The effect of S-adenosyl-l-methionine on ischemia-induced disturbances of brain phospholipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries with reported procedures. Changes in the energy status of brain demonstrated that carotid ligation was effective. At different time intervals from ligation, groups of gerbils were given either saline of S-Adenosyl-L-methionine (SAMe) by the intraventricular (i.v.) route (1.6 mg/Kg body wt. twice, at each 10 min interval), or by the intraperitoneal (i.p.) administration (200 mg/Kg body wt.) or subcutaneously (s.c.) with 40 mg/Kg body wt, daily, for two weeks. Control animals, with and without SAMe, together with the ischemic groups, were decapitated directly into liquid nitrogen, 10 min after ligation. Brain neutral and polar lipid, together with free fatty acids, which were all labeled in vivo by the intraventricular injection of [1-14C]arachidonic acid 2 hr prior to ligation, were extracted, purified and separated by conventional procedures. SAMe when injected i.v. or i.p. noticeably corrected the changes in polar lipid by reversing the decrease of brain phosphatidylcholine and choline plasmalogen, as well as of their labeling, which was due to ischemia. Concurrently with this action, SAMe treatment (i.v. and i.p.) also provided to some extent to re-establish the normal level of labeling of ethanolamine lipids. When SAMe was given s.c., no effect was present. SAMe had no effect on the increase of free fatty acid and diglyceride due to ischemia. The prevention by SAMe of the changes of choline lipids suggests that a stimulation of the methyltransferase reaction may occur in the ischemic brain, due to increased substrate (SAMe) availability. This effect may be important for cell survival, since membrane phospholipid derangements alter the properties of the membrane.     

Generation of reactive oxygen species by beta amyloid fibrils and oligomers involves different intra/extracellular pathways

A neuropathological characteristic of Alzheimer’s disease is the extracellular accumulation of amyloid beta peptide (Aβ) in neuritic plaques. Recent evidences suggested that soluble Aβ oligomers are the predominant neurotoxic species for neurons. Thus, considerable attention has been paid to discriminate the cytotoxic pathways of Aβ pre-fibrillar aggregates and mature fibrils. We showed that the mechanisms by which Aβ oligomers and fibrils generated reactive oxygen species differ in terms of site of production and kinetics, suggesting the involvement of different intra/extracellular pathways.     

Increased expression of Interleukin-18 receptor in blood cells of subjects with Mild Cognitive Impairment and Alzheimer’s disease

Inflammation has been proposed as a leading force in neurodegeneration and Interleukin (IL)-18 is a pro-inflammatory cytokine which is suggested to be implicated in Alzheimer’s disease (AD). However, the meaning of the IL-18 participation in this disease is still unclear.Since IL-18 activity is mediated by its heterodimeric receptor complex IL-18Rα/β, we evaluated the presence of both IL-18R chains on peripheral blood cells of AD patients, as well as in individuals with Mild Cognitive Impairment (MCI), at increased risk to develop AD. More specifically, we compared the levels of CD14⁺ monocytes and CD3⁺ T-lymphocytes bearing IL-18Rα and β chains in the two groups of patients with those in healthy control subjects, both before and after in vitro cell treatment with lipopolysaccharide (LPS).While no differences in the levels of monocytes and T-lymphocytes bearing IL-18Rα chain were found among the three groups, either in untreated and LPS-treated conditions, the IL-18Rβ chain expression appeared differently regulated in MCI and AD patients, as compared to controls. In particular, the amount of IL-18Rβ-bearing monocytes was similar among the three groups at unstimulated conditions, while after LPS treatment it was increased in MCI vs. controls. A significant increase of IL-18Rβ-bearing T-lymphocytes was also observed in MCI and AD vs. controls, both in untreated and LPS-stimulated conditions.Our findings indicate that the expression of IL-18R complex on blood cells is perturbed in AD and even more markedly in its preclinical state of MCI, confirming that an increased peripheral activity of IL-18 may be involved in the early phase of AD pathophysiology.     

Identification of a novel Baeyer‐Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA

This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long‐chain alkanes as the sole energy source expresses almA gene coding for a Baeyer‐Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer‐Villiger reactions as well as oxidation of the prodrug ethionamide.     

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the α2,6-sialyltransferase (α2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both α2,6- and α2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing α2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the α2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-γ and the tissue inhibitor of metalloproteinases-1. Interferon-γ purified from the universal host carried 40.4% α2,6- and 59.6% α2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-γ produced by normal CHO cells.     

Molecular analysis of the cystinuria disease gene: identification of four new mutations,one large deletion,and one polymorphism

A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria choromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26). Received: 15 March 1996 / Revised: 17 May 1996