Intermolecular hydrogen bonding of the two independent molecules of N-3,5-dinitrobenzoyl-L-leucine.

The title compound, C(13)H(15)N(3)O(7), crystallizes as two independent molecules which differ in their conformation. Intermolecular hydrogen bonding between the amide and carboxylic acid groups as N-H.O=C interactions results in the formation of one-dimensional chains with N.O distances of 2.967 (6) and 3.019 (6) A. Neighbouring chains are linked by C=O.H-O interactions to form a two-dimensional network, with O.O distances of 2.675 (6) and 2.778 (6) A.

SP-303, an antiviral oligomeric proanthocyanidin from the latex of Croton lechleri (Sangre de Drago).

SP-303, a large proanthocyanidin oligomer isolated from the latex of the plant species Croton lechleri (Eupborbiaceae) has demonstrated broad activity against a variety of DNA and RNA viruses. In cell culture, SP-303 exhibits potent activity against isolates and laboratory strains of respiratory syncytial virus (RSV), influenza A virus (FLU-A) and parainfluenza virus (PIV). Parallel assays of SP-303 and ribavirin showed comparable activity against these viruses. SP-303 also exhibits significant inhibitory activity against herpesvirus (HSV) types 1 and 2, including herpesviruses resistant to acyclovir and foscarnet. Inhibition was also observed against hepatitis A and B viruses. The antiviral mechanism of SP-303 seems to derive from its direct binding to components of the viral envelope, resulting in inhibition of viral attachment and penetration of the plasma membrane. Antiviral effects of SP-303 were measured by three distinct methods: CPE, MTT and precursor uptake/incorporation. Cytotoxicity endpoints were markedly greater than the respective antiviral endpoints. SP-303 exhibited activity in RSV-infected cotton rats and African green monkeys, PIV-3-infected cotton rats, HSV-2 infected mice and guinea pigs and FLU-A-infected mice. The most successful routes of SP-303 administration for producing efficacy were: topical application to HSV-2- genital lesions in mice and guinea pigs, aerosol inhalation to FLU-A-infected mice and PIV-3-infected cotton rats, and oral dosage to RSV-infected cotton rats. A variety of toxicological evaluations demonstrated the safety of SP-303, particularly orally, which was predictable, since condensed tannins are a common dietary component. It is notable that the larger proanthocyanidins as a class have high antiviral activity, whereas most of the monomers are inactive. Clinical trials are ongoing to evaluate SP-303 as a therapeutic antiviral agent.

Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra.

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimer's disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimer's disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.

The FMRFamide-related decapeptide of Mytilus contains a D-amino acid residue.

1. An FMRFamide-related decapeptide isolated from the anterior byssus retractor muscle (ABRM) of the bivalve mollusc, Mytilus edulis, was shown to have D-Leu as the second amino acid residue. 2. The excitatory effects of the peptide (Mytilus-FFRFamide) on the ABRM were not changed appreciably by substituting an L-Leu residue for the D-Leu residue.

Covalent modification of Alzheimer's amyloid beta-peptide in formic acid solutions.

beta-peptide is a normal component of amyloid deposits in Alzheimer's disease. In aqueous solution, beta-peptide is extremely insoluble and rapidly aggregates forming oligomeric beta-sheet structures that eventually precipitate from solution. Presumably, this process is related to the production of amyloid deposits in Alzheimer's disease. Formic acid is commonly used to dissolve the beta-peptides and prevent aggregation in biological and biophysical studies. However, a side-reaction which covalently modifies beta-peptide is encountered with formic acid. In this report, fast atom bombardment mass spectrometry and tandem mass spectrometry demonstrate that both Ser8 and Ser26 become O-formylated in 70% aqueous formic acid solutions. The implications of this O-formylation upon the aggregational properties of beta-peptide are discussed.

Tandem mass spectrometric analysis of peptides at the femtomole level.

In the sequential analysis of peptides by fast atom bombardment (FAB) tandem mass spectrometry (MS/MS), the principal obstacle to decreasing sample quantities was determined to be the signal-to-background ratio of the ionization/desorption process. By decreasing the background ion current, continuous-flow FAB allows complete analysis (both MS and MS/MS) to be performed on 25-75% less sample than required for a conventional FABMS/MS experiment alone. The combination of CF-FAB with array detection permitted sequential analysis of several peptides (900-2000 Da) at the 900 fmol to 5.8 pmol level, without interference from the background. These levels do not produce a molecular ion species easily discernible above the background in conventional FAB.

Fragmentation studies of peptides: the formation of y ions.

The mechanism for the formation of y ions in the collision-induced dissociation (CID) spectra of protonated peptides produced by fast-atom bombardment was investigated by tandem mass spectrometry and deuterium labelling studies. The results show that a hydrogen atom attached to nitrogen and not to carbon migrates during cleavage of the amide bond. A mechanism based on these results is presented.

Mytilus-inhibitory peptide analogues isolated from the ganglia of a pulmonate mollusc, Achatina fulica.

1. Ten peptides that showed an inhibitory effect on phasic contraction of the ABRM of Mytilus were isolated from the ganglia of the African giant snail, Achatina fulica. 2. Seven of the peptides were shown to be hexapeptides having -Pro-Xaa-Phe-Val-NH2 as a common structure, which was previously shown to be a characteristic of Mytilus inhibitory peptides (MIPs). 3. The remaining three were pentadecapeptides, each of which consisted of two MIP-related hexapeptides linked by -Gly-Arg-Arg-.

Novel hypotensive peptides from the body of Elaphe climacophora.

Four hypotensive peptides called elapherine-A, -B, -C and -D were isolated from the body of Elaphe climacophora after removal of the internal organs. Elapherine-A, which had the lowest molecular weight, exhibited a prolonged fall for 5 min in the blood pressure of spontaneously hypertensive rats, whereas elapherin-B, -C and -D displayed a transient fall in the blood pressure. The molecular weights of elapherine-A---D estimated from sodium dodecyl sulfate (SDS) gel disk electrophoresis were 6600, 6900, 7000 and 7200, respectively. The isoelectric points of these peptides were 10.4, 10.6, 10.7 and 10.5, respectively. They possessed a single polypeptide chain of 34-35 (elapherine-A), 56-58 (elapherine-B), 56-57 (elapherine-C) and 52-53 (elapherine-D) amino acid residues.

Philanthotoxins. A mass spectrometric investigation.

A method employing liquid secondary-ion mass spectrometry (SIMS) in conjunction with metastable-ion measurements (linked scanning at constant B/E) to obtain sequence-specific information for three synthetic polyamine isomers was developed. The normal liquid SIMS spectra gave molecular weight information, but important sequence ions were of low intensity or obscured by the background. The metastable-ion spectra contained important fragment ions in particular due to cleavage along the polyamine chain. One of the three synthetic isomers was identical with a toxin present in the venom of the digger wasp. In conjunction with nuclear magnetic resonance spectroscopic studies, this should be a powerful method for the structural characterization of other closely related toxins present in the venom of this wasp.

Achatin-I, an endogenous neuroexcitatory tetrapeptide from Achatina fulica Férussac containing a D-amino acid residue.

A tetrapeptide named achatin-I was purified from the suboesophageal and cerebral ganglia of the African giant snail Achatina fulica Férussac, and evoked a potent neuroexcitatory effect. The amino acid sequence of achatin-I is Gly-D-Phe-Ala-Asp. Achatin-I induced a voltage-dependent inward current, due to Na+, on the identifiable giant neuron, periodically oscillating neuron (PON), of the same snail. All possible isomers of achatin-I were synthesized using the solid-phase method. The sensitivity of the neuron to achatin-I and its isomers was strictly stereospecific; among the various isomers, only achatin-I showed marked effects (ED50 = 2.29 x 10(-6)M), while Gly-D-Phe-D-Ala-Asp, the synthetic D-Ala-isomer, was less than 10(-3) active.

Free sialooligosaccharides found in the unfertilized eggs of a freshwater trout, Plecoglossus altivelis. A large storage pool of complex-type bi-, tri-, and tetraantennary sialooligosaccharides.

Unfertilized eggs of Plecoglossus altivelis (a kind of freshwater trout, "Ayu" in Japanese) were found to contain a relatively large pool of three sialooligosaccharides. These free oligosaccharides were accumulated in the cytoplasm up to concentrations of about 35 ng per egg, which correspond to about 107 micrograms/g. Their structures were determined by a combination of chemical (composition and methylation analysis and Smith degradation) and instrumental (fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and 400-MHz1H NMR) analyses. The structures established represent a typical type of bi-, tri-, and tetraantennary sialooligosaccharides all of which end with di-N-acetylchitobiose structure (-GlcNAc beta 1-4GlcNAc) and alpha -2,3-linked NeuAc at reducing and nonreducing termini, respectively. These data show that these free sialooligosaccharides, which were originally protein-linked components, must be released during oogenesis. Although a specific functional requirement for their release is not known, the occurrence of such free sialooligosaccharides in normal animal cells or tissues has not been previously reported.

Chemical association in symbiosis sterol donors in planthoppers.

The role of intracellular symbionts contributing to their host has been investigated in the planthoppers,Nilaparvata lugens Stal andLaodelphax striatellus Fallen. We have found that the isolated yeastlike symbionts, identified as a member of the genusCandida, from the host's egg produce ergosterol when cultured. A comparative study of sterols in the cultured symbionts, the host insects, aposymbiotic host insects, and dietary plants demonstrated that ergosterol produced in the symbiotes is provided to the host insects and possibly transformed in the host insects into cholesterol via 24-methylenecholesterol. The conversion of injected 24-methylenecholesterol-d3 into cholesterol has been shown in the brown planthopper (N. lugens).