor of protein phosphatase 2A; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; ERK1/2: extracellular signal-regulated kinases ½; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; HM: human mesothelial cells; Hr: hours; MM: malig- nant mesothelioma; SphK1: sphingosine kinase 1; PARP: poly(ADP-Ribose) polymerase; PI: propidium iodide; PS: phosphatidylserine; PP2A: protein phosphatase 2A; PVDF: polyvinylidenefluoride; TRIS: trisaminomethane; WT-1: Wilms tumor 1; VDAC: voltage-dependent anion channel.
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receptor-mediated phenomena at the cell surface (Garcia- Romeu et al. 1988). This decline in activity also does not reflect a simple activation/deactivation transition of the antiporter, but a transition from an active to a refractory state. βNHE can no longer be immediately reactivated by a fresh challenge with catecholamine or cyclic AMP. Several hours without stimulation are necessary for the exchanger to recover its ability to respond to cyclic AMP or catecholamines (Guizouarn et al. 1993). The decline in activity thus reflects a desensitisation of the transport system itself (Garcia-Romeu et al. 1988). This βNHE desensitisation is blocked and reversed by OA, indicating control by an OA-sensitive phosphatase of the phosphorylation level of a site critical for the desensitisation process. Phosphorylation of this site is also mediated by a cyclic-AMP-dependent protein kinase (Guizouarn et al. 1993). In conclusion, the activation of βNHE would result from the phosphorylation by PKA of at least two different sites, one being insensitive to OA, switching the antiporter on, and the other sensitive to this inhibitor, being necessary to maintain the antiporter in a ‘transporting’ state. Desensitisation would be due to the dephosphorylation of the OA-sensitive site by a protein phosphatase 1 (PP1) (Guizouarn et al. 1993, 1995). Experiments designed to evaluate the phosphorylation state of the antiporter, immunoprecipitated from red cells, at different times after stimulation are in progress.
The Epstein-Barr virus (EBV)-encoded leader protein, EBNA-LP, strongly activates the EBNA2-mediated transcriptional activation of cellular and viral genes and is therefore important for EBV-induced B-cell transformation. However, a truncated form of EBNA-LP is produced in cells infected with variant EBV strains lacking EBNA2 due to a genetic deletion. The function of this truncated form is unknown. We show here that some Burkitt’s lymphoma cells harboring defective EBV strains are specifically resistant to the caspase- dependent apoptosis induced by verotoxin 1 (VT-1) or staurosporine. These cells produced low-molecular- weight Y1Y2-truncated isoforms of EBNA-LP, which were partly localized in the cytoplasm. The transfection of sensitive cells with constructs encoding truncated EBNA-LP isoforms, but not full-length EBNA-LP, induced resistance to caspase-mediated apoptosis. Furthermore, VT-1 induced protein phosphatase 2A (PP2A) acti- vation in sensitive cells but not in resistant cells, in which the truncated EBNA-LP interacted with this protein. Thus, the resistance to apoptosis observed in cells harboring defective EBV strains most probably results from the inactivation of PP2A via interactions with low-molecular-weight Y1Y2-truncated EBNA-LP isoforms.
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concluded that PMA induced receptor down-regulation could be the result of an inhibition of G -protein activity. Other studies report changes in receptor affinity and/or number in response to phorbol ester treatment as explanation for the observed receptor down-regulation (Chen et al., 1995; Turner et al., 1996). Phosphorylation is an important step in receptor regulation, and the fact that OA effects were not additive with PMA, as results presented here demonstrate, suggests that OA and PMA influence the same component in this process. Results also show that the effect of PMA on NA-evoked [^H]-IP accumulation was reversed completely by staurosporine. Although this evidence strongly indicate that PKC is the PK involved in regulating these receptors, confirmation was sought by the use of PKC depleted ghal cultures. Young et al. (1987) have shown that prolonged phorbol ester treatment causes down regulation of PKC. The authors demonstrated a progressive loss of PKC as a result of chronic PMA exposure of rat ghoma ceUs and that the consequence of this was an increased rate of degradation of the polypeptide. Consistent with that observation, they were also unable to detect any change in the amounts of mRNA for PKC. Pearce et al. (1993) also showed that prolonged PMA treatment results in PKC down-regulation in gha. Results presented here show the effect of OA was not observed in PKC depleted ghal cultures which, having demonstrated that the effects of OA on NA-stimulated pH]-IP accumulation are reversed by a PKC inhibitor, suggests that PKC is involved in this response.
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␣⬘ catalytic subunit of the CK2 kinase, thus indicating that CK2 kinase is a novel determinant of a fully functional ceramide synthase. Deletion of DPL1 was previously demonstrated to be lethal in strains lacking Lcb3 phosphatase (18, 48). A single LCB3 or DPL1 deletion results in a 5- to 40-fold increase of the steady-state levels of the LCBPs and does not have a measurable effect on cell growth. However, elimination of both the Lcb3 phosphatase and the Dpl1 lyase activities from the cell causes massive accumulation of LCBPs, over 400-fold above the wild-type levels, which in turn leads to cell death. Recent detailed analysis of LCBP accumulation in yeast indi- cates that DHS-1-P species are more toxic than PHS-1-Ps and that the phosphatase rather than the lyase is involved in the removal of the toxic excess of these intermediates (48). Over FIG. 7. The ⌬lcb3 synthetically lethal mutants accumulate LCBs and LCBPs. LCBs and LCBPs were extracted from 12 OD units of Y388 background cells and from 30 OD units of BY4741 background cells, derivatized with AQC, and treated with KOH to deacylate glycerophos- pholipids as described in Materials and Methods. (A) Ten microliters of each extract was resolved by TLC in solvent I under conditions separating AQC-LCBs. (B) Fifty microliters of each extract was absorbed on a C 18 column, and AQC-LCBPs that eluted from the column were separated
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