Kinetic analysis of artificial peptide self-replication. Part II: the heterochiral case

A kinetic model has been designed to describe and to analyze the stereoselective behavior of a recently discovered heterochiral template-directed peptide self-replicator by Ghadiri and co-workers [Nature 409 (2001) 797-801]. It turned out that previous assumptions stating that exclusively homochiral species participate in a stereoselective and autocatalytic pathway and that heterochiral species originate only from uncatalyzed background reactions could not be validated by our model. On the contrary, excellent fitting of experimental data indicated that the whole combinatorial variety of possible cross-catalytic processes involving L- and D- peptide species play an important role and need to be taken into account. The system shows no net creation of chiral matter but only a redistribution of the initially present chiral material. Both, the separation of an optically inactive meso-type template dimer and a slight chiroselective autocatalytic effect, contribute to a predicted amplification of enantiomeric excess that, in some cases, can simultaneously result in a substantial amount of optically active matter.     

Expression of cholera toxin under non-AKI conditions in Vibrio cholerae El Tor induced by increasing the exposed surface of cultures

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.     

Kinetic Analysis of Self-Replicating Peptides: Possibility of Chiral Amplification in Open Systems

A simplified kinetic model scheme is presented that addresses the main reactions of two recently reported peptide self-replicators. Experimentally observed differences in the autocatalytic efficiency between these two systems-- caused by variations in the peptide sequences--and the possible effect of chiral amplification under heterochiral reaction conditions were evaluated. Our numerical simulations indicated that differences in the catalytic performance are exclusively due to pronounced variations in the rate parameters that control the reversible and hydrophobic interactions in the reaction system but neither to alterations in the underlying reaction network nor to changes in the stoichiometry of the involved aggregation processes. Model predictions further demonstrated the possible existence of chiral amplification if peptide self-replication is performed under heterochiral reaction conditions. Pointing into the direction of a possible cause for biomolecular homochirality, it was found that in open flow reactors, keeping the system under non-equilibrium conditions, a remarkable amplification of enantiomeric excess could be achieved. According to our modeling, this is due to a chiroselective autocatalytic effect and a meso-type separation process both of which are assumed to be intrinsic for the underlying dynamics of heterochiral peptide self-replication.     

Lipid modification during cytodifferentiation of Tetrahymena vorax. Whole cell phospholipids and triacylglycerols of microstomal and macrostomal phenotypes

Microstomal cells of the ciliate Tetrahymena vorax V2S can be induced to undergo cytodifferentiation to form an alternate phenotype known as the macrostomal cell; however, sublines of T. vorax exist that respond differently to methods that induce macrostomal cell formation. The phospholipid- and triacylglycerol-bound fatty acid compositions of microstomal and macrostomal cells of a high-transforming subline (designated 3-C) were determined and compared to similar data from cells of a low-transforming subline (designated Ala). Differences in fatty acid composition were found between the two phenotypes as well as between the different sublines. Some change in the distribution of radioactive acetate and lauric acid into phospholipid classes of the different subline was observed, and evidence was also obtained that indicated changes in the relative amounts of the sterol-like pentacyclic triterpenoid tetrahymanol. A limited analysis of the lipid composition of stomatin revealed the presence of small amounts of tetrahymanol, phospholipid and free fatty acid. Stomatin is the naturally produced material obtained from T. pyriformis that triggers differentiation in T. vorax. The existence of a low-transforming subline provides a powerful experimental tool for elucidating the underlying biochemical and molecular mechanisms that control cytodifferentiation in T. vorax and possibly in other eukaryotic cells.     

Polar Profile of Antiviral Peptides from AVPpred Database

Diseases of viral origin in humans are among the most serious threats to health and the global economy. As recent history has shown the virus has a high pandemic potential, among other reasons, due to its ability to spread by air, hence the identification, investigation, containment, and treatment of viral diseases should be considered of paramount importance. In this sense, the bioinformatics research has focused on finding fast and efficient algorithms that can identify highly toxic antiviral peptides and to serve as a first filter, so that trials in the laboratory are substantially reduced. The work presented here contributes to this effort through the use of an algorithm already published by this team, called polarity index method, which identifies with high efficiency antiviral peptides from the exhaustive analysis of the polar profile, using the linear sequence of the peptide. The test carried out included all peptides in APD2 Database and 60 antiviral peptides identified by Kumar and co-workers (Nucleic Acids Res 40:W199–204, 2012), to build its AVPpred algorithm. The validity of the method was focused on its discriminating capacity so we included the 15 sub-classifications of both Databases.     

Localization by indirect immunofluorescence of tetrin, actin, and centrin to the oral apparatus and buccal cavity of the macrostomal form of Tetrahymena vorax

ABSTRACT. We have taken advantage of the size of the macrostomal oral apparatus of Tetrahymena vorax to investigate the immunofluorescent localization of three cytoskeletal proteins—tetrin, actin, and centrin. Tetrin and actin antibodies co-localize to cross-connectives that anchor the membranelles. These antibodies also recognize the coarse filamentous reticulum, a filament associated with the undulating membrane. Actin-specific localization extends beyond the coarse filamentous reticulum-undulating membrane complex into a region called the specialized cytoplasm. A centrin antibody localizes to the fine filamentous reticulum which, along with micro-tubules of the oral ribs, circumscribes the cytostomal opening. Models of phagocytic contraction based on these data are presented.     

FIP Position Paper on Qualification of Paddle and Basket Dissolution Apparatus

The qualification process for ensuring that a paddle or basket apparatus is suitable for its intended use is a highly debated and controversial topic. Different instrument qualification and suitability methods have been proposed by the pharmacopeias and regulatory bodies. In an effort to internationally harmonize dissolution apparatus suitability requirements, the International Pharmaceutical Federation's (FIP) Dissolution/Drug Release Special Interest Group (SIG) reviewed current instrument suitability requirements listed in the US, European, and Japanese pharmacopeias and the International Conference on Harmonization (ICH) Topic Q4B on harmonization of pharmacopoeial methods, in its Annex 7, Dissolution Test General. In addition, the SIG reviewed the Food and Drug Administration (FDA) Draft Guidance for Industry, “The Use of Mechanical Calibration of Dissolution Apparatus 1 and 2—Current Good Manufacturing Practice (CGMP)” and the related ASTM Standard E2503-07. Based on this review and several in-depth discussions, the FIP Dissolution/Drug Release SIG recommends that the qualification of a dissolution test instrument should be performed following the calibration requirements as indicated in the FDA (draft) guidance. If additional system performance information is desired, a performance verification test using US Pharmacopeia Reference Standard tablet or an established in-house reference product can be conducted. Any strict requirement on the use of a specific performance verification test tablet is not recommended at this time.