The human placental transferrin receptor: Reconstitution into liposomes and electron microscopy

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptortransferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon, detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w) Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed ographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5×10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2–3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - PAGE polyacrylaminde gel electrophoresis - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - WGA wheat germ agglutinin     

Effect of chloramphenicol,antimycin A and hydroxamate on the morphogenetic development of the dimorphic ascomyceteEndomycopsis capsularis

Mitochondrial protein synthesis, primary (antimycin-sensitive) respiration and secondary (antimycin-insensitive, salicyl-hydroxamate-sensitive) respiration, have been characterized in the dimorphic yeastEndomycopsis capsularis. The inhibition by chloramphenicol (CAP) of the morphogenetic development from the yeast-like form to the mycelial structure in this yeast could represent the intervention in the morphogenetic process of mitochondrial protein synthesis, since chloramphenicol blocks in vivo and in vitro mitochondrial protein synthesis. In fact, other functions such as primary and secondary respiration, do not seem to play a role in the morphogenetic development since their inhibition by antimycin A (AA) or by salicyl-hydroxamic acid (SHAM) does not affect the process. In addition, mitochondrial protein synthesis has been shown to be uninhibited by the two respiratory inhibitors.     

Induction of type C virions from normal rat kidney cells by 2-deoxy-D-glucose.

The sugar 2-deoxy-D-glucose (2-DG) induced the release of type C virions from an established line of normal rat kidney (NRK) cells. Within 20 h after the addition of 5 mg of 2-DG per ml to exponentially growing NRK clutures, more than 80% of the cells expressed the mammalian type C virus interspecies-specific antigen (p30) as determined by indirect cytoplasmic immunofluorescence. Maximal virion release occurred 1 to 2 days after 2-DG was added for 24 h to the growth medium although a low level of virion production was detected as early as 2.5 h after 2-DG treatment. Studies with inhibitors of RNA synthesis indicated a requirement for de novo RNA synthesis after the addition of 2-DG. Sensitivity of NRK cells to type C virion induction was limited to a relatively short period of in vitro growth and preceded spontaneous virion release by 8 to 10 subculture generations. A model is presented for the sequential derepression of latent type C virus information in serially propagated NRK cells.     

The Treatment with Interleukin 17 Inhibitors and Immune-Mediated Inflammatory Diseases

IL-17 inhibitors (IL-17i) are medicines used to treat dermatological and rheumatic diseases They belong to a class of medicines called biological disease-modifying anti-rheumatic drugs (bDMARDs). This class of drugs has had a major impact on the therapy of autoimmune diseases, being much safer and more effective than treatment with small molecules. At the same time, they have highly beneficial effects on skin and joint changes, and their efficacy has been extensively monitored and demonstrated in numerous clinical trials. More and more such drugs are still being discovered today to ensure the best possible treatment of these patients, but more frequently and relatively constantly three agents are used. Two of them (Secukinumab and Ixekizumab) inhibit IL-17A directly, and the third, Brodamulab, inhibits the IL-17A receptor. Although they are extremely effective in the treatment of these diseases, sometimes their administration has been associated with paradoxical effects, i.e., there is an exacerbation of the inflammatory process. Tough, clinical trials of IL-17i have described cases of exacerbation or even onset of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, after administration of these drugs in patients previously diagnosed with psoriasis (PS), psoriatic arthritis (PsA), or ankylosing spondylitis (AS). The pathophysiological mechanism of action is not well understood at present. One explanation would be that this hyperreactive inflammatory process would be triggered by Interferon 1 derived from dendritic plasma cells. Even though there are many reports in the recent literature about the role of IL17i in the onset of IBD, conclusions of studies do not converge. Some of them show an increased incidence of IBD in patients treated with IL17i, while some others affirm their safety of them. In the near future we will surely have more data emerging from ongoing meta-analyses regarding safety of use IL17i in patients who are at risk of developing IBD. Clinical and paraclinical evaluation (inflammatory intestinal markers) are carefully advised before recommending treatment with IL-17i and after initiation of treatment, and prospective surveillance by clinical and biomarkers of patients treated with IL-17i is absolutely essential to capture the onset of IBD.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Biofilm induced tolerance towards antimicrobial peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.     

Effect of Acute and Chronic Administration of Methylphenidate on Mitochondrial Respiratory Chain in the Brain of Young Rats

Methylphenidate is commonly used for the treatment of attention deficit/hyperactivity disorder. There are still few works regarding the effects of methylphenidate on brain energy metabolism. Thus, in the present study we evaluated the effect of chronic administration of methylphenidate on the activities of mitochondrial respiratory chain complexes I and III in the brain of young rats. The effect of acute administration of methylphenidate on mitochondrial respiratory chain complexes I, II, III and IV in the brain of young rats was also investigated. For acute administration, a single injection of methylphenidate was given to rats on postnatal day 25. For chronic administration, methylphenidate injections were given starting at postnatal day 25 once daily for 28 days. Our results showed that complexes I and III were not affected by chronic administration of methylphenidate. Moreover, the acute administration of methylphenidate decreased complex I activity in cerebellum and prefrontal cortex, whereas complexes II, III and IV were not altered.