Germinal centres and the B-cell system. V

Summary Thoracic duct lymphocytes (TDL) were studied with respect to their capacity to give rise to germinal centres (GC) and to form primary antibody in an adoptive transfer system of the rat. Challenge with sheep erythrocytes (SRBC) 24h after lethal irradiation (900 rads) and syngeneic TDL reconstitution (10⁸) lead to conspicuous GC activity already 7 days after transfer. In contrast, using syngeneic bone marrow (BM) in the adoptive transfer system, no GC formation was observed over the period studied (14 days after reconstitution). Reconstitution experiments using in vivo-separated T-TDL (1–5 % s-Ig⁺) and B-TDL (>90% s-Ig⁺) subpopulations, either separately or in combination, indicated that GC originate from B-TDL but require T-TDL for induction.Abbreviations BM Bone Marrow - TDL Thoracic Duct Lymphocytes - GC Germinal Centre - SRBC Sheep Red Blood Cells - GCDC Germinal Centre-Derived Cells - GCPC Germinal Centre-Precursor Cells - DAB Mineral Salt Solution Dulbecco A + B - FCS Fetal Calf Serum - PALS Peri-Arteriolar Lymphocyte Sheath - AFCP Antibody-Forming Cell Precursors     

Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells.

The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre- and post-embedding EM immunocytochemistry with a variety of anti-tubulin antibodies. The 6-11B-1 monoclonal antibody, specific for the post-translational acetylation of Lys 40 of alpha-tubulin, and a polyclonal antibody raised against Paramecium axonemal tubulin (anti-PA tubulin antibody) both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti-PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti-tubulin antibody and the anti-PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti-PA tubulin one. This provided a strong indication that the anti-PA tubulin antibody is directed against a post-translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol-treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti-PA tubulin antibody however, suggesting that in Metazoa the post-translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post-translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes.     

Efficacy and safety of long-term continuous growth hormone treatment of children born small for gestational age

Twelve years of growth hormone (GH) therapy of short children born small for gestational age (SGA) have demonstrated that GH is an effective and well-tolerated therapy. Most children will reach a normal adult height (AH). AH of 55 SGA adolescents was comparable for those treated with a GH dose of 1 or 2 mg/m2 (approximately 0.033 or 0.066 mg/kg) per day, mean (SD) AH SDS being -1.2 (0.7) and -0.8 (0.7), respectively. GH therapy had no influence on the age at onset, the progression of puberty, duration of puberty and pubertal height gain. GH therapy induced higher fasting and glucose-stimulated insulin levels after 1 and 6 years, but 6 months after GH stop, all levels returned to normal. At baseline mean systolic blood pressure was significantly increased, but both systolic and diastolic blood pressure decreased significantly during 6 years of GH and remained so after GH stop. GH therapy demonstrated a beneficial effect on serum lipid profiles, body composition, bone mineral density and head growth. Treatment with 2 mg GH/m2 per day induced mean serum IGF-I levels of +2 SDS, whereas IGF-I levels remained within the normal range with 1 mg GH/m2 per day. In conclusion, long-term GH therapy of short SGA children with 1 mg/m2 per day appears to be effective and safe. Since the future consequences of high serum IGF-I levels during long-term GH therapy with 2 mg/m2 per day are as yet unknown, it seems safer to treat short prepubertal SGA children with a GH dose of 1 mg/m2 per day when children are to be treated continuously for many years.     

Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.     

Arginine deiminases: Therapeutic tools in the etiology and pathogenesis of Alzheimer's disease

There is, at present, no definitive pre-mortem diagnostic tool for Alzheimer's disease, (AD) which relates to a poor understanding of its etiology. Brains of AD patients contain large amounts of the toxic plaque-forming β-amyloid_1–42 fragment in addition to elevated concentrations of the amino acid l-arginine. This work proposes that lowering levels of arginine in the astrocytes surrounding amyloid plaques may serve as a therapeutic tool in this neurodegenerative disorder. Arginine deiminase (ADI), from Pseudomonas aeruginosa, and peptidylarginine deiminase [PAD II], from bovine brain, are inhibited by amyloid peptides that contain arginine (amyloid_1–42) and those that have no arginine (amyloid_{12–28/22–35}). Enhanced activity of PAD II is noted with free l-arginine.     

Photobiomodulation reduces oxidative stress in diabetic wounded fibroblast cells by inhibiting the FOXO1 signaling pathway

This study aimed to elucidate the underlying molecular mechanism of photobiomodulation (PBM) in attenuating oxidative stress in diabetic wounded fibroblast cells. Cell models were exposed to PBM at a wavelength of 660 nm (fluence of 5 J/cm², and power density of 11.2 mW/cm²) or 830 nm (fluence of 5 J/cm², and power density of 10.3 mW/cm²). Non-irradiated cell models were used as controls. Cellular migration was determined at regular time intervals (0, 12, 24 and 48 h) using inverted light microscopy. Cell viability was determined by the Trypan blue exclusion assay. The levels of enzymic antioxidants superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HMOX1) were determined by the enzyme linked immunosorbent assay (ELISA). The alteration in the levels of AKT and FOXO1 was determined by immunofluorescence and western blotting. Upon PBM treatment, elevated oxidative stress was reversed in diabetic and diabetic wounded fibroblast cells. The reduced oxidative stress was represented by decreased FOXO1 levels and increased levels of SOD, CAT and HMOX1. This might be due to the activation of the AKT signaling pathway. This study concluded that treatment with PBM progressed diabetic wound healing by attenuating oxidative stress through inhibition of the FOXO1 signaling pathway.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00588-x) contains supplementary material, which is available to authorized users.     

Aubergine is a component of a nanos mRNA localization complex

Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far. Through a sensitized genetic screen, we have now identified the Argonaute family member Aubergine (Aub) as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. Our results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex.