alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow

A variety of biologic response modifiers (BRM) can potently augment NK activity in nonlymphoid organs. By using the liver as a model organ, we have shown that this augmentation of organ-associated NK activity is coincident with a 10- to 15-fold increase in the number of large granular lymphocytes (LGL) which can be isolated. The present study was designed to investigate the mechanism by which BRM induce this increase in liver-associated LGL and the coincident increase in hepatic NK activity. Initial studies confirm that a single dose of the pyran copolymer, maleic anhydride divinyl ether (MVE-2), augmented hepatic NK activity and increased the number of liver-associated LGL from 3 x 10(4)/liver to 5 x 10(5)/liver (a 17-fold increase). Multiple injections of MVE-2 further augmented total liver-associated NK activity and LGL number (to 13 x 10(5)/liver). As expected, both the NK activity and detectable LGL were eliminated by treatment of the mice with antiasialo GM1 (asGM1) serum. Three possible mechanisms for the BRM-induced increase in liver-associated LGL have been investigated, including 1) the rapid proliferation of resident hepatic LGL, 2) the redistribution of mature LGL from peripheral sites such as the spleen, or by 3) a rapid output and subsequent hepatic localization of LGL or their precursors recently derived from the bone marrow (BM). Our results demonstrated that the contribution of in situ proliferation to the BRM-induced increase in liver-LGL was relatively small, since the number of cells expressing NK-associated markers (i.e., asGM1, Thy-1.2, and NK1.1) and in G2/M phase (as assessed by propidium iodide uptake) was only 4 to 8%. Further experiments demonstrated that splenectomy before the administration of MVE-2 did not inhibit the augmentation of liver-associated NK activity. This result argued against a recruitment of mature LGL from the spleen. In contrast, selective depletion of the BM following administration of 89Sr decreased the ability of MVE-2 to augment liver-associated NK activity by greater than 80%. This procedure also significantly decreased the ability of Propionibacterium acnes (85%) and multiple doses of IL-2 (49%) to augment liver-associated NK activity. These results demonstrate that the rapid augmentation of liver-associated NK activity by BRM is largely due to localization and accumulation in the liver of LGL recently derived from the BM.     

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft?Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, were port an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at ～4 °C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85%glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchangedmechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.     

Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids

Burkholderia sp. accumulates polyhydroxyalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbüchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389–7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp. However, when total genomic DNA from Burkholderia sp. was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase gene, based on high sequence homology, genes encoding a β-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R. eutropha poly-β-hydroxybutyrate biosynthesis operon. Received: 3 September 1999 / Received revision: 29 October 1999 / Accepted: 5 November 1999     

Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli

An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E. coli led to the purification of the enzyme to homogeneity. The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction. However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E. coli strain DH5α. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate). Received: 16 June 1999 / Received revision: 19 August 1999 / Accepted: 19 August 1999     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Mesenchymal stem cells in autoimmune diseases: hype or hope?

Intervention with mesenchymal stem cells (MSCs) represents a promising therapeutic tool in treatment-refractory autoimmune diseases. A new report by Schurgers and colleagues in a previous issue of Arthritis Research & Therapy sheds novel mechanistic insight into the pathways employed by MSCs to suppress T-cell proliferation in vitro, but, at the same time, indicates that MSCs do not influence T-cell reactivity and the disease course in an in vivo arthritis model. Such discrepancies between the in vitro and in vivo effects of potent cellular immune modulators should spark further research and should be interpreted as a sign of caution for the in vitro design of MSC-derived interventions in the setting of human autoimmune diseases.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.