NMR studies of an exocyclic 1,N2-propanodeoxyguanosine adduct (X) located opposite deoxyadenosine (A) in DNA duplexes at basic pH: simultaneous partial intercalation of X and A between stacked bases

The NMR parameters for the 1,N2-propanodeoxyguanosine (X) opposite deoxyadenosine positioned in the center of the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A 16-T17-G18) X.A 9-mer duplex are pH dependent. A previous paper established protonated X5(syn).A14(anti) pairing in the X.A 9-mer duplex at pH 5.8 [Kouchakdjian, M., Marinelli, E., Gao, X., Johnson, F., Grollman, A., & Patel, D. J. (1989) Biochemistry 28, 5647-5657]; this paper focuses on the pairing alignment at the lesion site at pH 8.9. The observed NOEs between specific exocyclic CH2 protons and both the imino proton of G6 and the sugar H1' protons of C13 and A14 establish that X5 is positioned toward the G6.C13 base pair with the exocyclic ring directed between C13 and A14 on the partner strand. The observed NOE between the H2 proton of A14 and the imino proton of G4, but not G6, establishes that A14 at the lesion site is directed toward the G4.C15 base pair. NOEs are detected between all exocyclic CH2 protons of X5 and the H2 proton of A14, confirming that both X5 and A14 are directed toward the interior of the helix. The X5(anti).A14(anti) alignment at pH 8.9 is accommodated within the helix with retention of Watson-Crick pairing at flanking G4.C15 and G6.C13 base pairs. The energy-minimized conformation of the (G4-X5-G6).(C13-A14-C15) segment at pH 8.9 establishes that X5 and A14 are directed into the helix, partially stack on each other, and are not stabilized by intermolecular hydrogen bonds. The X5 base is partially intercalated between C13 and A14 on the unmodified strand, while A14 is partially intercalated between G4 and X5 on the modified strand. This results in a larger separation between the G4.C15 and G6.C13 base pairs flanking the lesion site in the basic pH conformation of the X.A 9-mer duplex. The midpoint of the transition between the protonated X5(syn).A14(anti) and X5(anti).A14(anti) conformations occurs at pH 7.6, establishing an unusually high pKa for protonation of the A14 ring opposite the X5 exocyclic adduct site. Thus, the interplay between hydrophobic and hydrogen-bonding contributions modulated by pH defines the alignment of 1,N2-propanodeoxyguanosine opposite deoxyadenosine in the interior of DNA helices.     

Characterization of an inducible mannitol-binding protein fromPseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO formed a mannitol-binding protein that was coinducible with mannitol uptake activity. One species of mannitol-binding protein was isolated from disrupted cell suspensions and the protein also was released from mannitol-grown cells by osmotic shock treatment. The mannitol-binding protein )molecular weight 37,000) was purified 49-fold and resolved from the enzyme mannitol dehydrogenase (molecular weight 85,000), which did not exhibit mannitol-binding activity. Mannitol-binding protein had an isoelectric point (pI) of 8.3 and an apparent dissociation constant (K _D ) of 2.3 M for mannitol. Mannitol binding was unaffected by glucose and glycerol in competitive inhibition studies, although mannose, fructose, and sorbitol caused 5 to 20% inhibition of binding at concentrations equimolar with mannitol.     

Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex.

The gene A protein cleaves phi X174 single-stranded DNA (ssDNA). The cleavage appears to be stoichiometric, whereby a gene A protein molecule breaks a phosphodiester bond and binds to the 5' end. The enzyme introduces mostly a single break in a circular ssDNA molecule. However, at high enzyme-to-DNA ratios, more than one break in the DNA could be observed. The cleavage of the ssDNA by gene A protein renders the DNA sensitive to the action of terminal transferase to incorporate [alpha -32P]ATP. Thus, the 3'OH end is free. All attempts to label the 5' end by T4-induced polynucleotide kinase and [gamma-32P]ATP failed. The formation of a gene A-ssDNA complex was demonstrated directly by using 3H-labeled gene A protein and 32P-labeled ssDNA in the reaction. Such a complex is resistant to treatments with 0.2 M NaOH, banding in CsCl, and boiling in 2.5% sodium dodecyl sulfate. Only treatment with a nuclease released the bound protein. Also, after cleaving [32P]ssDNA by gene A protein, followed by either DNase I or micrococcal nuclease digestion, a fraction of the 32P label remained resistant to nuclease treatment and comigrated with gene A protein on polyacrylamide gels.     

Comparative structural analysis of glycoprotein gD of herpes simplex virus types 1 and 2.

We studied the synthesis and processing of the type-common glycoprotein gD in herpes simplex virus type 2 (HSV-2) and compared it structurally to glycoprotein gD of herpes simplex virus type 1 (HSV-1). We demonstrated that in HSV-2, gD undergoes posttranslational processing from a lower-molecular-weight precursor (pgD51) to a higher-molecular-weight product (gD56). Tryptic peptide analysis by cation-exchange chromatography indicated that this processing step altered neither the methionine nor the arginine tryptic peptide profile of gD of HSV-2. Comparative tryptic peptide analysis of gD of HSV-1 and HSV-2 showed that the methionine and arginine tryptic peptide profiles of these two proteins were very similar, but not identical. Some of the resolved peptides coeluted from the cation-exchange column, suggesting that some amino acid sequences of the two proteins might be very similar. However, each protein also appeared to possess several type-specific tryptic peptides. The structural similarity of these two glycoproteins correlates well with their antigenic cross-reactivity since monoprecipitin antibody to gD of HSV-1 also immunoprecipitates gD of HSV-2 and neutralizes the infectivity of both viruses to approximately the same extent.     

Properties of the foot inhibitor from hydra

Summary A substance was isolated from crude extracts of hydra that inhibits foot regeneration. This substance, the foot inhibitor, has a molecular weight of 500 daltons. It is a hydrophilic molecule, slightly basic in character and it has no peptide bonds. The pruified substance acts specifically and at concentrations lower than 10^–7 M. At this low concentration only foot and not head regeneration is inhibited. Hydra are sensitive to purified foot inhibitor between the second and eight hour after initiation of foot regeneration by cutting. In normal animals the foot inhibitor is most likely produced by nerve cells. A substance with similar biological and physico-chemical properties is found in other coelenterates.     

A new species of Eugenia (Myrtaceae) from Venezuela

Eugenia mcvaughii is described from a forest remnant of the Jardin Botánico of Caracas, Venezuela. Found also in the hills adjacent to Caracas, it may be considered as an endemic species of the Interior Coastal Cordillera.     

Apparent microviscosity of intact and post-lipolysis ("remnant") very low density lipoprotein particles.

The apparent microviscosity of intact rat plasma very low density lipoprotein (VLDL) and post-lipolysis very low density lipoprotein was determined by fluorescence depolarization measurements and flurorescence decay measurements using 1, 6-diphenylhexatriene. Post-lipolysis very low density lipoprotein was prepared in vitro after incubation of the intact lipoprotein with either purified bovine milk lipoprotein lipase or lipoprotein lipase rich (post-heparin) plasma. During lipolysis, an average of 88% of the triglycerides were hydrolyzed, and the lipoprotein became depleted in phospholipids, cholesterol and apolipoprotein C. The apparent microviscosity of the lipoprotein increased by three-fold from 0.63 to 1.88 poise. It is concluded that the compositional changes occurring during lipolysis affect the physical properties of the lipoprotein, as measured here by the fluidity (microviscosity) of the particles.     

Some self-consistent two-state sliding filament models of muscle contraction.

The general formalism required to treat two-state sliding filament models of muscle contraction, including free energy considerations, is first reviewed and amplified. This formalism is then used to examine, and modify as needed, three models studied previously by Podolsky and Nolan, in which cross-bridge attachment-detachment and ATP turnover are not tightly coupled. No attempt is made here to establish an optimal, self-consistent model of this type because our interest is primarily in methadology rather than in fitting experimental results. But it appears from this preliminary study that such a model, with satisfactory mechanical and thermodynamic properties, could be found. An extremely simple but unrealistic two-state model is also studied which is of interest because it demonstrates the fact that it is possible, in principle at least, for sliding filament models to work with very high thermodynamic efficiencies (50-100 percent). An appendix is included that is concerned with the form of the dependence of certain first-order rate constants on the concentrations of ATP, ADP, and P.     

Heavy metal-nucleoside interactions. Binding of methylmercury(II) to inosine and catalysis of the isotopic exchange of the C-8 hydrogen studied by 1-H nuclear magnetic resonance and raman difference spectrophotometry.

Raman difference spectrophotometry reveals that CH3HgII binds quantitatively to N(1) of inosine at pH 8, substituting for the proton. When N(1) is saturated, binding occurs at a second site. Measurements of the 1-H nuclear magnetic resonance spectra of both inosine and of CH3Hg-II are in agreement with the N(1) binding and indicate that the second site for mercuriation is N(7). This second binding reaction is observed to increase the rate of exchange of the C(8) hydrogen with solvent, consistent with results observed for alkylation at N(7). Coordination of the electrophilic CH3Hg-II to N(7) increases the acidity of H(8), facilitating OHminus--catalyzed proton abstraction and reprotonation by themedium. For comparison, the reaction of CH3Hg-II with [8-2-H]inosine has been studied. Displacement of the N(1) hydrogen upon mercuriation of inosine causes a significant electron delocalization into the ring, increasing the basicity of N(7), and accounting for the synergic effect in metal binding observed originally by Simpson. In contrast, 1-methylinosine interacts only slightly with CH3Hg-II at pH 8. Coordination appears to be at N(7), since H(8) again is observed to exchange rapidly with solvent protons. In acidic solution, pH less than 2, binding to inosine is almost quantitative and exclusively to N(7). The behavior of CH3Hg-II is compared with that of Pt(II) and with Ni(II), Co(II), AND Zn(II). A brief comparison is made among ultraviolet absorption spectrophotometry, nuclear magnetic resonance (NMR), and Raman difference spectrophotometry for studying reactions of nucleosides and nucleotides.     

Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (postheparin) plasma.

Incubation of 125I-labeled very low density lipoprotein (VLDL) with lipoprotein lipase-rich (postheparin) plasma obtained from intact or supradiaphragmatic rats resulted in the transfer of more than 80% of apoprotein C from VLDL to high density lipoprotein (HDL), whereas apoprotein B was associated with lipoprotein of density less than 1.019 g/ml (intermediate lipoprotein). The transfer of 125I-labeled apoprotein C from VLDL to HDL increased with time and decreased in proportion to the amount of VLDL in the incubation system. A relationship was established between the content of triglycerides and apoprotein C in VLDL, whereas the amount of apoprotein C in VLDL was independent of that of other apoproteins, especially apoprotein B. The injection of heparin to rats preinjected with 125I-labeled VLDL caused apoprotein interconversions similar to those observed in vitro. The intermediate lipoprotein was relatively rich in apoprotein B, apoprotein VS-2, cholesterol, and phospholipids and poor in triglycerides and apoprotein C. The mean diameter of intermediate lipoprotein was 269 A (compared with 427 A, the mean Sf rate was 30.5 (compared with 115), and the mean weight was 7.0 X 10(6) daltons (compared with 23.1 X 10(6)). From these data it was possible to calculate the mass of lipids and apoproteins in single lipoprotein particles. The content of apoprotein B in both particles was virtually identical, 0.7 X 10(6) daltons. The relative amount of all other constituents in intermediate lipoprotein was lower than in VLDL: triglycerides, 22%; free cholesterol, 37%; esterified cholesterol, 68%; phospholipids, 41%; apoprotein C, 7%, and VS-2 apoprotein, 60%. The data indicate that (a) one and only one intermediate lipoprotein is formed from each VLDL particle, and (b) during the formation of the intermediate lipoprotein all lipid and apoprotein components other than apoprotein B leave the density range of VLDL to a varying degree. Whether these same changes occur during the clearance of VLDL in vivo is yet to be established.