To establish an accurate molecular model of human branched-chain amino acid (BCAA) metabolism, the distribution, activity, and expression of the first 2 enzymes in the catabolic pathway--branched-chain-amino-acid aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) complex--were determined in human tissues. The same enzyme activities were measured in rat and African green monkey tissues. Overall, the activities of BCAT and BCKD were higher in rat than in human and monkey tissues; nevertheless, the ratio of the 2 activities was similar in most tissues in the 3 species. Total oxidative capacity was concentrated in skeletal muscle and liver (> 70%) with muscle having a higher proportion of the total in humans and monkeys. In humans, brain (10-20%) and kidney (8-13%) may contribute significantly to whole-body BCAA metabolism. Furthermore, in primates the high ratio of transaminase to oxidative capacity in the entire gastrointestinal tract serves to prevent loss of essential BCAA carbon and raises the possibility that the gastrointestinal tract contributes to the plasma branched-chain alpha-keto acid pool. Quantitative polymerase chain reaction was used to examine expression of human branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), the key enzyme that regulates the activity state of the human BCKD complex and human BCAT isoenzymes. To design the primers for the polymerase chain reaction, human BCKDK was cloned. BCKDK message was found in all human tissues tested, with the highest amount in human muscle. As in rats, there was ubiquitous expression of mitochondrial BCAT, whereas mRNA for the cytosolic enzyme was at or below the limit of detection outside the brain. Finally, the role of BCAA in body nitrogen metabolism is discussed.

Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of γ-interferon–inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILT's ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses.

The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal ␤-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes ␤-hydroxyisobutyryl-CoA. We demonstrated that a human ␤-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes ␤-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered ␤-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4+ T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.

Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)^positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC 50 , 12-24 Amol/L) and ER-negative (IC 50 , 45 Amol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-a levels notably in ER-positive lines. Ritonavir causes G 1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D 1 but not cyclin E, and depletes phosphorylated Rb and Ser 473 Akt. Ritonavir induces apoptosis independent of G 1 arrest, inhibiting growth of cells that have passed the G 1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum C max of 22 F 8 Amol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K D , 7.8 Amol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90a short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate is a common mechanism of stimulus transduction (1). Diacylglycerol (DAG) 1 released in this reaction activates protein kinase C (PKC) and is then rapidly metabolized back to phosphatidylinositol in a series of reactions initiated by a diacylglycerol kinase (DGK). As such, DGKs attenuate DAG-mediated PKC activation (2). Recent studies indicate that DGKs are also activated by mechanisms independent of phosphoinositide turnover (3, 4). Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of sn-1,2-diacylglycerol to form phosphatidic acid (PA), which is also a lipid mediator (5, 6). Several DGK isoforms have been cloned (7). All these sequences share a homologous catalytic domain and two or three C1 protein kinase C homology domains (7-9). Some DGKs contain EF hands, which are Ca 2ϩ -binding sites (7). These DGKs also have a domain at their N termini with homology to the recoverin family of neuronal calcium sensors (Fig. 1). We term this the recoverin homology (RVH) domain. In S-modulin, the frog orthologue of recoverin, this domain associates with the EF hands to mediate Ca 2ϩ -dependent inhibition of rhodopsin kinase (10).The varied structures of DGK regulatory domains suggest divergent mechanisms of regulation. Several studies have shown variation among DGKs with regard to activation by phospholipids, sphingosine, or Ca 2ϩ (11)(12)(13)(14)(15). Kanoh and coworkers (16 -18) have studied DGK␣, a Ca 2ϩ -activated isoform highly expressed in oligodendrocytes and thymocytes. They have shown that Ca 2ϩ binds the EF hand region of the enzyme and that deletion of the EF hands results in constitutive enzyme activation (19,20). We have now examined a series of DGK␣ mutants in which the RVH and EF hand domains are sequentially deleted. Our results indicate that the N-terminal RVH domain is required for Ca 2ϩ to activate this enzyme. In contrast to the constitutive activation seen with deletion of the EF hands, DGKs with deletions involving only the RVH domain expressed activity similar to that of wild-type enzyme in the absence of Ca 2ϩ . Sites within both the EF hands and RVH domain were protected from trypsin proteolysis by Ca 2ϩ , indicating that both domains participate in a Ca 2ϩ -induced conformational change. A cationic amphiphile, octadecyltrimethylammonium chloride, markedly stimulated DGK␣ activity in vitro. This effect, like Ca 2ϩ -dependent activation, was dependent on the RVH domain. The DGK␣ RVH domain does not itself bind Ca 2ϩ . However, it does appear to function together with the EF hands to couple Ca 2ϩ binding to release of EF handmediated autoinhibition of DGK␣. EXPERIMENTAL PROCEDURES Materials-Restriction

The metabolic syndrome is complicated by nephropathy in humans and rats, and males are more affected than females. We hypothesized that female rats had reduced expression of glomerular oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1), attendant glomerular oxidant injury, and renal inflammation. Three groups, obese males (OM), obese females (OF), and lean males (LM) of first-generation (F(1)) hybrid rats derived from the Zucker fatty diabetic (ZDF) strain and the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) were studied from 6 to 41 weeks of age. OM had severe renal oxidant injury and renal failure. Their glomeruli expressed the LOX-1, and exhibited heavier accumulation of the lipid peroxide 4-hydroxynonenal (4-HNE). OM had compromised mitochondrial enzyme function, more renal fibrosis, and vascular leakage. Younger LM, OM, and OF ZS (ZDF/SHHF F(1) hybrid rat) rats, studied from 6 to 16 weeks of age, showed that unutilized renal lipids were comparable in OM and OF, although young OM had worse nephropathy and inflammation. In conclusion, glomerular LOX-1 expression is coupled to deposits of 4-HNE and glomerulosclerosis in OM. We presume that LOX-1 enhances glomerular uptake of oxidized lipids and renal inflammation, causing greater oxidant stress and severe glomerulosclerosis. In OF, renal protection from lipid oxidants appears to be conferred by blunted glomerular LOX-1 expression and renal inflammation.

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.