Comparison of the effects of cationic porphyrins on DNA properties: influence of GC content of native and synthetic polymers

Interactions of meso-tetra(4-N-methylpyridyl)porphyrin [TMpyP(4)], meso-tetra(2-N-methylpyridyl)porphyrin [TMpyP(2)], and meso-tetra(para-N-trimethylanilinium)porphyrin (TMAP) with several native and synthetic DNAs were studied by a variety of physical techniques: nmr (³¹P and ¹H), absorption spectroscopy, viscosity, and flow dichroism (FD). Of the three porphyrins studied, only the interaction of TMpyP(4) with poly [d(G-C)₂] was fully consistent with intercalation. In particular, a large increase in viscosity, a downfield ³¹P-nmr signal (ca. -1 ppm), and upfield imino proton signals (11 to 12 ppm range) were observed. Comparison of the effects of TMpyP(4) on DNAs of different GC contents revealed larger changes in solution viscosity with increased GC content. However, the characteristic changes in ³¹P- and ¹H-nmr spectra were not observed. The viscosity increases observed in studies with poly[d(A-C)(G-T)] and C. Perf. DNA were much lower than with poly[d(G-C)₂], M. Lys. DNA, and calf thymus DNA. Thus, GC sequence and content are clearly important. The principal change in the ³¹P-nmr signal of native DNA is the appearance of a very broad shoulder centered at ca. -2.0 ppm, which is larger in M. Lys. DNA than in C. Perf. DNA. FD studies indicate highly ordered TMpyP(4) cations arranged perpendicular to the DNA axis of calf thymus DNA. Together, these results suggest the major effects of TMpyP(4) on DNA properties are due to strong GC-binding interactions that influence DNA structure. The data are consistent with combined intercalative and outside binding interactions of TMpyP(4) with GC regions of DNA. In contrast, similar studies with TMAP suggest that it influences AT regions of DNA by an outside binding mode. On the other hand, TMpyP(2) effects on DNA properties are consistent with nonselective outside binding.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

Site-specific mutants of oncomodulin. 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59----Glu59 substitution in the calcium-specific site

High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.     

Immunological and genetic characterization of 2-deoxygalactose-resistant, galactokinase-deficient mutants of Chinese hamster cells: evidence for structural mutations at the galK locus.

Ten independent mutants resistant to 2-deoxygalactose and without any detectable galactokinase activity (null-galactokinase mutations) were isolated from mutagenized Chinese hamster somatic cells. They were analyzed for the presence of serologically cross-reacting material (CRM) with antiserum generated against highly purified Chinese hamster galactokinase. All 10 mutants contain cross-reacting material (i.e., were CRM+), indicating that all the mutations affect the correct expression of a product of the galactokinase structural gene. Complementation analysis among them shows that the 10 mutations fall in one functional genetic unit.     

NMR investigation of the interaction of mithramycin A with d(ACCCGGGT)2.

The binding of mithramycin A to d(ACCCGGGT)2 has been investigated by one- and two-dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.     

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.     

Structural analysis of d(GCAATTGC)2 and its complex with berenil by nuclear magnetic resonance spectroscopy

The structures of d(GCAATTGC)2 and its complex with berenil in solution were analyzed by two-dimensional 1H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10 degrees C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC)2 system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC)2. The aromatic drug protons are placed within 5 A of the H2 proton of both adenines, the H1', H5', and H5" of both thymidines, and the H4', H5', and H5" of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18 degrees C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45 degrees C. Two molecular models are proposed for the berenil-(GCAATTGC)2 complex; one has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.     

Galactokinase Mutants of Chinese Hamster Somatic Cells Resistant to 2-Deoxygalactose

The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase(EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & kmax of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possible a missence mutation, in the galactokinase gene.- A simple diagnostic test for juvenile cataract is proposed.     

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG_2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG₁ anti-BCII antibody activity and increased splenocyte secretion of IFN-γ and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.