Antibody-based diagnosis of small ruminant lentivirus infection in seminal fluid.

Antibody-based diagnosis of small ruminant lentiviruses (SRLVs) has been efficiently achieved using serum and milk, but not semen, for which polymerase chain reaction (PCR) has been proposed as a confirmatory technique. This work, involving 296 ovine (Ovis aries) and caprine (Capra hircus) semen donors, investigates whether seminal fluid (SF) can be reliably used in antibody-based SRLV diagnosis. First, a gold standard was established to assess the infection status and determine the sensitivity and specificity of three commercial enzyme-linked immunosorbent assays (ELISAs) in serum testing using Western blot and PCR as confirmatory tests. For SF testing, both gold standard and serum testing results were used as reference. The performance of SF testing was affected not only by the ELISA assay sensitivity (related to antigen spectrum) compared with that of the gold standard (as it occurred in serum testing) but also by SF sample quality and SF working dilution. Nonturbid SF samples, commonly collected in artificial insemination centers (AICs), were required. Compared with serum, SF testing had a decreased sensitivity in two of the ELISA assays (with original serum working dilutions

4-Guanidino-2-pyrimidinone nucleobases: synthesis and hybridization properties.

N-Alkylated 4-guanidino-2-pyrimidinone-containing nucleosides, in which the guanidine group mimics the double hydrogen bond donor pattern of protonated cytosine, were introduced in polypyrimidine sequences to explore their triple-helix forming capabilites. UV and CD melting experiments showed that strands containing these base analogues did not form triplex complexes.

Synthesis of thymine-modified oligonucleotides.

Oligonucleotide-resins containing N-nitrothymidine residues yield N3-thymine modified oligonucleotides by reaction with a variety of amines followed by the standard ammonia treatment.

Towards a better understanding of the cisplatin mode of action.

We have studied how platinum(II) complexes [Pt(dien)Cl]Cl, [Pt(en)Cl2] and cisplatin react with hybrid molecules that contain sulfur and nitrogen ligands, in particular Phac-Met-linker-p5'dG (Phac = phenylacetyl), Phac-His-linker-p5'dG, Phac-His-Met-linker-p5'dG and Phac-His-Gly-Met-linker-p5'dCATGGCT. The progress of the reactions was monitored by HPLC, and by [1H,15N]-HSQC NMR when 15N-cisplatin was used. The products were isolated and characterised by using enzymatic and chemical reactions and spectroscopic techniques (UV and/or NMR spectroscopy, electrospray or MALDI-TOF mass spectrometry). The combined use of digestion with proteases and reaction with hydrogen peroxide followed by mass spectrometric analysis indicated the platinum coordination positions on the peptide moiety of the largest hybrid. Monofunctional Pt-S adducts were transformed into Pt-N complexes in which Pt-N7 bonds were formed preferentially. Most of the chelates isolated had Pt-S bonds, and, in the case of cisplatin complexes, loss of the ammine trans to sulfur gave rise to the formation of tricoordinate species with platinum-mediated peptide-nucleotide cross-links. 1,2-Intrachain platinum GpG adducts were only obtained in very small amounts (1-4%).

Study of the interaction between a histidine-deoxyguanosine hybrid and cisplatin.

A histidine-2'-deoxyguanosine hybrid, Phac-Hse(p5'dG)-His-OH (I), was synthesized, and its reaction with cisplatin was monitored by reversed-phase HPLC and 1HNMR. Two new compounds, II and III, were observed to be simultaneously formed, II in larger proportion than III. These products were isolated after HPLC purification and extensively characterized. Both II and III contained platinum, had the same mass and showed a bathochromic displacement of their absorption maxima with respect to that of I. Both remained undegraded upon enzymatic digestion and yielded I when treated with NaCN. From these data and the information provided by 1HNMR analysis, we inferred that II and III were constitutional isomers, in particular chelates in which platinum was coordinated to the N7 of guanine and either the N pi or the N tau imidazole nitrogens, respectively. No preference of the metal for either of these N-donors was observed.

Synthesis and enzymatic stability of phosphodiester-linked peptide-oligonucleotide hybrids.

Nucleopeptides Ac-Tyr(p3' dACGT)-Ala-Phe-Gly-NH2, Ac-Thr(p3'dACGT)-Ala-Phe-Gly-OH, Ac-Ser(p3'dACGT)-Ala-Phe-Gly-OH, and Phac-Hse(p3'dACGT)-Ala-Phe-Gly-OH, in which the 3'-end of a tetradeoxyribonucleotide is linked by a phosphodiester bond to a hydroxylated amino acid, were synthesized using a stepwise solid-phase methodology to study the influence of the linking amino acid on their stability to 3'-exonucleases. HPLC analysis of the reaction crudes after treatment of each nucleopeptide with snake venom phosphodiesterase showed that the lability of the amino acid-nucleoside linkage increases in the order Thr < Ser < Hse < Tyr.

The bi-loop, a new general four-stranded DNA motif.

The crystal structure of the cyclic octanucleotide d contains two independent molecules that form a novel quadruplex by means of intermolecular Watson-Crick A.T pairs and base stacking. A virtually identical quadruplex composed of G.C pairs was found by earlier x-ray analysis of the linear heptamer d(GCATGCT), when the DNA was looped in the crystal. The close correspondence between these two structures of markedly dissimilar oligonucleotides suggests that they are both examples of a previously unrecognized motif. Their nucleotide sequences have little in common except for two separated 5'-purine-pyrimidine dinucleotides forming the quadruplex, and by implication these so-called "bi-loops" could occur widely in natural DNA. Such structures provide a mechanism for noncovalent linking of polynucleotides in vivo. Their capacity to associate by base stacking, demonstrated in the crystal structure of d(GCATGCT), creates a compact molecular framework made up of four DNA chains within which strand exchange could take place.

Solid-phase synthesis of a nucleopeptide from the linking site of adenovirus-2 nucleoprotein, -Ser(p5'CATCAT)-Gly-Asp-. Convergent versus stepwise strategy.

The synthesis of a nucleopeptide with the sequence -Ser(p5'CATCAT)-Gly-Asp- has been undertaken by either convergent or stepwise solid-phase strategies, both of which use base-labile permanent protecting groups. The coupling of phosphitylated protected peptides onto oligonucleotide-resins did not afford the desired nucleopeptide, which was nevertheless obtained after oligonucleotide elongation at the hydroxyl group of the resin-bound peptide and deprotection under mild basic conditions. A preliminary study on the stability of different nucleopeptides to bases is also reported.

Preparation of an aspartic acid-containing protected peptide. Alternatives for overcoming a side reaction during HF treatment.

Treatment of the peptide-resin Ac-Asn-Ser(Bzl)-Gly-Asp(OFm)-pMeBHA with HF/anisole cleaves the peptide-resin bond and removes the benzyl group, as expected, but also yields an impurity (ca. 25%) in which the benzyl group is linked to the fluorenylmethyl protecting group. Separation of the desired peptide from the impurity can be accomplished by reversed-phase medium-pressure liquid chromatography. The side reaction does not take place when the gamma-carboxyl of aspartic acid is protected with the 2-(4-acetyl-2-nitrophenyl)ethyl group.

Reversed-phase high-performance liquid chromatography of protected peptide segments.

There is little evidence that reversed-phase high-performance liquid chromatography can be successfully used in the analysis of protected peptide segments. The use of C18 and CN packings and mobile phases containing water-acetonitrile with or without propionic acid in the separation of complex mixtures of synthetic protected peptides is reported. CN packings show a lower efficiency and exhibit poorer resolution than C18 packings but provide different separations. The addition of propionic acid to the mobile phase increases the retention time of peptides but also provides dramatic and useful changes in selectivity. Retention is not related to the molecular mass of the protected peptides but mainly to their hydrophobicity.

Amino-acids condensations in the preparation of N alpha-9-fluorenylmethyloxycarbonylamino-acids with 9-fluorenylmethylchloroformate.

Synthesis of N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) amino-acids by reaction of free amino-acids (glycine and alanine) with 9-fluorenylmethylchloroformate leads to formation of small amounts of Fmoc-dipeptide which are difficult to eliminate by crystallization. The alternative way to prepare Fmoc-amino-acids by reacting the Fmoc-chloride first with sodium azide and then with the free amino-acid eliminates this side reaction, at least for glycine and alanine.