Inhibition of deoxyribonuclease activity associated with soybean chloroplasts

The effects of spermidine, pH, ethylene diamine tetracetic acid (EDTA), and adenosine triphosphate (ATP) on deoxyribonuclease (DNase) activity associated with the chloroplasts of soybean (Glycine max (L.) Merr.) were investigated. Chloroplast DNase activity was found to be partially inhibited by either 10 mM spermidine, 20 mM EDTA, or 20 mM ATP. DNase activity was also partially inhibited at non-neutral pH's. Nearly complete inhibition was achieved with use of 30 mM EDTA, pH 10, or a combination of 10 mM spermidine and 10 mM EDTA.     

The comparison of rat liver rough endoplasmic reticulum membrane proteins before and after in vitro removal of its bound ribosomes

The comparison of the proteins of rat liver rough membrane after stripping with EDTA or KCl-puromycin by two dimensional gel electrophoresis is described. By stripping the membrane with EDTA, most of the basic ribosomal proteins are still attached to the membrane; in contrast to the EDTA stripping method, treatment with KCl-puromycin removes most of the ribosomal proteins and does not remove any of the membranal proteins.     

Regulation of androgenesis inNicotiana tabacum L. cv. White Burley andDatura innoxia Mill. Effect of bivalent and trivalent iron and chelating substances

The effect of FeSO₄.7H₂O, Fe₂(SO₄)₃.9H₂O, disodium salt of ethylene-diaminotetraacetic acid, dihydrate (EDTA) and N-(2-acetamido) iminodiacetic acid (ADA) and their combinations on the androgenesis was studiedin vitro in tobacco (cv. White Burley) and datura (Datura innoxia Mill.). Simultaneously the reversibility and irreversibility of the morphogenic process leading to the conversion of the pollen embryoid into complete plant was followed. Complete plants developed in anthers on media with trivalent iron, chelated trivalent iron, chelated bivalent iron, bivalent iron in the presence of ADA and of media with EDTA. The number of androgenic plants in anthers increased in the following order: Fe₃₊ < Fe₃₊ EDTA ≦ ≦ EDTA < Fe²⁺ EDTA. The marked brown colour of cultured anthers was due to the presence of trivalent iron in the medium. The androgenic development was most rapid on the medium containing only trivalent iron, slower on media with chelated iron and slowest on medium with EDTA. The viability of cultures with complete plants decreased in the reverse order. No complete plants grew on media without trivalent iron and without EDTA and on media containing only bivalent iron whereas globular embryoids arose and developed continuously on these media. The anthers reacted in the same way on both complete and minimal media. Isolated embryoids formed complete plants in corresponding variants on complete media only. The development of pollen embryoids into complete plants was stopped by the transfer of globular and torpedo-shaped embryoids from medium with EDTA to the medium without EDTA. Isolated greenish cotyledonar embryoids continued to grow even on the medium without EDTA.     

Biological effects of nickel species and their determination in plant and soil

Chemical and biological behaviour of several Ni-forms was studied on different soil types with spinach as a test plant. For the positively charged forms, Ni-ions and Ni-TETREN-ions, extractability increased with decreasing pH and CEC values of the soil. On the other hand, Ni added as EDTA-complex resulted in an extremely high Ni mobility, enhancing extractable Ni in soils having higher pH and CEC values. Speciation of Ni in the saturation extracts of the soils revealed that the transformation of both added positive forms was dependent on the soil characteristics, while Ni added as EDTA-complex mainly remained in the negative form. This altered mobility, accompanied by variable modification of the original chemical form in the soil solution, affected plant uptake and appearance of phytotoxic effects. Speciation in the plant extracts indicated that regardless of the chemical form added to the soil, Ni was only found in neutral and negative complexes. Finally, using ion-pair reversed phase HPLC, Ni-EDTA-ions were quantified in both the soil solution and the plant extract.     

Simple assay of serum copper fractions by ultrafiltration and flameless atomic absorption

The authors present a simple and rapid method for assaying ultrafilterable copper (Cu UF) and albumin-bound copper (Cu ALB). It is based on the ultrafiltration of serum in the presence of ethylenediaminetetraacetic acid (EDTA), used to prevent adsorption on the membranes. EDTA at 0.4 g/L has no effect on the equilibrium of serum copper vectors and enables Cu UF to be assayed by flameless atomic absorption. EDTA at 2 g/L, is used to assay total exchangeable copper (CU EXC) (ultrafilterable+albumin-bound). The evaluation criteria of the method are furnished, as are the normal values in healthy subjects: 14.6 μg/L for Cu UF and 87 μg/L for Cu EXC. Finally, the usefulness of the methods described here for the diagnosis of Wilson's and Menkes' disease was demonstrated.     

The influence of EDTA on the composition of alginate synthesized by Azotobacter vinelandii

1. During an investigation of the physiology of Azotobacter vinelandii with particular reference to polysaccharide formation, a suitable medium which was precipitate-free was developed by adding EDTA at a concentration of 50 mg/l to a basal medium containing one of eight different carbohydrates as sole carbon source.
2. Acetylated alginate was always produced by the organism when cultured under defined conditions, regardless of the carbohydrate source incorporated in the basal medium.
3. When EDTA was added to the medium, the bacteria produced acetylated polyuronides with a preponderance of mannuronic acid residues.
4. A comparison of the infrared spectra of the alginate produced by Azotobacter vinelandii and the affect of EDTA upon the mannuronic acid/guluronic acid ratios of the alginate are reported.

     

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.     

EDTA- and puromycin-derived duck- and rabbit globin-messenger ribonucleoprotein complexes isolated by oligo(dT)-cellulose chromatography

Duck- and rabbit globin messenger ribonucleoprotein complexes isolated by oligo(dT) cellulose chromatography reveal an identical protein pattern—two main proteins of molecular weights of 73000 and 49000 daltons and minor components—whether the complexes have been liberated from polyribosomes with the EDTA-or the puromycin-high-salt method. In the globin messenger ribonucleoprotein particles of both species predominantly the protein with a molecular weight of 73000 daltons is attached to poly(A)-containing regions of the messenger RNAs.     

Studies on choline acetyltransferase isolated from human brain

The purified choline acetyltransferase from human striatal tissue was found to have aK _m value of 8 μM for acetyl-coenzyme A and 250 μM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimatedS-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCl, NaCl, (NH₄)₂SO₄, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 mM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

The nucleolus organizer region of maize (Zea mays L.)

Maize plants were produced partially triploid for the heterochromatic segment of the nucleolus organizer region (NOR) or partially triploid or tetraploid for the site giving rise to the secondary constriction of the NOR. These partially hyperploid plants were characterized in terms of chromosome and/or nucleolar constitution by light microscopy at pachytene, diakinesis, and quartet stages of microsporogenesis. DNA's of the various partially hyperploid plants and appropriate controls were extracted and hybridized with ³H-rRNA. The heterochromatic segment of the NOR was found to contain most of the rRNA cistrons, but has little or no interaction with the nucleolus. In contrast with the heterochromatic segment, the site giving rise to the secondary constriction contains few rRNA cistrons but is active in nucleolar formation as viewed at pachytene, diakinesis and quartet stages.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.