A rapid method of gene detection using DNA bound to Sephacryl

A rapid method of gene detection has been developed utilising DNA fragments immobilized on resins and a sandwich hybridization assay. This method permits the detection of restriction fragment length polymorphisms (RFLPs) without the need to immobilize sample DNA. The method is based on the use of two non-overlapping DNA restriction fragments, one of which is attached to a resin (fragment A) and the other 32P-labelled (fragment B). Fragments A and B will not hybridize to each other unless there is a DNA or RNA fragment capable of hybridizing to both A and B present in the same reaction. Hybridization in this instance will result in the resin being radioactively labelled. The RFLP associated with the mutation causing sickle-cell anaemia was used as a model to develop the method. The resin Sephacryl S-500 appeared most suited to our method for two reasons: (i) DNA immobilization experiments using two coupling procedures and four resins indicated that Sephacryl S-500 bound the most DNA with very little non-covalent coupling. (ii) Hybridization experiments with DNA bound to a number of resins showed that DNA bound to Sephacryl S-500 hybridized most efficiently with a low level of nonspecific hybridization. Using optimum hybridization conditions 5 X 10(-18) mol of beta-globin DNA could be detected. The method has been used to distinguish between DNA from sickle, heterozygote and normal patients.     

DNA binding of aflatoxin B1 by co-oxygenation in mouse embryo fibroblasts C3H/10T1/2

The mechanism of activation of aflatoxin B1 to ultimate metabolites capable of DNA binding was investigated in mouse embryo fibroblasts C3H/10T1/2. The contribution of co-oxygenation reactions which are coupled to arachidonic acid metabolism was assessed by the use of inhibitors of prostaglandin endoperoxide synthetase and lipoxygenase. Indomethacin and 5,8,11,14-icosatetraynoic acid inhibited AFB1-binding to maximally 60%. The antioxidant glutathione was also inhibitory while CuZn superoxide dismutase had no effect or slightly stimulated binding at high concentrations. These results indicate that co-oxygenation plays a major role in AFB1-metabolism in 10T1/2 cells. The observation that the phospholipase A2 inhibitor p-bromophenacylbromide diminished AFB1-DNA binding supports the notion that AFB1, because it is membrane-active, may enhance its own co-oxidative metabolism by stimulating the arachidonic acid cascade.     

Structural changes in the cell membrane of lambda-lysogenic Escherichia coli induced by colicin E2.

The structural changes in the cell membrane of λ-lysogenic $\text{Escherichia coli}$ induced by colicin E₂ were examined. The addition of colicin E₂ made the cells susceptible to various detergents and the transport rate of o-nitrophenyl-β-D-galactoside into the colicin-treated cells was stimulated markedly by adding a low concentration of sodium dodecyl sulfate. The fluorescence intensity of 8-anilino-1-naphthalenesulfonate bound to the cells was markedly increased by adding colicin E₂. Colicin E₂ stimulated the incorporation of ${}^{32}\text{P}$ from prelabeled phosphatidylglycerol to cardiolipin. All these changes probably suggesting the structural alteration of the cell membrane were dependent on the presence of the $\text{rex}$ gene of λ prophage in the cells.     

The gelation kinetics of platelet extracts

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca²⁺ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca²⁺ concentration up to about 2 μM. At 4–15 μM free Ca²⁺, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca²⁺ concentrations. Trifluoperazine (50 μM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca²⁺ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K⁺, ATP, cytochalasin E and colchicine. K⁺ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelations, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 μM) and colchicine (1 mM) interfered with the gelation process.     

The action of organic mercurials on the erythrocyte membrane

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although $\text{N-}\text{ethylmaleimide}$ and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ-³²P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg²⁺ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients

Due to the high reproducibility of pH gradient slope and width, immobilized pH gradients (IPG) have been used as the first dimension of two-dimensional techniques in order to generate maps of constant spot position in the $\text{p}\text{M}{}_{\mathrm{<mtext>r</mtext>}}$. However, when coupling IPG to SDS (sodium dodecyl sulphate) gels two problems were encountered: vertical streaking, due to incomplete zone solubilization in SDS, and horizontal streaking, due to spot fusion along the pH axis caused by the electroendosmosis of the charged Immobiline gels. Two methodical modifications are herewith described to overcome these drawbacks: (a) the SDS equilibrium time of the first-dimension gel has been prolonged to at least 30 min; (b) the SDS electrophoresis gel has been cast together with a starting gel, containing 2.5 mM of each Immobiline species used in the first dimension, which serves as a transition from the charged to the uncharged gel.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.