Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution.

Thioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic beta/A4-derived peptides beta(1-28) and beta(1-40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys the signal. There was no effect of high salt concentrations. Binding to the beta(1-40) is of lower affinity, Kd 2 microM, while it saturates with a Kd of 0.54 microM for beta(1-28). Insulin fibrils converted to a beta-sheet conformation fluoresce intensely with ThT. A variety of polyhydroxy, polyanionic, or polycationic materials fail to interact or impede interaction with the amyloid peptides. This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.     

Solid-phase synthesis of oligopurine deoxynucleic guanidine (DNG) and analysis of binding with DNA oligomers

The first stepwise solid-phase synthesis of deoxynucleic guanidine (DNG), a positively charged DNA analog, using controlled pore glass as the solid support is reported. For the first time, purine bases have been incorporated into the DNG oligomer and DNG has been synthesized using a solid-phase method, proceeding in the 3′→5′ direction, that is compatible with the cleavage conditions used in the solid-phase synthesis of DNA. A DNG sequence containing a pentameric tract of adenosine nucleosides has been synthesized and the thermal denaturation temperature of its complexes with complementary thymidyl DNA oligomers was 79°C. Binding of thymidyl DNA oligomers to adenyl DNG oligomers is 2:1, as seen in thymidyl and adenyl DNA triplexes. No binding of adenyl DNG with octameric cytidyl DNA was observed, indicating that the positive charge does not overcome base pairing fidelity.     

Incorporation of positively charged ribonucleic guanidine linkages into oligodeoxyribonucleotides: Development of potent antisense agents

Oligodeoxynucleic acid (21-mer) containing both negatively charged phosphate and positively charged ribonucleic guanidine linkages (RNG/DNA chimera) have been synthesized. DNA binding characteristics and nuclease resistance of RNG/DNA chimeras have been evaluated. Using the bcr-abl oncogene (cause of chronic myeloid leukemia) as a target, the binding of a 21-mer RNG/DNA chimera that includes six RNG's is more than 103.5 stronger than the binding of 21-mer composed solely of DNA.     

Effects of spermidine and ATP on stabilities of chromatosomes and histone H1-depleted chromatosomes

It is shown in our FRET studies that both chromatosomes and histone H1-depleted chromatosomes exist in their arm-closed forms in the absence of spermidine. In the presence of spermidine, however, these two types of structural assemblies are converted into their arm-open forms. In addition, ATP as polyanion is capable of suppressing the polycationic effect of spermidine, thus facilitating re-formation of arm-closed forms of these two types of structural assemblies. Our studies therefore illustrate that conversion between arm-closed and arm-open forms of chromatosomes and histone H1-depleted chromatosomes can be manipulated by varying concentrations of polycationic spermidine and polyanionic ATP.     

Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus (opaE1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5' region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.     

Glycosaminoglycans and a newly purified aortic chondroitin proteoglycan block polycationic modulation of protein phosphatase activity

Recently, we described a bovine aortic phosphatase which we called PCM-phosphatase (polycation modulable) because its activity in vitro can be modulated by polycations such as polylysine and histone-H1 (Di Salvo J, Gifford D, Kokkinakis A. Modulation of aortic protein phosphatase activity by polylysine. Proc Soc Exp Biol Med 177:24-32, 1984). We We suspected that polycationic modulation might be inhibited by polyanionic glycosaminoglycans. Accordingly, an aortic anionic substance was purified by sequential steps including (a) heating aortic extracts at 90 degrees C, (b) precipitation of protein with (NH4)2 SO4, and (c) anionic-exchange chromatography on a Mono Q HR 5/5 column using the Pharmacia fast protein liquid chromatography system. Electrophoresis (polyacrylamide-agarose) of the purified substance revealed one band which stained metachromatically with toluidine blue; however, no staining occurred with Coomassie blue. Electrophoretic mobility increased following proteolytic digestion of the substance with papain. The substance produced concentration-dependent reversal of polylysine-mediated inhibition of myosin light chain dephosphorylation, and it also reversed polylysine-mediated stimulation of phosphorylase phosphatase activity expressed by PCM-phosphatase. Its ability to inhibit or reverse polycationic modulation was abolished after incubation with either chondroitinase AC or chondroitinase ABC. Based on these properties the substance was identified as a chondroitin proteoglycan. Commercially available glycosaminoglycans (heparin and chondroitin sulfates) also reversed polycationic modulation. The results show that modulation of phosphatase activity may be significantly modified by naturally occurring glycosaminoglycans. These studies may also have an important bearing on the purported roles of phosphatase(s) and glycosaminoglycans in calcification of soft tissues.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.     

The essential DNA-binding protein sap1 of Schizosaccharomyces pombe contains two independent oligomerization interfaces that dictate the relative orientation of the DNA-binding domain.

The sap1 gene from Schizosaccharomyces pombe, which is essential for mating-type switching and for growth, encodes a sequence-specific DNA-binding protein with no homology to other known proteins. We have used a reiterative selection procedure to isolate binding sites for sap1, using a bacterially expressed protein and randomized double-strand oligonucleotides. The sap1 homodimer preferentially selects a pentameric motif, TA(A/G)CG, organized as a direct repeat and spaced by 5 nucleotides. Removal of a C-terminal dimerization domain abolishes recognition of the direct repeat and creates a new specificity for a DNA sequence containing the same pentameric motif but organized as an inverted repeat. We present evidence that the orientation of the DNA-binding domain is controlled by two independent oligomerization interfaces. The C-terminal dimerization domain allows a head-to-tail organization of the DNA-binding domains in solution, while an N-terminal domain is involved in a cooperative interaction on the DNA target between pairs of dimers.     

Improved preparation of perallylated cyclodextrins: facile synthesis of cyclodextrin-based polycationic and polyanionic compounds

An improved procedure for the perallylation of cyclodextrins allowed the preparation of O-perallylated alpha-, beta-, and gamma-cyclodextrins in 89, 91, and 88% yields, respectively. These were converted into two cyclodextrin-based functionalized compounds, the polycationic heptakis[2,3,6-tri-O-(6-amino-3-thiahexyl)]-beta-cyclodextrin hydrochloride (3), and the polyanionic heptakis[2,3,6-tri-O-(sodium 5-carboxyl-3-thiapentyl)]-beta-cyclodextrin (4), a potential inhibitor of HIV-1 replication.     

NO signal at the crossroads: polyamine-induced nitric oxide synthesis in plants?

Polyamines, such as spermine, spermidine and putrescine, are ubiquitous polycationic compounds that are produced by almost all living organisms, including plants, animals, fungi and bacteria. Polyamines are multifunctional and interact with polyanionic biomolecules such as DNA or protein. However, despite their potential significance, the polyamine-dependent signal transduction system has not been revealed yet. Ni Ni Tun and colleagues have recently reported a possible linkage between polyamine and nitric oxide (NO), another ubiquitous signalling molecule.     

Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities.

A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the detection of polycationic ribonuclease activity rather than simply the presence of protein. Because use is made of the catalytic properties of ribonucleases, polynucleotide/polyacrylamide-gel electrophoresis is apparoximately 10(5) times more sensitive for the detection of these enzymes than conventional gel electrophoresis with the use of protein-staining dyes. Initial studies showed that the poor migration, in the gels, of highly charged polycationic ribonucleases in the presence of negatively charged synthetic polynucleotides could be overcome by high concentrations of spermine. The positively charged polyamine, by neutralizing the polyanionic polynucleotide, enabled these basic enzymes to migrate considerable distances in the gel. Electrophoresis of the RNAases under conditions of low pH, and incubation of the gel at neutral pH followed by staining for polynucleotide, resulted in coloured gels containing clear bands that define regions of enzyme activity. Alterations in spermine concentration or substrate identity caused changes in the positions of these bands, suggesting a dynamic interaction among the enzyme, polyamine and polynucleotide. Because of the advantages, in terms of selectivity and sensitivity of polynucleotide/polyacrylamide-gel electrophoresis, this technique was used to demonstrate the nuclease homogenity of three purified bovine muscle enzymes, and to compare these enzymes with each other, as well as with bovine pancreatic ribonuclease A.     

Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions.

Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration. The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group. This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA. We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions. Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining. However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions. The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity. A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position. Single-stranded DNA served as an in vitro integration target for HIV IN. The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated.     

The highly variable pentameric repeats of the AT-rich germline limited DNA in Parascaris univalens are the telomeric repeats of somatic chromosomes.

We have characterized the organization of the germline limited DNA of P. univalens by means of sequence analysis. The repeat unit of this satellite DNA is the pentanucleotide 5'TTGCA, although there is a high degree of sequence variation. Repeat variants are not arranged in tandem but in a disperse, nonrandom manner. In the somatic genome which arises from the germline genome through extensive genomic rearrangement early in development, copies of these pentamers represent the telomeric repeats, indicated by their sensitivity to Bal 31 and their presence in a somatic endlibrary. Unlike telomeric sequences from other species the P. univalens telomeres do not display consecutive guanines and no strand bias for that base, recently suggested as universal features of eukaryotic telomeres. Investigation of fragments that carry pentameric repeats along with sequences of different type identifies a 5 bp consensus sequence at the junction point. We suggest a model in which pentameric repeats originate via amplification by a terminal transferase (telomerase) in both the germline and the somatic genome.     

Chemical characterization and purification of the beta subunit of the DNA polymerase III holoenzyme from an overproducing strain

We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.     

Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus.

Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration.     

Ribonucleic guanidine demonstrates an unexpected marked preference for complementary DNA rather than RNA

Replacement of the phosphodiester linkages of DNA and RNA by guanidinium linkages provides DNG and RNG. We report here the order of stability of mixed duplexes (RNG-U5.DNA-A5>RNA-U5.RNA-A5>RNG-U5.RNA-A5>RNA-U5.DNA-A5>DNA-T5.DNA-A5). The considerable stability of RNG.DNA compared to RNG.RNA is shown to be due to the rigid backbone of RNG existing only in B-form and therefore lowering its affinity for A-RNA. RNG oligomers are putative antigene agents which are specific for DNA and would have minimal competitive binding to ncRNA.     

Effect of the methyl group on DNA bending and curvature: structure of d(GA4U4C)2 in solution

NMR studies on d(GA4T4C)2 and d(GT4A4C)2 indicated two important factors that contribute to intrinsic DNA bending in polymers containing A/T tracts [Sarma, M.H., et al. (1988) Biochemistry 27, 3423; Gupta, G., et al. (1988) Biochemistry 27, 7909]. They are (i) propeller-twisted A.T pairs with associated bifurcated H bonds inside the A/T tract and (ii) the base sequence that joins the two neighboring A/T tracts. As an extension of our bending project, we carried out quantitative NMR studies on the decamer d(GA4U4C)2, a structural analogue of d(GA4T4C)2, to examine the effect of the methyl group on DNA bending. On the basis of quantitative NMR analysis, we arrive at the following results. (i) The decamer d(GA4U4C)2 adopts the gross morphology of a right-handed B-DNA duplex with A and U nucleotides belonging to C2'-endo,anti domain. (ii) A.U pairs are propeller twisted and hence can result in an array of interstrand bifurcated H bonds involving N6 of A and O4 of U (one base pair apart) inside the A/U tract. (iii) The orientations of A and U with respect to the long axis of the molecule are different; as a result, at the A5-U6 sequence that joins the two A/U tracts, two neighboring frames of reference do not exactly coincide in space and a junction is created at A5-U6. (iv) Inside the A/U tract, intrastrand stacking is more compact (average separation between secessive base planes being 3.2 A) than at the A5-U6 junction, where average separation between the base planes of A5 and U6 is 3.6 A.(ABSTRACT TRUNCATED AT 250 WORDS)