Purification and properties of carnitine acetyltransferase from human liver

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.     

Allosteric regulation of Bacillus subtilis NAD kinase by quinolinic acid

NADP is essential for biosynthetic pathways, energy, and signal transduction. In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase. We expressed, purified, and characterized Bacillus subtilis NAD kinase. This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors. NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis. We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme. Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Volume-regulated Cl- channels in human pleural mesothelioma cells

A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.     

Structure of human NMN adenylyltransferase. A key nuclear enzyme for NAD homeostasis.

Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta-phosphodiesterases superfamily, catalyzes a universal step (NMN + ATP = NAD + PP(i)) in NAD biosynthesis. Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy. By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-A resolution, revealing a hexamer that is composed of alpha/beta-topology subunits. The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change. We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs. Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.     

Evaluation of immune responses raised against Tc13 antigens of Trypanosoma cruzi in the outcome of murine experimental infection

We have previously reported that genetic immunization with Tc13Tul antigen of Trypanosoma cruzi, the aetiological agent of Chagas' disease, triggers harmful effects and non-protective immune responses. In order to confirm the role of Tc13 antigens during T. cruzi infection, herein we studied the humoral and cellular immune responses to the Tc13Tul molecule and its EPKSA C-terminal portion in BALB/c T. cruzi-infected mice or mice immunized with recombinant Tc13Tul. Analysis of the antibody response showed that B-cell epitopes that stimulate a sustained IgM production along the infection and high levels of IgG in the acute phase are mainly located at the Tc13 N- and C-terminal domains, respectively. DTH assays showed that T-cell epitopes are mainly at the Tc13 N-terminal segment and that they do not elicit an efficient memory response. Recombinant Tc13Tul did not induce IFN-gamma secretion in either infected or immunized mice. However, a putative CD8+Tc13Tul-derived peptide was found to elicit IFN-gamma production in chronically infected animals. Immunization with recombinant Tc13Tul did not induce pathology in tissues and neither did it protect against the infection. Our results show that in the outcome of T. cruzi infection the Tc13 family protein mainly triggers non-protective immune responses.     

Changes in HDL-cholesterol, subfractions HDL2 and HDL3 and apo-A, apo-A1 and apo-B lipoproteins in patients with chronic renal failure subjected to maintenance hemodialysis

Hyperlipoproteinemia was found in 200 patients affected by chronic renal failure on maintenance hemodialysis, due to increase in Total Cholesterol, VLDL-TG, LDL, TG, LDL and VLDL. In 30 of these patients, the analysis was extended to the dosing of decreased Total Serum HDL-Cholesterol, HDL2, HDL3, Apoprotein A and Apoprotein A1 values, and of increased Apoprotein B values. These findings further contribute to proving accelerated atherogenesis in the patients examined.     

Membrane thickness changes ion-selectivity of channel-proteins.

The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become.     

In silico study of structural determinants modulating the redox potential of Rigidoporus lignosus and other fungal laccases

Laccases are multicopper oxidases in which substrate oxidation takes place at the type-1 (T1) copper site. The redox potential (E (0)) significantly varies amongst members of the family and is a key parameter for substrate specificity. Despite sharing highly conserved features at the T1 copper site, laccases span a large range of E (0), suggesting that the influence of the metal secondary coordination sphere is important. In silico analysis of structural determinants modulating the E (0) of Rigidoporus lignosus and other fungal laccases indicated that different factors can be considered. First, the length of the T1 copper coordinating histidine bond is observed to be longer in high E (0) laccases than in low E (0) ones. The hydrophobic environment around the T1 copper site appeared as another important structural determinant in modulating the E (0), with a stronger hydrophobic environment correlating with higher E (0). The analysis of hydrogen bonding network (HBN) around the T1-binding pocket revealed that the amino acids building up the metal binding site strongly interact with neighbouring residues and contribute to the stabilization of the protein folds. Changes in these HBNs that modified the Cu1 preferred coordination geometry lead to an increase of E (0). The presence of axial ligands modulates the E (0) of T1 to different extent. Stacking interactions between aromatic residues located in the second coordination shell and the metal ion coordination histidine imidazole ring have also been identified as a factor that modulates the E (0). The electrostatic interactions between the T1 copper site and backbone carbonyl oxygen indicate that Cu1-CO=NH distance is longer in the high E (0) laccases. In short, the in silico study reported herein identifies several structural factors that may influence the E (0) of the examined laccases. Some of these are dependent on the nature of the coordination ligands at the T1 site, but others can be ascribed to the hydrophobic effects, HBNs, axial ligations, stacking and electrostatic interactions, not necessary directly interacting with the copper metal.     

BMI and Severity of Clinical and Radiographic Signs of Hip Osteoarthritis

Obesity might be involved in the pathogenesis of osteoarthritis (OA) not only via increased mechanical loading, but also via an inflammatory component possibly causing increased pain and functional disability. The study aim was to examine the relationship between BMI and clinical symptoms as well as radiographic severity of OA in patients scheduled for primary total hip arthroplasty (THA). We conducted a cross‐sectional study of 855 patients scheduled for a first THA for primary OA at a single centre between November 2001 and December 2006. The primary outcome was clinical and radiographic severity of OA, which was evaluated in four BMI categories (18.5–24.9, 25–29.9, 30–34.9 and ≥35 kg/m²). We used the Harris Hip Score (HHS) and the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) to assess pain and function. The severity of radiographic hip joint damage was evaluated using the Kellgren–Lawrence classification. Multivariate analyses were performed to adjust for potential confounders. In patients scheduled for THA, increasing BMI was associated with significantly higher levels of pain and functional disability on both HHS (P for trend <0.001) and WOMAC (P for trend <0.001). However, the degree of radiographic joint damage remained similar across BMI categories. These findings emphasize the need to further investigate the potential pathogenic role of obesity and low‐grade inflammation in OA and underscore the importance of obesity prevention to avoid early prosthetic replacement of the hip.