Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the 13-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH is a complex process catalyzed by the fatty acid synthase (FAS). In animal tissues, the active synthase is a homodimer of a multifunctional protein that is organized in a head-to-tail fashion, generating two active catalytic centers (1). The seven partial activities and the site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein), are arranged on the multifunctional protein subunit from the amino to carboxyl termini in the following order: f3-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase (1). Most information about the synthase is derived from nonhuman animal studies, so little is known about the human synthase. Investigators who isolated the enzyme from human biopsy tissues (2-5) or cell lines (6) have shown that, except for the enzyme from the SKBR3 cell line (6), all the human FAS preparations had lower activity than the FASs of other animals. This lower activity of the human FAS is comparable to the activities of related human enzymes that are involved in lipogenesis (e.g., pyruvate dehydrogenase, citrate lyase, and glucose-6-phosphate dehydrogenase), which are lower (by a factor of 4-7) in human tissues than in other animal tissues, implying that lipogenesis in humans is highly repressed (7-9).Here we describe the purification and catalytic properties of Assays of FAS and Its Partial Activities. The synthase activity was assayed by measuring the rate of oxidation of NADPH or the incorporation of radiolabeled acetyl-CoA or malonyl-CoA into palmitate (10). The partial activities of FAS were assayed as described earlier: the acetyl/malonyl transacylases (11), dehydratase (12), ,B-ketoacyl synthase (12), f3-ketoacyl reductase (13), ,B-hydroxyacyl enoyl reductase (12), and thioesterase (14). The f3-ketoacyl synthase activity was also determined by measuring the increase in absorbance at 280 nm due to formation of triacetic acid lactone (15).4'-Phosphopantetheine Content. To determine the 4'-phosphopantetheine content of FAS, we developed a simple and sensitive method based on spectrophotometric measurement of the phenylthiocarbamoyl (PTC) derivative of taurine released after performic acid oxidation and hydrolysis of the protein. PTC-taurine can be separated from other PTC-amino acids by using reverse-phase high-performance liquid chromatography (HPLC) and following the absorbance at 254 nm. In this system,...

Gamma-aminobutyric acidergic (GABAergic) interneurons (INs) in the dentate gyrus (DG) provide inhibitory control to granule cell (GC) activity and thus gate incoming signals to the hippocampus. However, how various IN subtypes inhibit GCs in response to different excitatory input pathways remains mostly unknown. By using electrophysiology and optogenetics, we investigated neurotransmission of the hilar commissural pathway (COM) and the medial perforant path (MPP) to the DG in acutely prepared mouse slices. We found that the short-term dynamics of excitatory COM-GC and MPP-GC synapses was similar, but that the dynamics of COM- and MPP-mediated inhibition measured in GCs was remarkably different, during theta-frequency stimulation. This resulted in the increased inhibition-excitation (I/E) ratios in single GCs for COM stimulation, but decreased I/E ratios for MPP stimulation. Further analysis of pathway-specific responses in identified INs revealed that basket cell-like INs, total molecular layer- and molecular layer-like cells, received greater excitation and were more reliably recruited by the COM than by the MPP inputs. In contrast, hilar perforant path-associated and hilar commissural-associational pathway-related-like cells were minimally activated by both inputs. These results demonstrate that distinct IN subtypes are preferentially recruited by different inputs to the DG, and reveal their relative contributions in COM-mediated feedforward inhibition.

In earlier studies, the neural cell adhesion molecule, N-CAM, was found to inhibit the proliferation of rat astrocytes both in vitro and in vivo. To identify the gene targets involved, we used subtractive hybridization to examine changes in gene expression that occur after astrocytes are exposed to N-CAM in vitro. While the mRNA levels for N-CAM decreased after such treatment, the levels of mRNAs for glutamine synthetase and calreticulin increased. Since glutamine synthetase and calreticulin are known to be involved in glucocorticoid receptor pathways, we tested a number of steroids for their effects on astrocyte proliferation. Dexamethasone, corticosterone, and aldosterone were all found to inhibit rat cortical astrocyte proliferation in culture in a dose-dependent manner. RU-486, a potent glucocorticoid antagonist, reversed the inhibitory effects of dexamethasone. These observations prompted the hypothesis that the inhibition of proliferation by N-CAM might be mediated through the glucocorticoid receptor pathway. Consistent with this hypothesis, the inhibition of astrocyte proliferation by N-CAM was reversed in part by a number of glucocorticoid antagonists, including RU-486, dehydroepiandrosterone, and progesterone. In transfection experiments with cultured astrocytes, N-CAM treatment increased the expression of a luciferase reporter gene under the control of a minimal promoter linked to a glucocorticoid response element. The enhanced activity of this construct stimulated by N-CAM was abolished in the presence of RU-486. The combined data suggest that astrocyte proliferation is in part regulated by alterations in glucocorticoid receptor pathways.

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress‐inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA‐A*0201. These peptides were able to trigger a CTL response in vivo in HLA‐A*0201‐transgenic HHD mice and in vitro in HLA‐A*0201+ healthy donors. p391‐ and p393‐specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA‐A*0201‐restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA‐A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70‐derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins. © 2003 Wiley‐Liss, Inc.