Mechanism of methaemoglobin breakdown by the lysine-specific gingipain of the periodontal pathogen Porphyromonas gingivalis

Abstract The R- and K-gingipain proteases of Porphyromonas gingivalis are involved in proteolysis of haemoglobin from which the defensive dimeric haem pigment is formed. Whilst oxyhaemoglobin is refractory towards K-gingipain, methaemoglobin is rapidly degraded. Ligation of methaemoglobin with N3-, which effectively blocks haem dissociation from the protein, prevented haemoglobin breakdown. Haem-free globin was rapidly degraded by K-gingipain. These data emphasise the need for haemoglobin oxidation which encourages haem dissociation and makes the haem-free globin susceptible to proteolytic attack.     

Mixed model approaches for the identification of QTLs within a maize hybrid breeding program

Two outlines for mixed model based approaches to quantitative trait locus (QTL) mapping in existing maize hybrid selection programs are presented: a restricted maximum likelihood (REML) and a Bayesian Markov Chain Monte Carlo (MCMC) approach. The methods use the in-silico-mapping procedure developed by Parisseaux and Bernardo (2004) as a starting point. The original single-point approach is extended to a multi-point approach that facilitates interval mapping procedures. For computational and conceptual reasons, we partition the full set of relationships from founders to parents of hybrids into two types of relations by defining so-called intermediate founders. QTL effects are defined in terms of those intermediate founders. Marker based identity by descent relationships between intermediate founders define structuring matrices for the QTL effects that change along the genome. The dimension of the vector of QTL effects is reduced by the fact that there are fewer intermediate founders than parents. Furthermore, additional reduction in the number of QTL effects follows from the identification of founder groups by various algorithms. As a result, we obtain a powerful mixed model based statistical framework to identify QTLs in genetic backgrounds relevant to the elite germplasm of a commercial breeding program. The identification of such QTLs will provide the foundation for effective marker assisted and genome wide selection strategies. Analyses of an example data set show that QTLs are primarily identified in different heterotic groups and point to complementation of additive QTL effects as an important factor in hybrid performance.     

Sequential action of R- and K-specific gingipains of Porphyromonas gingivalis in the generation of the haem-containing pigment from oxyhaemoglobin

The arginine- and lysine-specific gingipains of Porphyromonas gingivalis have been implicated in the degradation of haemoglobin from which the black mu-oxo haem dimer-containing pigment is generated. Here, we examined interactions of oxyhaemoglobin (oxyHb) with the Arg-(R)-specific (HRgpA) and Lys-(K)-specific (Kgp) gingipains. Incubation of oxyHb with HRgpA resulted in formation of methaemoglobin (metHb), which could be prevented by the R-gingipain specific inhibitor leupeptin. oxyHb-Kgp interactions resulted in formation of a haemoglobin haemichrome. This was inhibited by the lysine-specific protease inhibitor Z-Phe-Lys-acyloxymethylketone (Z-FKck). metHb, formed by treatment of oxyHb with either NaNO(2) or by pre-incubation with HRgpA, was rapidly degraded by Kgp compared to oxyHb. metHb degradation by Kgp was also inhibited Z-FKck. Together these data show that R-gingipain activity is crucial for converting oxyHb into the metHb form which is rendered more susceptible to Kgp degradation for the eventual release of iron(III) protoporphyrin IX and production of the mu-oxo haem dimer. This explains previous observations [J.W. Smalley, M.F. Thomas, A.J. Birss, R. Withnall, J. Silver, Biochem. J. 379 (2004) 833-840.] of the requirement for a combination of both R- and K-gingipains for pigment production from oxyhaemoglobin by P. gingivalis.     

Zostera marina population genetics in Barnegat Bay, New Jersey, and implications for grass bed restoration

Within Barnegat Bay, New Jersey, Zostera marina populations have declined by 62% over the last 20 years, and restoration efforts have met with mixed success. We have completed a microsatellite-based genetic investigation of eight populations of Z. marina within Barnegat Bay to determine whether the genetic stock origins of the plants used in management projects may affect restoration success. Additionally, we assessed the genetic diversity of Z. marina in Barnegat Bay to better understand its population structure. Clonal diversity ranged from 0.70 to 0.95 for the populations studied. Individually, Barnegat Bay populations are not genetically diverse, and there is also little divergence among populations. The Atlantic populations had mean Hobs values (0.20–0.34) that were far lower than the Hexp values (0.69–0.83). Also, the F _IS values in all of the eastern populations indicate a surfeit of homozygotes over heterozygotes, suggesting a low degree of outcrossing in the Barnegat Bay populations. Six of the ten populations studied (Ham Island, Manahawkin Bay, Shelter Island, Marsh Elder, Harvey Cedar Sedge, and Long Island) show evidence of historical bottlenecks. Mean estimated F _ST values would suggest that most alleles are undergoing moderate genetic differentiation, with values that range from 0.06 to 0.13. Oyster Creek and Sedge Island demonstrate the largest estimated effective population sizes and may be the most appropriate populations for use in future eelgrass restoration projects.     

Study of the structure of troponin-T by measuring the relative reactivities of lysines with acetic anhydride

The structure of troponin-T has been studied by measuring the relative reactivity of lysines with acetic anhydride using a competitive labeling method. Troponin-T was acetylated free and complexed with -I and -C in the native state with [³H]acetic anhydride, purified, and then combined with ¹⁴[C]troponin-T that had been acetylated in 6 m-guanidine · HCl. Peptides containing labeled lysines were isolated following chymotryptic and tryptic digestion and identified in the published sequence. The ${}^3\text{H}{}^{14}\text{C}$ ratio of these peptides was used as a measure of relative accessibility of the lysines. Troponin-T contains 39 lysines; we have identified 35 of these in 22 different peptides. The region of troponin-T influenced by binding to the other troponin components is extensive and includes the C-terminal half of the molecule as well as some residues in the N-terminal half. The lysines showing the greatest change in reactivity are concentrated between residues 114 to 223. The reactivities of the troponin-T lysines labeled in native troponin were not significantly influenced by the binding of calcium to the calcium-specific binding sites of troponin-C. A model for the structure of troponin-T is proposed based on the present and previous studies.     

Lectin-binding activity and antibody response with Pseudomonas paucimobilis and Flavobacterium multivorum

Pseudomonas paucimobilis and Flavobacterium multivorum (formerly Group IIK biotype 1 and biotype 2, respectively) showed lectin-binding activity with Helix aspersa and no activity with eight other well-characterized lectins. Microagglutination titrations and immunodiffusion precipitation revealed specific antibody activity to immunizing antigens. However, no major antigens were found to be common from crude antigen preparations of P. paucimobilis and F. multivorum when tested against opposing antisera.     

Molar Growth Yields as Evidence for Oxidative Phosphorylation in Streptococcus faecalis Strain 10C1

During the aerobic growth of Streptococcus faecalis strain 10C1, with limiting levels of glucose as the substrate, a molar growth yield (Y) of 58.2 g (dry weight) per mole of glucose was obtained. Under these conditions of growth, glucose was dissimilated primarily to acetate and CO(2). The incorporation of (14)C-glucose into cell material was no greater under aerobic conditions than during anaerobic growth. Assuming an adenosine triphosphate coefficient of 10.5, the aerobic Y cannot be explained solely on the basis of substrate phosphorylation and would appear to substantiate previous enzymatic evidence for oxidative phosphorylation in this cytochromeless species. With mannitol as the substrate, an aerobic Y of 64.6 was obtained. Extracts of mannitol-grown cells contained a nicotinamide adenine dinucleotide (NAD)-linked mannitol-1-phosphate (M-1-P) dehydrogenase. The difference in aerobic Y values with mannitol and glucose as substrates would indicate that the in vivo P/O ratio from the oxidation of reduced NAD generated by the oxidation of M-1-P approximates 0.6. The Y values with pyruvate and glycerol as substrates under aerobic conditions were 15.5 and 24.7, respectively.