Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells

Summary The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min.1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with K_i values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a K_i = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 µM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 µM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.Abbreviations ODC Ornithine Decarboxylase - TLC Thin Layer Chromatography - DNEM Dulbecco's Modified Essential Medium - PBS Phosphate Buffered saline - PEG Polyethyleneglycol     

l-carnitine and its propionate: improvement of endothelial function in SHR through superoxide dismutase-dependent mechanisms

To clarify the mechanism underlying the antioxidant properties of l-carnitine (LC) and propionyl-l-carnitine (PLC) on spontaneously hypertensive (SHR) and normotensive WKY, animals were treated with either PLC or LC (200 mg kg(- 1)). Aorta was dissected and contraction to (R)-( - )-phenylephrine (Phe) and relaxation to carbachol (CCh) were assessed in the presence or not of the NO synthase (NOS) inhibitor, l-NAME. [image omitted] production was evaluated by lucigenin-enhanced chemiluminescence and its participation on relaxation was observed after incubation with superoxide dismutase (SOD) plus catalase. Protein expressions of eNOS, Cu/Zn-SOD and Mn-SOD were studied by western blot. Both LC and PLC treatments improved endothelial function of SHR through increasing NO participation and decreasing [image omitted] probably involving higher Cu/Zn-SOD expression. PLC treatment augmented eNOS expression in SHR. Surprisingly, LC increased [image omitted] produced by aorta from WKY and thus diminished NO and damaged endothelial function. Conversely, PLC did not affect CCh-induced relaxation in WKY. These results demonstrate that LC and PLC prevent endothelial dysfunction in SHR through an antioxidant effect.     

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD⁺ dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.     

The Obestatin/GPR39 System Is Up-regulated by Muscle Injury and Functions as an Autocrine Regenerative System

The maintenance and repair of skeletal muscle are attributable to an elaborate interaction between extrinsic and intrinsic regulatory signals that regulate the myogenic process. In the present work, we showed that obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor are expressed in rat skeletal muscle and are up-regulated upon experimental injury. To define their roles in muscle regeneration, L6E9 cells were used to perform in vitro assays. For the in vivo assays, skeletal muscle tissue was obtained from male rats and maintained under continuous subcutaneous infusion of obestatin. In differentiating L6E9 cells, preproghrelin expression and correspondingly obestatin increased during myogenesis being sustained throughout terminal differentiation. Autocrine action was demonstrated by neutralization of the endogenous obestatin secreted by differentiating L6E9 cells using a specific anti-obestatin antibody. Knockdown experiments by preproghrelin siRNA confirmed the contribution of obestatin to the myogenic program. Furthermore, GPR39 siRNA reduced obestatin action and myogenic differentiation. Exogenous obestatin stimulation was also shown to regulate myoblast migration and proliferation. Furthermore, the addition of obestatin to the differentiation medium increased myogenic differentiation of L6E9 cells. The relevance of the actions of obestatin was confirmed in vivo by the up-regulation of Pax-7, MyoD, Myf5, Myf6, myogenin, and myosin heavy chain (MHC) in obestatin-infused rats when compared with saline-infused rats. These data elucidate a novel mechanism whereby the obestatin/GPR39 system is coordinately regulated as part of the myogenic program and operates as an autocrine signal regulating skeletal myogenesis.     

The NMR Structure of Human Obestatin in Membrane-Like Environments: Insights into the Structure-Bioactivity Relationship of Obestatin

The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6–23)-obestatin (3), (11–23)-obestatin (4), and (16–23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels.     

Improved bioavailability of inhibitors of Trypanosoma cruzi trans-sialidase: PEGylation of lactose analogs with multiarm polyethyleneglycol

The trans-sialidase of Trypanosoma cruzi (TcTS) catalyzes the transfer of sialic acid from host glycoconjugates to terminal β-galactopyranosides in the mucins of the parasite. During infection, the enzyme is actively shed by the parasite to the bloodstream inducing hematological alterations. Lactitol prevents cell apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. Linear polyethyleneglycol (PEG) conjugates of lactose analogs were prepared but their clearance from blood was still quite fast. With the aim of improving their circulating half-lives in vivo, we now synthesized covalent conjugates of eight-arm PEG. The star-shape of these conjugates allows an increase in the molecular weight together with the loading of the active sugar. Two approaches were used for PEGylation of disaccharide derivatives containing β-d-Galp as the non-reducing unit. (1) Amide formation between benzyl β-d-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-d-glucopyranoside and a succinimide-activated PEG. (2) Conjugation of lactobionolactone with amino end-functionalized PEG. Two 8-arm PEG derivatives (20 and 40?kDa) were used for each sugar. Substitution of all arms was proved by (1)H nuclear magnetic resonance (NMR) spectroscopy. The bioavailability of the conjugates in mice plasma was considerably improved with respect to the 5?kDa linear PEG conjugates retaining their inhibitory properties.     

Computational RNA secondary structure design: empirical complexity and improved methods

Background  

We investigate the empirical complexity of the RNA secondary structure design problem, that is, the scaling of the typical difficulty of the design task for various classes of RNA structures as the size of the target structure is increased. The purpose of this work is to understand better the factors that make RNA structures hard to design for existing, high-performance algorithms. Such understanding provides the basis for improving the performance of one of the best algorithms for this problem, RNA-SSD, and for characterising its limitations.     

Conformational Effects of a Common Codon 399 Polymorphism on the BRCT1 Domain of the XRCC1 Protein

The X-ray cross-complementing-1 (XRCC1) protein functions as a scaffold that coordinates the activity of the cellular machinery involved in base excision repair (BER) of DNA damage. The BRCT1 domain of XRCC1 is responsible for interacting with several of the key components of the BER machinery, and it is also the site of a common genetic polymorphism in XRCC1 at amino acid residue 399 (Arg → Gln). Experimental and epidemiologic evidence suggest that this polymorphism may alter BER capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XRCC1 induced by the polymorphism. Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the BRCT1 domain of XRCC1, and differences in structure produced by the polymorphic substitution were determined. The results indicate that, although the general configuration of both proteins is similar and there is little actual deviation at the site of the polymorphism itself, the substitution produces significant conformational changes at several other sites in the BRCT1 domain, including the loss of secondary structural features such as α helices that may be critical for protein–protein interactions. These results provide support for the hypothesis that this polymorphism in XRCC1 could affect DNA repair capability by altering the structure of the BRCT1 domain and thus the ability of XRCC1 to coordinate BER.