Coordinate replication of dispersed repetitive sequences in Physarum polycephalum

The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.C pairing in dodecanucleotide duplexes

The pairing of O6etG with C located four base pairs in from either end of the self-complementary d(C1-G2-C3-O6etG4-A5-G6-C7-T8-C9-G10-C11-G12) duplex (designated O6etG.C 12-mer) has been investigated from an analysis of proton and phosphorus two-dimensional NMR experiments. The structural consequences of increasing the alkyl group size were elucidated from a comparative study of the pairing of O6meG4 with C9 in a related sequence (designated O6meG.C 12-mer). The NMR parameters for both O6alkG-containing dodecanucleotides are also compared with those of the control sequence containing G4.C9 base pairs (designated G.C 12-mer). The NOE cross-peaks detected in the two-dimensional NOESY spectra of the O6alkG.C 12-mer duplexes in H2O solution establish that the O6etG4/O6meG4 and C9 bases at the lesion site stack into the helix between the flanking C3.G10 and A5.T8 Watson-Crick base pairs. The amino protons of C9 at the O6alkG4-C9 lesion site resonate as an average resonance at 7.78 and 7.63 ppm in the O6etG.C 12-mer and O6meG.C 12-mer duplexes, respectively. The observed NOEs between the amino protons of C9 and the CH3 protons of O6alkG4 establish a syn orientation of the O6-alkyl group with respect to the N1 of alkylated guanine. A wobble alignment of the O6alkG4.C9 base pair stablized by two hydrogen bonds, one between the amino group of C9 and N1 of O6alkG and the other between the amino group of O6alkG and N3 of C9, is tentatively proposed on the basis of the NOEs between the amino protons of C9 at the lesion site and the imino protons of flanking Watson-Crick base pairs. The proton and phosphorus chemical shift differences between the O6etG.C 12-mer and O6meG.C 12-mer duplexes are small compared to the differences between these O6alkG-containing duplexes and the control G.C 12-mer duplex.     

Solution structure of the chromomycin-DNA complex

The structure of the chromomycin-DNA complex at the deoxyoctanucleotide duplex level has been determined from one- and two-dimensional proton NMR studies in Mg-containing aqueous solution. The NMR results demonstrate that the antitumor agent binds as a symmetrical dimer to the self-complementary d[T-T-G-G-C-C-A-A] duplex with retention of the 2-fold symmetry in the complex. A set of intermolecular nuclear Overhauser enhancements (NOEs) establishes that two chromomycin molecules in the dimer share the minor groove at the G-G-C-C.G-G-C-C segment in such a way that each hydrophilic edge of the chromophore is located next to the G-G.C-C half-site and each C-D-E trisaccharide chain extends toward the 3'-direction of the octanucleotide duplex. In addition, the A-B disaccharide segment and the hydrophilic side chain of the antitumor agent are directed toward the phosphate backbone. The observed changes in nucleic acid NOEs and coupling patterns on complex formation establish a transition to a wider and shallower minor groove at the central G-G-C-C.G-G-C-C segment required for accommodating the chromomycin dimer. The present demonstration that chromomycin binds as a dimer and switches the conformation of the DNA at its G.C-rich minor groove binding site provides new insights into antitumor agent design and the sequence specificity of antitumor agent-DNA recognition.     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite acyclic lesions

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated APP 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated APE 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the APP 9-mer and APE 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution between -5 and 5 degrees C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and E14) and stacks between the flanking G4.C15 and G6.C13 Watson-Crick base pairs in both the APP 9-mer and APE 9-mer duplexes. The helix is right-handed at and adjacent to the abasic site, and all glycosidic torsion angles are anti in both 9-mer duplexes. Proton NMR parameters for the APP 9-mer and APE 9-mer duplexes are similar to those reported previously for the APF 9-mer duplex (F = furan) in which a cyclic analogue of deoxyribose was embedded in an otherwise identical DNA sequence [Kalnik, M. W., Chang, C. N., Grollman, A. P., & Patel, D. J. (1988) Biochemistry 27, 924-931]. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the APP 9-mer and APE 9-mer duplexes (5 degrees C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles. These same torsion angles are also distorted in the APF 9-mer but assume a different conformation than those in the APP 9-mer and APE 9-mer duplexes.     

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.     

NMR studies of DNA (R+)n.(Y-)n.(Y+)n triple helices in solution: imino and amino proton markers of T.A.T and C.G.C+ base-triple formation

High-resolution exchangeable proton two-dimensional NMR spectra have been recorded on 11-mer DNA triple helices containing one oligopurine (R)n and two oligopyrimidine (Y)n strands at acidic pH and elevated temperatures. Our two-dimensional nuclear Overhauser effect studies have focused on an 11-mer triplex where the third oligopyrimidine strand is parallel to the oligopurine strand. The observed distance connectivities establish that the third oligopyrimidine strand resides in the major groove with the triplex stabilized through formation of T.A.T and C.G.C+ base triples. The T.A.T base triple can be monitored by imino protons of the thymidines involved in Watson-Crick (13.65-14.25 ppm) and Hoogsteen (12.9-13.55 ppm) pairing, as well as the amino protons of adenosine (7.4-7.7 ppm). The amino protons of the protonated (8.5-10.0 ppm) and unprotonated (6.5-8.3 ppm) cytidines in the C.G.C+ base triple provide distinct markers as do the imino protons of the guanosine (12.6-13.3 ppm) and the protonated cytidine (14.5-16.0 ppm). The upfield chemical shift of the adenosine H8 protons (7.1-7.3 ppm) establishes that the oligopurine strand adopts an A-helical base stacking conformation in the 11-mer triplex. These results demonstrate that oligonucleotide triple helices can be readily monitored by NMR at the individual base-triple level with distinct markers differentiating between Watson-Crick and Hoogsteen pairing. Excellent exchangeable proton spectra have also been recorded for (R+)n.(Y-)n.(Y+)n 7-mer triple helices with the shorter length permitting spectra to be recorded at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.