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 distribution of aquatic oligochaetes in the south and eastern Mediterranean area

On the basis of the material collected in Morocco, Algeria, Tunisia, Lebanon, Syria and Turkey, and published data, mainly on Israel and Egypt, we try to establish a synthesis of the geographical distribution of the poorly known fauna of the South and Eastern Mediterranean basin. A first analysis shows that the fauna of this region is characterized by a high number of species with a widespread distribution (cosmopolite, holarctic, palaearctic ...) and therefore, with great affinities with the European fauna. Nevertheless, three distinct communities can be distinguished:

- The south western basin (North Africa; Morocco, Algeria, Tunisia), where the community is very poor, and which shows great affinities with south European, but none with the Ethiopian region;

- The south eastern basin (Egypt), with some species of African origin;

- The eastern basin (Near East, mainly Lebanon and Israel) with a very diversified community, partly due to its geographical situation, and partly due to a more intensive collecting pressure. This community includes a group of species with more eastern affinities (East Siberian ...), but none typically Ethiopian or exclusively Sino-Indian.

     

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.     

ATP-driven succinate oxidation in the catabolism of Desulfuromonas acetoxidans

The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained:

1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD).
2. The membrane preparation also catalyzed the reduction of fumarate with H₂S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD.
3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H₂S and fumarate and between NADH and fumarate.
4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H₂S, or NADH, and the oxidation of DMNH₂ by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP.
5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H₂S and NADH oxidation by DMN were not affected by the inhibitor.

It is concluded that the catabolism of D. acetoxidans involves the energy-driven oxidation of succinate with elemental sulphur or NAD as electron acceptors and that menaquinone is a component of the electron transport chain catalyzing these reactions.     

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.     

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.     

On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

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.     

Fumarate reduction inProteus mirabilis

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorateresistant mutant strains of the wild type, fumarate reductase appeared to be affected.
2. Cytoplasmic membrane suspensions isolated from anaerobically grownP. mirabilis oxidized formate and NADH with oxygen and with fumarate, too.
3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochromeb, cytochromea ₁ and cytochromed. Cytochromeb was reduced by NADH as well as by formate to approximately 80%.
4. 2-n-Heptyl-4-hydroxyquinoline-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state.
5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochromeb.
6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen.
7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochromeb as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grownP. mirabilis is presented.

     

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.     

ATPase activities in partially purified membranes of Acetabularia

Partly purified membranes (with plasmalemma material) of Acetabularia mediterranea were studied with respect to ATPase activity in alkali- and Ca⁺⁺-free media and its sensitivity to pH (5 – 9), oligomycin (200 ?g/mg protein), 100 ?M N-N′-dicyclohexylcarbodiimide (DCCD), and 50 ?M vanadate. Besides activities which may originate from mitochondrial H⁺ ATPase (oligomycin-sensitive, alkaline pH optimum) and tonoplast H⁺ ATPase (DCCD-sensitive, pH optimum 7.5), there is ATPase activity with a pH optimum around pH 6.5, sensitive to vanadate and insensitive to DCCD. These results strongly suggest that the electrogenic Cl^? pump in the plasmalemma of Acetabularia is an ATPase. Effects of Mg⁺⁺, Mg-ATP, ADP, GTP, UTP, CTP and HCO₃ ^? versus Cl^? on this ATPase activity are described.     

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.