Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).     

Changes in cyclic AMP binding and protein kinase activities during growth and differentiation of Blastocladiella emersonii.

Multiple protein kinases in the water mould Blastocladiella emersonii are described. A cyclic AMP-independent protein kinase which prefentially phosphorylates casein remains unchanged during vegetative growth of the cells and in the two phases of differentiation: germination and sporulation. In contrast, cyclic AMP-dependent protein kinase activity and cyclic AMP binding components are induced during the sporulation.     

H-2 antigens,on mast cell membrane,as target antigens for anaphylactic degranulation

When single mast cells were isolated by micromanipulation, specific H-2 antigen-bearing mast cells were degranulated upon incubation with alloimmune sera (DAAD). When specific alloantigens were presented by lymphoid cells only, no degranulation occurred. Only antigen-bearing mast cells were degranulated, irrespectively of the presence of antigen-bearing lymphoid cells. Therefore, in DAAD, anaphylactic alloantibodies can and must recognize specific H-2 antigens on the mast cell membrane and simultaneously deliver the degranulation signal, through an Fc-Fc receptor interaction on the surface of the same mast cell.     

Baclofen and γ-hydroxybutyrate: similar effects on cerebral dopamine neurones

Baclofen (20 mg/kg) caused an increase in the content of homovanillic acid (HVA) and dopamine (DA) in rat brain 2–3 h after drug injection without appreciable changes in the level of other monoamines and their main metabolites. Six and eight hours after baclofen, the content of HVA but not that of DA was reduced. Moreover, baclofen initially (20 min after injection) reduced, but later (105 min post drug) enhanced the accumulation of HVA induced by probenecid. The shortlasting (20 min) initial reduction of HVA elevation in probenecid-pretreated animals as well as the longlasting (6–8 h) decrease of HVA levels in rats injected with baclofen alone are interpreted to be due to a decreased release and metabolism of DA, probably as a consequence of the blockade of impulse flow in mesolimbic and nigro-striatal DA neurones. The increase in HVA and DA seen during the first few hours is thought to result from enhanced DA synthesis similar to that known for γ-hydroxybutyrate (GHB). This initial rise in HVA due to synthesis stimulation probably masked a reduction of HVA to be expected immediately after baclofen injection. The similarity between baclofen and GHB is stressed by the finding that baclofen counteracted the increase of HVA occuring after chlorpromazine and D-amphetamine but not that induced by the benzoquinolizine derivative, Ro 4-1284.     

Chemically defined media for growing anaerobic bacteria of the genus Veillonella

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirement for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition.No specific requirements for single amino acids were observed. A combination of l-cysteine, dl-aspartic acid, l-glutamic acid, l-serine and l-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source.Decarboxylation of l-ornithine, l-lysine and l-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, l-lysine, l-ornithine and l-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate l-ornithine while forming putrescine.     

Kinetics of reaction of (nitrotyrosyl)cytochrome c with ligands.

The reaction of [nitrotyrosyl]cytochrome c with ligands was studied by stopped-flow techniques. At pH 7.0 the reaction with imidazole shows two distinct phases, one fast phase being concentration-dependent and a slow phase being concentration-independent. The results are consistent with the existence of two forms of [nitrotyrosyl]cytochrome c in solutions [Schejter et al. (1970) Biochemistry 9, 5118-5122]; form I, the smaller fraction, seems to be responsible for the slow first-order process.     

Diterpenoids from Micrandropsis scleroxylon

The structure of micrandrol-C from Micrandropsis scleroxylon (Euphorbiaceae) is revised to 2,6-dihydroxy-7-methyl-1-methylthiophenanthrene. This and other micrandrols are probably diterpenes in view of their co-occurrence with micrandrol-D, the hemiketal of 1,2,3,4,9,10-hexahydro-6-hydroxy-4a-hydroxymethyl-1,1,7-trimethy-2-oxophenanthrene.     

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.Subject terms: Stem cells, Diseases