Si RNA inhibition of GRP58 associated with decrease in mitomycin C-induced DNA cross-linking and cytotoxicity

The anti-cancer drug mitomycin C is metabolically activated to bind and cross-link DNA. The cross-linking contributes significantly to the cytotoxicity. The complex chemical structure of mitomycin C allows its metabolism by several known (cytosolic NAD(P)H:quinone oxidoreductase and microsomal NADPH:cytochrome P450 reductase) and unknown enzymes. The identification of new enzymes/proteins that metabolize mitomycin C and like drugs is an area of significant research interest since these studies have direct implications in drug development and clinical usage. In the present studies, we have investigated a role of cytosolic glucose regulatory protein GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. The control and GRP58 siRNA were transfected in human colon carcinoma HCT116 cells in culture. The transfection of GRP58 siRNA but not control siRNA significantly inhibited GRP58 in human colon carcinoma HCT116 cells. The inhibition of GRP58 led to decrease in mitomycin C-induced DNA cross-linking and cytotoxicity. These results establish a role of GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. Site-directed mutagenesis of cysteines to serines in thioredoxin domains of GRP58 and cross-linking assays revealed that both N- and C-terminal thioredoxin domains are required for GRP58-mediated mitomycin C-induced DNA cross-linking. These results suggest that GRP58 might be an important target enzyme for further studies on mitomycin C and similar drug therapy.     

A novel cell-surface protein CSP82 on bone marrow stem cells and a cytosolic phosphoprotein DP58 (ankyrinRD 34B) are involved in promyeloid progenitor induction

The molecular events associated with the development of common myeloid progenitor (CMP) remain largely unknown. This study reports that a novel glycosylphosphatidylinositol (GPI)-anchored lactoferrin CSP82 on uninitiated mouse bone marrow cells (BMC) may be involved in inducing pro-DC from CMP.By peptide mass fingerprinting, CSP82 has been identified as the mouse lactoferrin precursor, but unlike the latter, it occurs as a GPI-linked cell-surface protein. The GPI-linkage was demonstrated on BMC-derived immunoprecipitates and by other techniques. Furthermore, BMC and hematopoietic stem BM cells following incubation with either CSP82 peptide antibody or purified Reagent A yielded CMP-like progenitors (BM4 cells). These progenitors expressed a previously reported cytosolic phosphoprotein DP58 (AnkRD 34B protein). Continued cultivation of BMC in media containing only anti-CSP82 antibody led to DC-like cells, that bore phenotypic and endocytic resemblance with those obtained using GM-CSF. The results suggest that a receptor lactoferrin on BMC may be an important non-cytokine mechanism for early promyeloid progenitor differentiation.     

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of β₂-microglobulin (β₂m) predominantly in the musculoskeletal system, but the initiating events leading to β₂m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of β₂m, ΔK58-β₂m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-β₂m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of β₂m amyloid by a scaffold mechanism.     

Different effects of p58PITSLRE on the apoptosis induced by etoposide,cycloheximide and serum-withdrawal in human hepatocarcinoma cells

Minimal overexpression of the p58^PITSLRE protein kinase in Chinese hamster ovary cells induces telephase delay, abnormal cytokinesis, retarded cell growth and apoptosis. Fas mediated T cell death is correlated with p58^PITSLRE proteolysis and an increase in its histone H1 kinase activity. In this study, it was found that p58^PITSLRE had different effects on the apoptosis induced by etoposide, cycloheximide and serum-withdrawal in human hepatocarcinoma cells. The ectopic expression of p58^PITSLRE in human hepatocarcinoma cells suppressed apoptosis induced by etoposide, while enhancing the apoptosis induced by cycloheximide and serum-withdrawal respectively. Elevated expression of p58^PITSLRE was found during the apoptosis induced by etoposide, whereas most of p58^PITSLRE was proteolytically processed during apoptosis induced by cycloheximide and serum-withdrawal. Furthermore, transient transfection of p50^PITSLRE resembling the proteolytic form of p58^PITSLRE enhanced the 7721 cells susceptibility to apoptosis induced by all the three stimuli. These findings suggest that the full-length p58^PITSLRE might protect the cells from the apoptosis induced by etoposide and its proteolysis might contribute to and enhance the apoptosis induced by cycloheximide and serum-withdrawal respectively.     

Purification and properties of a novel β-galactosidase or exo-(1 → 4)-β-d-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds

The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1 58kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 4)-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-linked d-galactan, which is mobilised after germination (L.A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449–454). The isolation from the germinated cotyledons of a 58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactosidase or exo-(1 58kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 4)-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited 58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase action, catalysing the hydrolysis of p-nitrophenyl-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactopyranoside and (158kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 4)- and (1 58kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 6)-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-linked galactobioses. Lactose [58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactopyranosyl-(1 58kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 4)-d-glucose] was hydrolysed only very slowly and methyl-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing 58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactopyranosyl residues were not substrates. A linear (1 58kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 4)-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, 58kq4n5325/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-galactonolactone and Cu⁺² were inhibitory. No endo-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 58kq4n5325/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0"> 4)-58kq4n5325/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactan component of the cell wall.Abbreviations CM carboxymethyl - DEAE diethylaminoethyl - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TLC thin-layer chromatography We wish to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the award of a studentship to M.S. Buckeridge, and the Government of São Paulo State, Brazil for granting him leave of absence. We are grateful to Dr. Amanda Heyller (Unilever Research Laboratory, Colworth House, Bedford, UK) for N-terminal sequence determinations, to Dr. Stuart Wilson (Stirling) for preparing gelatin SDS-gels and to Cristina Fanutti (Stirling) for purifying the xyloglucan oligosaccharide.     

Radiotracer method in the study of environmental speciation and migration of contaminants

Principles, advantages, and limitations of the use of radiotracer method for the analysis of speciation and migration of contaminants in the environment are briefly discussed. Several recent examples of use in the author's laboratory are given: development of the separation method for methylmercury and inorganic mercury in hair, analysis of the speciation of cadmium in soil solutions, study of the interaction of¹³⁷Cs and⁵⁸Co with suspended sediments in river water, and determination of input data for mathematical modeling of radiocesium migration in a small river.     

The Non-Ionic Detergent Brij 58 Conserves the Structure of the Tonoplast H+-ATPase of Mesembryanthemum crystallinum L. During Solubilization and Partial Purification

The effects of solubilization with Triton X-100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H⁺-ATPase of plants of Mesembryanthemum crystallinum performing C₃ photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H⁺-ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent K_M value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C₃ and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H⁺-ATPase due to the Triton X-100 treatment. After solubilization with Brij 58 the tonoplast H⁺-ATPase was partially purified by Superose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H⁺-ATPase was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H⁺-ATPase is important for enzyme structure and function.     

Evaluation and control of the nutritional status of cereals

Summary A quantitative therapy method was developed for predicting and controlling grain yields of oats and spring wheat based on methods of diagnosis and yield pronosis and on effects of supplementary applications of nutrients on the chemical composition of the young plant at a fixed Dry Matter weight-level.The characteristic interactions integrated in the models of therapy and depending on kind, source, amount and combination of the nutrient applications on the chemical composition of the young plant allow selection of the best possible nutrient therapy under the given circumstances.The therapy method, tested by comparing predicted with experimentally obtained nutrient concentrations in the young plant, was proved reliable by the high and highly significant correlation coefficients (r>0.9;p<0.001). The correctness of the basic concepts underlying the therapy method, was thus indirectly confirmed and the possibility to use the method in agricultural practice would appear promising.     

Epstein-Barr virus RNA in Burkitt tumor tissue.

Analysis of the viral RNA in four Burkitt tumor biopsies indicates that tumor tissue contains RNA homologous to at least 3–6% of the DNA of Epstein-Barr virus (EBV). Most of these RNA species accumulate in the polyadenylated RNA fraction of Burkitt tumor tissue. Two approaches have been used to determine the location within the EBV genome of the DNA sequences which encode stable RNA in two Burkitt tumor biopsies, F and S, which contain 6–10 copies per cell of at least 80% of the EBV genome. With the first approach, ³²P-EBV DNA homologous to polyadenylated or nonpolyadenylated RNAs from the F, S or R tumors was hybridized to blots of fragments of EBV DNA. With the second approach, polyadenylated or nonpolyadenylated RNAs from the F or S tumors were hybridized to separated, labeled fragments of EBV DNA in solution. The results indicate that first, most of the viral RNA in Burkitt tumor tissue is encoded by approximately 20% of the Hsu I D fragment, 20% of the Eco RI A/Hsu I A double-cut fragment and 3% of the Hsu I B fragment of EBV DNA; second, an abundant RNA species in tumor tissue is homologous to the “additional DNA” present in the W91 and Jijoye/HR-I Burkitt tumor isolates of EBV and absent in the B95-8 virus, an isolate of EBV from outside the Burkitt endemic region; and third, there is little or no homology to other regions of the EBV genome.