ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium . This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum . Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)-fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA-fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum . According to our molecular analysis, P. caudatum is not a species complex, but just one single species.     

Potentiation of the proliferative response of human B lymphocytes to low molecular weight B cell growth factor (LMW-BCGF) by fibroblast growth factors (FGFs)

Both acidic and basic fibroblast growth factor (FGF), although devoid alone of growth-promoting ability on resting or activated human lymphoid B cells, were found to markedly increase the proliferative response of anti-mu-chain or SAC preactivated B cell blasts to the low molecular weight B cell growth factor (LMW-BCGF) and to enhance the costimulatory response of resting B cells to anti-mu-chain and LMW-BCGF. This potentiating effect was also observed for a LMW-BCGF-dependent B cell tumor derived from a lymphocytic nodular lymphoma. Other growth factors acting on fibroblasts, such as epidermal growth factor, alpha-thrombin, platelet-derived growth factor, and insulin-like growth factor-I did not display such enhancing effect on LMW-BCGF-driven proliferation. Activated, but not resting B cells were found to bear receptor sites for FGFs and from kinetics experiments, it is suggested that LMW-BCGF induces competence expression for FGFs in those cells. Moreover, the LMW-BCGF-elicited generation of inositoltrisphosphate resulting from polyphosphoinositides hydrolysis was increased in the presence of FGF.     

Co-existence of immunoreactivity to anti-dopamine,anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis

Summary Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with antivasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.     

Phosphorylation by liver glucokinase of D-glucose anomers at anomeric equilibrium

The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate.     

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16^INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.     

Toxicity of chemically generated nitric oxide towards pancreatic islet cells can be prevented by nicotinamide.

Previous studies have indicated that nitric oxide is involved in the lysis of pancreatic islet cells by inflammatory macrophages. Here we show that the incubation of islet cells with chemical NO-donors leads to cell lysis in a concentration and time dependent way. Islet cell death could be prevented by nicotinamide and 3-aminobenzamide, which are known to inhibit ADP-ribosylation, while several scavengers of oxygen radicals, N-acetylcysteine, dihydrolipoic acid, dimethylthiourea and citiolone, provided no protection.     

Ganglioside therapy of type I diabetes: enhancement of hyperglycemia in the low dose streptozotocin model.

The therapeutic potential of bovine brain gangliosides on the development of insulin deficient diabetes was analysed. Daily ganglioside administration (50 mg/kg body weight) caused a more pronounced rise of blood glucose levels (p less than 0.05) in low dose streptozotocin treated mice, a model of human type I diabetes. Hyperglycemia induced by the injection of a single high dose of streptozotocin was slightly increased by ganglioside administration (not significant). The previously reported protective effect of bovine brain gangliosides on the development of diabetes in NOD mice was thus not found in a second mouse model.