C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by Lactococcus lactis

C nuclear magnetic resonance (C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-C]citrate, [3-C]pyruvate, and [2-C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. alpha-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the alpha-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.     

A robotics-based automated assay for inorganic and organic phosphates.

Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine.     

Diet and locomotion of the armadillo Peltephilus: a new view

The Santacrucian armadillos Peltephilus (Mammalia, Dasypodidae, Peltephilinae) are reanalysed in order to test the traditional hypothesis that they were the most cursorial and active hunters of the dasypodids. The masticatory musculature is reconstructed, the form and distribution of teeth are studied, and a model of the jaw movements is proposed. Furthermore, the limb anatomy as well as their index of fossorial ability are compared with those of other armadillos. It is concluded that Peltephilus , and very likely other peltephilines, are poorly designed as carnivorous, actively cursorial mammals. An alternative habit is proposed, in which Peltephilus and the other peltephilines are viewed as having the conservative armadillo structure of diggers, feeding on soft but tough items, probably plant material of underground origin.     

Development of a robust transient expression screening system in protoplasts of Cannabis

In Vitro Cellular & Developmental Biology - Plant - Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function...     

Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system.     

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F₂(I.4R×I.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

L-methionine degradation pathway in Kluyveromyces lactis: identification and functional analysis of the genes encoding L-methionine aminotransferase

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.     

Synthesis and pharmacological activity of aminoindanone dimers and related compounds

A series of N-substituted 3-aminoindanones were synthesised and evaluated for smooth muscle relaxant activity and mediator release inhibition effects. A low level of smooth muscle relaxant activity has been identified in all derivatives. Data have revealed that the significant mediator release inhibition effects observed are related to the nature of the amine substituents. A structure activity relationship is proposed.     

Two-Fluid Model of Biofilm Disinfection

We consider a dynamic model of biofilm disinfection in two dimensions. The biofilm is treated as a viscous fluid immersed in a fluid of less viscosity. The bulk fluid moves due to an imposed external parabolic flow. The motion of the fluid is coupled to the biofilm inducing motion of the biofilm. Both the biofilm and the bulk fluid are dominated by viscous forces, hence the Reynolds number is negligible and the appropriate equations are Stokes equations. The governing partial differential equations are recast as boundary integral equations using a version of the Lorenz reciprocal relationship. This allows for robust treatment of the simplified fluid/biofilm motion. The transport of nutrients and antimicrobials, which depends directly on the velocities of the fluid and biofilm, is also included. Disinfection of the bacteria is considered under the assumption that the biofilm growth is overwhelmed by disinfection. Supported by NSF award DMS-0612467.