A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa)

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate.     

Distribution of epiplanktonic Chaetognatha along a transect in the Indian Ocean

Chaetognaths were identified and counted in 23 samples collectedin the west Indian Ocean along a transect from the Gulf of Adento the Cape of Good Hope, in February–March 1967. Althoughthe hydrographic front located at 5°S does not seem to representa barrier for the distribution of the majority of the species,some of them are in a preferential area south or north of thefront. The principal component analysis indicates the locationswhere the more intense overlapping of species takes place. Itis important to mention the occurrence of S.pseudoserratodentatain the proximity of Madagascar Island.     

Immunoregulatory effects of a suppressor factor from healthy pregnant women's lymphocytes after progesterone induction

Progesterone-treated pregnancy lymphocytes release an immunologic blocking factor. The mode of action of this substance was investigated. The supernatant of progesterone-treated pregnancy lymphocytes was highly suppressive of natural cytotoxicity toward human embryonic fibroblast target cells as well as of natural killer cell activity. The effect was not observed when progesterone induction was performed in the presence of RU 486, a progesterone receptor blocking agent. The factor was able to inhibit mixed lymphocyte reactions (MLRs), and transfer coculture experiments revealed that this effect was dependent on major histocompatibility complex nonspecific, nonrestricted suppressor T cells. The activation/expansion of suppressor inducer and suppressor effector T cells was further proved by fluorescence-activated cell sorter analysis of the populations from MLRs cultured in the presence of the inhibitory factor. These changes were not observed with MLRs performed in the presence of supernatants from progesterone + RU 486-treated peripheral blood lymphocytes. The inhibitory material, on the other hand, did not affect either production or function of IL-2. We conclude that in the presence of high local concentrations of progesterone, a suppressive pathway dependent on specific progesterone-CD8+ lymphocyte interaction might be established. This mechanism might play an important role in the maintenance of pregnancy.     

Ultrastructure of the sperm and spermatogenesis and spermiogenesis of Dina lineata (hirudinea,erpobdellidae)

The mature sperm of Dina lineata is of the modified type. The sperm are 48 μm long and 0.3 μm wide. The sperm are filiform and helicoidal cells with a distinct head, a midpiece, and a tail. There are two distinct regions in the head: the acrosome and the posterior acrosome, each with its own characteristic morphology. The midpiece is the mitochondrial region and has a single mitochondrion. Two distinct portions can be observed in the tail: the axonematic region and the terminal piece. In the process of spermatogenesis the early spermatogonia divide to form a poliplast of 512 spermatic cells. In the spermiogenesis the following sequential stages can be distinguished: elongation of the flagellum; reciprocal migration of mitochondria and Golgi complex; condensation of chromatin and formation of the posterior acrosome; spiralization of nuclear and mitochondrial regions; and, finally, formation of the anterior acrosome. The extreme morphological complexity of the Dina spermatozoon is related to the peculiar hypodermal fertilization which characterizes the erpobdellid family. Correlation between sperm morphology and fertilization biology in the Annelida is revised.     

Occurrence of a capsule in Aeromonas salmonicida

Aeromonas salmonicida grown in a medium with excess glucose as carbon source produces both capsular and exocellular polysaccharides. The capsular polysaccharide is composed of glucose, mannose, rhamnose, N-acetylmannosamine and mannuronic acid in the molar ratios of approximately 5:3:0.75:2:1. The extracellular polysaccharide is similarly constituted, but in the molar ratios of approximately 4.75:10.5:1.5:2:1. The capsular and exocellular polysaccharides did not cross-react with monoclonal antibodies against the A-layer or the O-antigen lipopolysaccharide.     

Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects

The mechanism of action of cecropin was studied by using liposomes as a model system. The bilayer was efficiently destroyed if the liposome net charge was zero or negative. Cecropin analogues with an impaired N-terminal helix had reduced membrane disrupting abilities that correlate with their lower antibacterial activity. The reduced bactericidal activity of the analogues was rationalized in terms of reduced binding to bacteria. The stoichiometry of cecropin killing of bacteria suggests that amounts of cecropin sufficient to form a monolayer strongly modify the bacterial membrane. Although some bacteria were resistant to cecropin they did bind large amounts in a non-productive manner. In contrast, mammalian erythrocytes achieve resistance by avoiding the binding of cecropin.     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.