In the complete absence of K+ and phosphate (Pi), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated K m for ATP, and this activity is insensitive to ...effector regulation. We find that K+ or 5-fold lower levels of NH4 + markedly enhanced quenching of Trp383 fluorescence of PDHK2 by ADP and ATP. K+ binding caused an ∼40-fold decrease in the equilibrium dissociation constants (K d) for ATP from ∼120 to 3.0 μM and an ∼25-fold decrease in K d for ADP from ∼950 to 38 μM. Linked reductions in K d of PDHK2 for K+ were from ∼30 to ∼0.75 mM with ATP bound and from ∼40 to ∼1.7 mM with ADP bound. Without K+, there was little effect of ADP on pyruvate binding, but with 100 mM K+ and 100 μM ADP, the L 0.5 of PDHK2 for pyruvate was reduced by ∼14 fold. In the absence of K+, Pi had small effects on ligand binding. With 100 mM K+, 20 mM Pi modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L 0.5 for pyruvate was specifically decreased ∼125-fold with 100 μM ADP. Pi effects were minimal when NH4 + replaced K+. We have quantified coupled binding of K+ with ATP and ADP and elucidated how linked K+ and Pi binding are required for the potent inhibition of PDHK2 by ADP and pyruvate.
A facile metal-catalyzed diversification step for the synthesis of novel bi- and tricyclic scaffolds from enyne substrates is reported in this study. From a single starting material, topologically ...diverse scaffolds for library synthesis can be generated and decorated in a few steps. The methodology was used to produce a library of 490 compounds within the European Lead Factory (ELF) Consortium.
We studied the prognostic value of angiogenesis grading and microvessel
density estimation in newly diagnosed multiple myeloma. Seventy-five
patients with newly diagnosed myeloma, treated on Eastern ...Cooperative
Oncology Protocol E9486 and Intergroup study 0141 (S9321) at the Mayo
Clinic, were studied. Bone marrow microvessels were examined using
immunohistochemical staining for von Willebrand factor. Determination
of microvessel density and angiogenesis grading was done in a blinded
manner. There was a strong correlation between microvessel density and
the plasma cell labeling index, rho 0.42, P <
0.001. Angiogenesis grade was also significantly associated with the
plasma cell labeling index. Fifteen % of patients with low-grade
angiogenesis had a high labeling index (>1%). In contrast, 47% of
patients with intermediate or high-grade angiogenesis had high labeling
indices ( P = 0.02). Overall survival was
significantly different among those with high-, intermediate-, and
low-grade angiogenesis, with median times of 2, 4, and 4.4 years,
respectively ( P = 0.02). Similarly, patients with
microvessel density >50/×400 field had poorer survival compared with
those with 50 or fewer microvessels/field, median survival 2.6
versus 5.1 years, respectively ( P =
0.004). There was a strong association between angiogenesis grade and
microvessel density ( P < 0.001). We conclude that
bone marrow angiogenesis is a predictor of poor survival in newly
diagnosed myeloma. Angiogenesis is correlated with the plasma cell
labeling index but not the bone marrow plasma cell percentage. A simple
visual grading of angiogenesis is an efficient alternative to
microvessel density estimation.
Association of the PDHK2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2)) dimers was enhanced by K+ with higher affinity K+ binding ...than occurs at the PDHK2 active site. Supporting a distinct K+ binding site, the NH4 + ion did not effectively replace K+ in aiding GST-L2 binding. With 50 mM K+, Pi enhanced interference by ADP, ATP, or pyruvate of PDHK2 binding to GST-L2. The inclusion of Pi with ADP or ATP plus pyruvate greatly hindered PDHK2 binding to GST-L2 and promoted PDHK2 forming a tetramer. Reciprocally, GST-L2 interference with ATP/ADP binding also required elevated K+ and was increased by Pi. Potent inhibition by Nov3r of E2-activated PDHK2 activity (IC50 of ∼7.8 nM) required elevated K+ and Pi. Nov3r only modestly inhibited the low activity of PDHK2 without E2. By binding at the lipoyl group binding site, Nov3r prevented PDHK2 binding to E2 and GST-L2. Nov3r interfered with high-affinity binding of ADP and pyruvate via a Pi-dependent mechanism. Thus, GST-L2 binding to PDHK2 is supported by K+ binding at a site distinct from the active site. Pi makes major contributions to ligands interfering with PDHK2 binding to GST-L2, the conversion of PDHK2 dimer to a tetramer, and Nov3r (an acetyl-lipoate analog) interfering with binding of ADP and pyruvate. Pi is suggested to facilitate transmission within PDHK2 of the stimulatory signal of acetylation from the distal lipoyl-group binding site to the active site.
Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is ...required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: ∼53%/3μm for ATP; ∼49%/15μm for ADP; and ∼71%/∼590 μm for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and ≥80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 μm pyruvate, ≥150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2red) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2red was modestly hindered by 200 μm level of ATP or ADP or 5.0 mm pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 μm levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2red may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.
Pyruvate dehydrogenase phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by ...Ca2+-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca2+ was evaluated in activity and L2 binding studies. An increase in the Mg2+ concentration decreased the Ca2+ concentration required for half-maximal activation of PDP1 from 3 to 1 μM, but this parameter was unchanged at 3 μM with PDP1c. Near 1 μM Ca2+, tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg2+. With just Ca2+ included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c·Ca2+·L2 complex is supported by micromolar Ca2+ concentrations and becomes sensitive to the Mg2+ level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 ± 2.5 μM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K d of ∼0.3 μM. Isothermal titration calorimetry and 45Ca2+ binding studies failed to detect binding of Ca2+ (<100 μM) to L2 or PDP1c, alone, but readily detected binding to L2 and PDP1c. Therefore, both proteins are required for formation of a complex with tightly held Ca2+, and complex formation hinders the tendency of PDP1c to form a dimer.
The objective of the present study was to determine how low progesterone (P4) affects the endometrial transcriptome, with specific emphasis on those changes that may impact conceptus elongation. ...Following estrous synchronization and detection (estrus = Day 0, n = 40), heifers were randomly assigned to a control group (n = 12) or a low P4 group (n = 28). Heifers in the low P4 group had consistently lower P4 concentrations compared to controls (P < 0.05). Microarray analysis of endometrial gene expression revealed low P4 altered the expression of 498 differentially expressed genes (DEGs; 215 up- and 283 down-regulated) on Day 7 and 351 DEGs (272 up- and 79 down-regulated) on Day 13. A similar number of temporal changes occurred between Day 7 and Day 13 in both groups (2212 in heifers with normal P4 compared with 2247 in heifers with low P4); of these DEGs, 1278 were common to both groups. Little overlap in the number of DEGs affected by high or low P4 was observed across days. Comparison of the temporal changes that occur during normal estrous cycle progression (i.e., from Day 7 to Day 13) to those affected by altered P4 found significant numbers of genes were modulated by elevated (4157) and decreased (809) P4 alone. Analysis of selected genes by quantitative real-time PCR and in situ hybridization revealed that expression of MEP1B, NID2, and PRSS23 increased on Day 13 compared to Day 7 (P < 0.05) and that the magnitude of increase was significantly diminished in heifers with low P4 compared to controls. MEP1B predominantly localized to the both the superficial and deep glandular epithelium (GE), NID2 localized to the deep GE, whereas PRSS23 localized only to the luminal epithelium. In conclusion, we have determined the global changes in the endometrial transcriptome induced by decreasing the output of P4 from the corpus luteum in vivo using a unique animal model. Placing these data into context with previous data in which P4 was supplemented or elevated after ovulation, we have identified a panel of genes that are truly regulated in the endometrium by circulating concentrations of P4 in vivo and that likely impact conceptus elongation.
Control of the plasma-wall interaction during transient events will be a critical issue in ITER. A new ITER-like wide-angle infrared and visible diagnostic, allowing to observe plasma wall ...interaction in the main chamber, has been installed on JET. The design and the manufacture of the diagnostic and the test results are presented. ELM interaction with the JET main chamber has been observed. ELM interaction with the walls occurs mainly on the divertor but a part of the ELMs energy impacts also on the upper and outer limiters.
The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding ...framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd ≃ 175 μm for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd ≃ 3 μm), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in ∼8-fold tighter binding of PDK2 to GST-L2red, which was ∼300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound ∼18 PDK2 dimers with a Kd similar to GST-L2. E2·E1 bound more PDK2 (∼27.6) than E2 with ∼2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2·E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3–5-fold and adenosine 5′-(β,γ-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2·E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed “hand-over-hand” movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.
Background & Aims
:
Although considerable effort has been directed toward the mapping of peptide epitopes by autoantibodies, the role of nonprotein molecules has been less well studied. The ...immunodominant autoantigen in primary biliary cirrhosis (PBC), E2 components of pyruvate dehydrogenase complexes (PDC-E2), has a lipoate molecule bonded to the domain to which autoantibodies are directed.
Methods
:
We examined sera from patients with PBC (n = 105), primary sclerosing cholangitis (n = 70), and rheumatoid arthritis (n = 28) as well as healthy volunteers (n = 43) for reactivity against lipoic acid. The lipoic acid hapten specificity of the reactive antibodies in PBC sera was determined following incubation of aliquots of the sera with human serum albumin (HSA), lipoylated HSA (HSA-LA), PDC-E2, lipoylated PDC-E2, polyethylene glycol (PEG), lipoylated PEG, free lipoic acid, and synthetic molecular mimics of lipoic acid.
Results
:
Anti-lipoic acid specific antibodies were detected in 81% (79 of 97) of antimitochondrial antibody (AMA)-positive patients with PBC but not in controls. Two previously unreported specificities in AMA-positive sera that recognize free lipoic acid and a carrier-conjugated form of lipoic acid were also identified.
Conclusions
:
We hypothesize that conjugated form(s) of native or xenobiotic lipoic acid mimics contribute to the initiation and perpetuation of autoimmunity by at first breaking self-tolerance and participating in subsequent determinant spreading. The variability in the immunoreactive carrier/lipoate conjugates provides an experimental framework on which potential mechanisms for the breakdown of self-tolerance following exposure to xenobiotics can be investigated. The data have implications for patients taking lipoic acid as a dietary supplement.
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