David J. Prescott

High Fatty Acid Content in Rabbit Serum Is Responsible for the Differentiation of Osteoblasts Into Adipocyte‐like Cells

Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte‐like differentiation in human osteosarcoma SaOS‐2/B10 and MG‐63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria‐derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS‐2/B10 and MG‐63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARγ mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG‐63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARδ/NUC‐1 but not PPARα, indicating that cells that do not express either PPARγ or PPARα are capable of differentiating into adipocyte‐like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR‐dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35‐fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte‐like differentiation of both ROS17/2.8 and SaOS‐2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte‐like cells.

[12] Acyl carrier protein synthetase

Publisher Summary This chapter discusses the determination of acyl carrier protein (ACP) synthetase. The conversion of apo-ACP to ACP is measured in a two-stage assay. In the first-stage synthesis of ACP is allowed to proceed, catalyzed by ACP synthetase. This stage is terminated, and the amount of ACP synthesized is determined in the second stage by means of the malonyl-CoA-CO2 exchange reaction. Apo-ACP can be prepared enzymatically from ACP or chemically using 60% HF to cleave the phosphodiester bond linking the hydroxyl of serine-36 to 4´-phosphopantetheine. In the course of purification, all operations are carried out at 4°. Escherichia coli strain B are thawed and suspended in 2 liters of 0.01 M Tris-HCl, pH 7.4 containing 0.01 M 2-mercaptoethanol and 0.0001 M ethylenediaminetetraacetic acid (EDTA). The suspension is ruptured in a Manton–Gaulin submicron dispersor at 9000 psi. To the viscous stirred suspension is added 800 ml of a 10% streptomycin sulfate solution and the suspension is centrifuged for 30 minutes at 27,500 g. The supernate is saved (3400 ml, 55 g protein).

Aggregation Properties of the Acetylcholinesterase from the Central Nervous System of Manduca sexta

Abstract: The CNS of the tobacco hornworm, Manduca sexta, provides a rich source of true acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7.). Optimal extraction of the enzyme was obtained with a nonionic detergent at high ionic strength (1% Triton X‐100, 0.5 M NaCl). Velocity sedimentation of the Triton + salt‐extracted enzyme demonstrated a single peak whose sedimentation coefficient was dependent upon the enzyme concentration layered on top of the gradient. When more than 20 units were applied to the gradient, a sedimentation coefficient of 8.6 S (205,000) was obtained, and extrapolation to zero units yielded a 5.7 S (110,500) species. Sedimentation in the absence of detergents (1.0 M NaCl or 10 mM phosphate buffer, pH 7.4) yielded pelleted enzyme and species with mean values of 18.6 S (650,000) and 17.5 S (600,000), respectively. The detergent‐extracted enzyme also demonstrated a concentration‐dependent size in gel filtration experiments. When less than 300 units were applied to the column, a single species was recovered, with a molecular radius of 40.15 ± 2.08 Å, (108,000) or 43.4 ± 2.38 Å, (117,000) calculated by different methods. If the sample contained 300 to 1300 units, two species were observed, with molecular radii of 40.15 ± 2.08 Å or 43.4 ± 2.38 Å and 78.4 ± 3.94 Å, (319,000) or 80.25 ± 3.01 Å (326,000). Velocity sedimentation and gel filtration of AChE have demonstrated that the enzyme has a minimum molecular weight of approximately 110,000 and also exists as higher‐molecular‐weight aggregates of this value.

Characterization of an unsaturated fatty acid auxotroph of tetrahymena thermophila

Abstract A mutant Tetrahymena thermophila RH179E1 was found to require an unsaturated fatty acid for growth in a defined medium. The mutant cannot be propagated on palmitic or stearic acid supplements; however, palmitoleic, oleic, cis -vaccenic, linoleic and γ-linolenic acid additions will sustain growth. The inability of the saturated acids, palmitate and stearate, to support growth indicated a deficiency in Δ 9 -desaturase activity and, thus, in the ability to form palmitoleic and oleic acids. This conclusion was supported by the low level of measurable Δ 9 -desaturase activity in the microsomal fraction of the mutant cells compared to the parental strain. Analysis of the fatty acyl composition of the glycerophospholipids from the unsaturated fatty acid-supplemented cells showed extensive conversion to Δ 6 derivatives: The major product of oleic acid (18:1 Δ 9 ) desaturation was 18:2 Δ 6,9 ; 16:1 Δ 9 gave rise to 18: 1 Δ 11 and 18:2 Δ 6,11 , and 18:1 Δ 11 18:2 Δ 6,11 (cilienic acid). Supplements of 18:2 Δ 9,12 were readily converted to γ-linolenic acid. The paucity of 18:2 Δ 9,12 and low levels of 18:3 Δ 6,9,12 in oleic acid-supplemented cells indicates that the Δ 12 -desaturase function is reduced in the mutant cells. The fatty acyl profiles of the parental strain were changed by palmitoleate, oleate, cis -vaccenate and 18:2 Δ 9,12 supplements, showing that large amounts of the supplemented acid are incorporated into the glycerophospholipids and that inhibition of the desaturase activities may occur. γ-Linolenate dramatically reduces the cellular content of all other unsaturated acids. In this case there appears to be strong regulation exerted on de novo unsaturated acid synthesis.

Preliminary characterization of the delta-9 desaturase of Tetrahymena pyriformis W

1. In vitro assay conditions have been defined for measurement of delta 9 desaturase activity in Tetrahymena pyriformis W. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30 degrees C. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or delta 12 desaturase activities have also been observed. 5. When the specificity of the delta 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16 degrees C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on delta 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.