Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

Interim use of Jarvik-7 and Novacor artificial heart: blood rheology and transient ischemic attacks (TIA's)

The rheological properties of blood were studied in patients supported by both the Jarvik-7 total artificial heart (TAH) and Novacor left ventricular assist device (LVAD) as a bridge to cardiac transplantation. Both groups of patients had abnormalities in blood rheology which differed according to the type of device implanted as well as on the clinical state of the patient. The rheology of individual patients correlated well with their clinical status and outcome, with incidences of TIA's and/or stroke being accompanied by marked increases in relative blood viscosity, erythrocyte rigidity, fibrinogen concentration and platelet aggregation in varying combination. Observed abnormalities in blood rheology were also crucial to thrombus formation on artificial heart valves as well. Our results show that the therapeutic management of rheological parameters should prove to be a unique and clinically rewarding approach to these patients.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway

The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate filament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cytoskeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKC-deficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies involving intact cells.     

Filamentous structures associated with Epstein-Barr virus-infected cells.

After the onset of Epstein-Barr virus DNA and protein synthesis 10 h after superinfection of Raji cells (a cell line containing Epstein-Barr virus DNA but not producing virus), filamentous structures 25 nm in diameter and 0.2 to 1.4 micrometers in length could be detected in the cell cytoplasm by electron microscopy. These structures banded in metrizamide gradients with viral DNA and proteins, but at a density different from that of virions or nucleocapsids. These filaments, enriched in a 155,000-dalton protein similar in size to a major nucleocapsid protein of Epstein-Barr virus, may represent intermediates in viral nucleocapsid assembly.     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Rapid identification of microorganisms by circular-intensity differential scattering

There is a pressing need in clinical medicine for rapid identification of microorganisms. We describe a method that has the potential for such rapid identification: circular-intensity differential scattering, which is based on the differential scattering of left and right circularly polarized light. The scanning time required to obtain the spectral signature of an organism is about 4 min. Using a commercial circular dichrograph modified to measure circular intensity differential scattering at 90 degrees, we obtained significantly different spectra for five different crude influenza viruses. Salmonella typhimurium TA98 and TA100, and Escherichia coli HB101, HB101(pBR322), and HB101(pMB9). Purified supercoiled plasmid pBR322 DNA was readily distinguishable from linear pBR322 DNA; such differences in nucleic acid packaging may be significant factors in the discriminatory power of this technique.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.     

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.