Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens

cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.     

Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

     

Transfer of 4‐hydroxynonenal from parasitized to non‐parasitized erythrocytes in rosettes. Proposed role in severe malaria anemia

Severe anaemia is a life‐threatening complication of falciparum malaria associated with loss of predominantly non‐parasitized red blood cells (npRBCs). This poorly elucidated process might be influenced by (i) rosettes, i.e. npRBCs cytoadherent to haemozoin‐containing parasitized RBCs (pRBCs) and (ii) generation in pRBCs of 4‐hydroxynonenal (4‐HNE) through haemozoin‐catalysed lipid peroxidation. We explored whether close proximity in rosettes may facilitate 4‐HNE transfer to npRBCs, which is likely to enhance their phagocytosis and contribute to malaria anaemia. Fluorescence microscopy and flow cytometry data indicated 4‐HNE transfer to npRBCs in rosettes. Rosettes were formed by 64·8 ± 1·8% varO‐expressing pRBCs, and 8·7 ± 1·1% npRBCs were positive for 4‐HNE‐protein‐conjugates, while low‐rosetting parasites generated only 2·4 ± 1·1% 4‐HNE‐conjugate‐positive npRBCs. 4‐HNE transfer decreased after blocking rosetting by monoclonal antibodies. A positive linear relationship between rosette frequency and 4‐HNE‐conjugates in npRBCs was found in 40 malaria patients, a first indication for a role of rosetting in npRBCs modifications in vivo. Children with severe malaria anaemia had significantly higher percentages of 4‐HNE‐conjugate‐positive npRBCs compared to children with uncomplicated malaria. In conclusion, 4‐HNE transfer from pRBCs to npRBCs in rosettes is suggested to play a role in the phagocytic removal of large numbers of npRBCs, the hallmark of severe malaria anaemia.     

Influence of substances affecting cell sulfhydryl/disulfide status on adherence of human monocytes

An in-vitro model is described for investigating the adherence of human monocytes. Mononuclear cells (MNC) isolated from peripheral blood were incubated in plastic multiwell cell culture plates. The adherence of monocytes was quantified on the basis of their DNA content. The cell adherence proceeds rapidly; after 30 min of incubation about 85% of maximum adherent cells were attached to the plastic surface. On testing the effects of various sulfhydryl-affecting substances on monocyte adherence we found that the thiol-oxidizing compound diamide (0.1-1 mmol/l) inhibited the adherence by about 60%. Incubation with an extract of the plant feverfew, which contains materials that neutralize cellular sulfhydryl groups, also diminished monocyte adherence. The soluble thiol 2-mercaptopropionylglycine, in a concentration range of 0.5 to 10 mmol/l, had no effect on adherence in this system. The results suggest that cellular sulfhydryl groups play an important role in the adherence of monocytes.     

Red Cell Glycolysis in a Case of 3-Phosphoglycerate Kinase Deficiency

Abstract. A severe deficiency in red cell 3-phosphoglycerate kinase was observed in a 62-year-old woman with haemolytic anaemia. Compared with a normal “young” red cell population with the same degree of reticulocytosis (6–7%) the 3-phosphoglycerate kinase activity was reduced to 27%. A concomitant decrease of hexokinase and pyruvate kinase (both reduced by about 30%) was observed. The activities of glucoses-phosphate dehydrogenase, phosphofructokinase, fructose-1,6-di-phosphate aldolase, gIyceraIdehyde-3-phosphate dehydrogenase and lactate dehydrogenase were slightly increased (7 to 15%). The total glycolytic output of the deficient cells was decreased by 28% at pH 7.0, by 36% at pH 7.4 and by 34% at pH 7.6. Compared with a normal “adult” red cell population the 3-phosphoglycerate kinase activity was reduced to 42% of the control values. Hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase activities were increased by approximately 20–50%. Phosphofructokinase activity was unchanged and pyruvate kinase only slightly increased. The steady state levels of the intermediates preceding the 3-phosphoglycerate kinase step were increased 2–3 fold. The subsequent metabolites were decreased or practically unmodified. ATP, ADP, NAD+, and NADH were not affected. The reduced glutathione level was increased and the ratio of reduced to oxidized glutathione was doubled. The glycolytic output and its pH-dependency were normal. The metabolic significance of the enzyme defect was assessed by the in vitro creation of cell stressing conditions, i.e. low pH and high pyruvate levels. In both cases, the 3-phosphoglycerate kinase activity became limiting at low pH, glucose-6-phosphate accumulated at a faster rate and fructose- 1,6-diphosphate and dihydroxyacetone phosphate disappeared more slowly in the deficient cells. After pyruvate loading these cells showed: a faster, more pronounced rise in 1,3-diphosphoglycerate and a decrease in 2,3-diphosphoglycerate (slightly increased in the controls): a drop in reduced glutathione (constant in the controls): constant ATP and slightly increased 3-phosphoglycerate concentrations (both strongly increased in the controls): a slight increase in NADH (dropped to nil in the controls). Steady state glycolysis under normal conditions seemed to be affected by the enzyme deficiency. 3-phosphoglycerate kinase however, became more severely limiting a low pH or after the addition of pyruvate. In these conditions, the flow was diverted to the 2,3-diphosphoglycerate bypass, less ATP was produced and the concentration of reduced glutathione decreased. This may be assumed to have led to impairment of the ionic pump and may thus explain the increased haemolysis.     

Regulation of nitric oxide synthesis in uraemia

Nitric oxide (NO) is a cell-to-cell mediator involved in theregulation of vascular tone and in the mechanisms of host defence.Since uraemic syndrome is characterized by abnormalities inblood pressure and flow and by impairment of white cell function,we studied the regulation of nitric oxide synthase (NOS) activityby uraemic plasma. We used three different cellular types havingdifferent levels of NOS activity: tEnd. 1 murine endothelialcell line transformed by mT oncogene of polyomavirus had a highNOS activityand expressed endothelial-NOS (eNOS) and inducible-NOS(iNOS) isoforms; human endothelial cells from cord umbilicalvein (HUVEC) had low enzymatic activity and expressed only eNOS;finally, J774 murine macrophage line was characterised by iNOSinduced after treatment with cytokines. We demonstrated thatmost (79%) of end-stage uraemic plasma studied inhibited NOSactivity in tEnd.1 and in cytokine induced -J774, whereas theywere ineffective on HUVEC. Twenty percent of plasma samples(14 of 67) activated NOS activity in tEnd.1 and in J774 cells,but not in HUVEC, suggesting the presence of molecule(s) whichinfluence iNOS. The effect of plasma was not dependent on thetype of haemodialysis treatment. A great number of plasmas frompatients with moderate renal failure also inhibited NOS activityin tEnd.1, suggesting that the accumulation of molecules affectingNOS was caused by the renal failure rather than the haemodialytictreatment. However, the haemodialysis modified the effect of plasmas onNOS activity. Plasma taken after haemodialysis session showeda reduced inhibitory activity in tEnd.1 and in some cases itenhanced NOS activity. Simultaneously, molecules reducing NOSactivity accumulated in the ultrafiltrate. The plasma concentrationof N^G-N^G dimethyl-L-arginine (asymmetrical dimethylarginine,ADMA), an inhibitor of NOS, increased in end-stage uraemic patientsand was reduced by haemodialysis. However, the concentrationsreached in uraemic plasmas were lower than the ADMA ICM₅₀ ontEnd.1 NOS, indicating that this compound contributes with othermolecules to the inhibitory effect of uraemic plasma. Haemodialysisreduced also the enhanced effect exerted by some plasmas onNOS in J774. Therefore, the effect of endstage uraemic plasmaon NOS activity derive from the balance between inhibitors andactivators.     

Chemical modification of cytoskeletal proteins of human blood platelets by diamide

Incubation of human blood platelets with diamide (azo-dicarboxylic acid-bis-dimethylamide, DIA) influences the aggregation behaviour considerably. Depending on concentration and incubation time DIA induces a reversible aggregation or brings about complete inhibition. The effect is reversible and may be due to the regeneration of reduced glutathione (GSH) which will be oxidized to GSSG by DIA. DIA causes disulfide-linked polymer formation of certain cytoskeletal proteins. At least three polymer families (P_a, P_b, P_c) with different molecular weights are formed depending on dosage and incubation time of DIA. The appearance of a double band in P_a correlates with reversible aggregation, the formation of P_c is always accompanied by a complete inhibition of aggregation. A disturbance of cytoskeleton-membrane interaction by polymer formation can be assumed. GSH serves as a reductant of disulfide-linked polymers whereby a direct link between the maintenance of SH/SS status of platelets and GSH can be established.     

Glycolytic enzyme activities in bovine hard dental tissue at different ages

The activity of the following enzymes was determined by improved spectrophotometrical methods in inely pulverized young and adult bovine hard dental tissue extracts: hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, pyruvate kinase, lactate dehydrogenase. The enzymes were solubilized from the calcified matrix by crushing the dentine at the temperature of liquid nitrogen. This technique led to substantial improvement in the extraction of many enzymes. The influence of the extracting fluids of enzyme activities was also examined. All the enzymes investigated were present in bovine dentine; the activities are consistent with a fast flux through the Embden-Meyerhof-pathway. Well-defined quantitative proportions between the maximal activities of functional correlated enzymes were observed, as had previously been reported in a number of quite different organisms and tissues. Most glycolytic activities increased on ageing; hexokinase, phosphofructokinase and aldolase were constant; lactate dehydrogenase showed a dramatic fall. A similar difference in aging pattern has been displayed by these enzymes in other avascular tissues.