Mechanism of action of choleragen

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside G_M1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A₁ fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A₁ fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.     

Group mating among Norway rats I. Sex differences in the pattern and neuroendocrine consequences of copulation

The copulatory pattern of groups of rats (Rattus norvegicus) was studied in the laboratory in a seminatural environment. In a given mating session, every oestrous female copulated with each male; likewise, every male copulated with each oestrous female. While individual males and females experienced similar amounts of copulation, there were dramatic sex differences in sequence and temporal pattern. Males mated in a multiple intromission pattern and had more ejaculatory series when several females were in oestrus. In contrast, females received intromissions and ejaculations in a random order, not in the sequence of a male ejaculatory series. Males copulated at shorter intervals than females did, a temporal sex difference that was determined by the pattern of female solicitations and male approaches. These sex differences are used to discuss the different units of analysis that are appropriate for male and female sexual behaviour in this species. Furthermore, the sex differences in the temporal pattern of copulation which emerged during group mating parallel the known sex differences in the temporal parameters of the neuroendocrine reflexes which mediate successful reproduction in the domestic strain.     

Group mating among Norway rats II. The social dynamics of copulation: Competition,cooperation, and mate choice

The social interactions within groups of Norway rats (Rattus norvegicus) had a strong impact on the individual pattern of copulation which, in turn, affects sperm precedence and the probability of implantation in this species. Males alternated uninterrupted ejaculatory series, augmenting each others' copulatory investment. Females took turns mating after receiving an intromission, collectively potentiating the males' copulatory behaviour; increasing the number of oestrous females increased the number of intromissions and ejaculations achieved by each male but did not affect the amount of copulation experienced by each female. These turn-taking patterns within each sex provided the opportunity to change partners and permitted the emergence of different sex-typical patterns of copulation. Furthermore, the dominant male contributed more intromissions and tended to give each female more ejaculations than the subordinates did. Dominant males were also more likely to inhibit the subordinates' sperm transport. Females competed among themselves for the opportunity to mate with a male as he approached ejaculation and were likely to protect more of the dominant male's sperm transport than the subordinate male's.     

Chemical and Physical Characterization of Interfacial-Active Lipids from Rhodococcus erythropolis Grown on n-Alkanes

Lipophilic compounds of the culture suspension containing Rhodococcus erythropolis DSM43215 had surfactant properties when the bacteria were cultivated with n-alkanes as the sole carbon source. Thirteen main components from a dichloromethane-methanol extract of the R. erythropolis cultures were isolated and characterized to specify quantitatively their surfactant properties, e.g., minimum surface and interfacial tensions and critical micelle concentrations. The interfacial activity of the organic extract was dominated by α,α-trehalose-6,6′-dicorynomycolates which reduced interfacial tension from 44 to 18 mN/m. Phosphatidylethanolamines which were also present in the organic extract reduced the interfacial tension below 1 mN/m. The trehalose corynomycolates had extremely low critical micelle concentrations in high-salinity solutions, and the interfacial properties were stabile in solutions with a wide range of pH and ionic strength.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status

The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.     

Evolutionary theory and systematics: relationships between process and patterns

The relevance of the Modern Evolutionary Synthesis to the foundations of taxonomy (the construction of groups, both taxa and phyla) is reexamined. The nondimensional biological species concept, and not the multidimensional, taxonomic, species notion which is based on it, represents a culmination of an evolutionary understanding. It demonstrates how established evolutionary mechanisms acting on populations of sexually reproducing organisms provide the testable ontological basis of the species category. We question the ontology and epistemology of the phylogenetic or evolutionary species concept, and find it to be a fundamentally untenable one. We argue that at best, the phylogenetic species is a taxonomic species notion which is not a theoretical concept, and therefore should not serve as foundation for taxonomic theory in general, phylogenetics, and macroevolutionary reconstruction in particular. Although both evolutionary systematists and cladists are phylogeneticists, the reconstruction of the history of life is fundamentally different in these two approaches. We maintain that all method, including taxonomic ones, must fall out of well corroborated theory. In the case of taxonomic methodology the theoretical base must be evolutionary. The axiomatic assumptions that all phena, living and fossil, must be holophyletic taxa (species, and above), resulting from splitting events, and subsequently that evaluation of evolutionary change must be based on a taxic perspective codified by the Hennig ian taxonomic species notion, are not testable premises. We discuss the relationship between some biologically, and therefore taxonomically, significant patterns in nature, and the process dependence of these patterns. Process-free establishment of deductively tested “genealogies” is a contradiction in terms; it is impossible to “recover” phylogenetic patterns without the investment of causal and processual explanations of characters to establish well tested taxonomic properties of these (such as homologies, apomorphies, synapomorphies, or transformation series). Phylogenies of either characters or of taxa are historical-narrative explanations (H-N Es), based on both inductively formulated hypotheses and tested against objective, empirical evidence. We further discuss why construction of a “genealogy”, the alleged framework for “evolutionary reconstruction”, based on a taxic, cladistic outgroup comparison and a posteriori weighting of characters is circular. We define how the procedure called null-group comparison leads to the noncircular testing of the taxonomic properties of characters against which the group phylogenies must be tested. This is the only valid rooting procedure for either character or taxon evolution. While the Hennig -principle is obviously a sound deduction from the theory of descent, cladistic reconstruction of evolutionary history itself lacks a valid methodology for testing transformation hypotheses of both characters and species. We discuss why the paleontological method is part of comparative biology with a critical time dimension ana why we believe that an “ontogenetic method” is not valid. In our view, a merger of exclusive (causal and interactive, but best described as levels of organization) and inclusive (classificatory) hierarchies has not been accomplished by a taxic scheme of evolution advocated by some. Transformational change by its very nature is not classifiable in an inclusive hierarchy, and therefore no classification can fully reflect the causal and interactive chains of events constituting phylogeny, without ignoring and contradicting large areas of corroborated evolutionary theory. Attempts to equate progressive evolutionary change with taxic schemes by Haeckel were fundamentally flawed. His ideas found 19th century expression in a taxic perception of the evolutionary process (“phylogenesis”), a merger of typology, hierarchic and taxic notions of progress, all rooted in an ontogenetic view of phylogeny. The modern schemes of genealogical hierarchies, based on punctuation and a notion of “species” individuality, have yet to demonstrate that they hold promise beyond the Haeckel ian view of progressive evolution.