Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats

The intestinal mucoprotein synthesis rate was measured in vivo for the first time. For this, a rapid, reproducible, and convenient method to purify mucoproteins from large numbers of intestinal samples at the same time was developed. The method takes advantage of both the high mucin resistance to protease activities due to their extensive glycosylations and the high mucin molecular size. Intestinal homogenates were partially digested with Flavourzyme. Nonprotected proteins partially degraded were easily separated from mucoproteins by small gel filtration chromatography using Sepharose CL-4B. Electrophoretically pure mucins were obtained. Their amino acid composition was typical of purified intestinal epithelial mucins. The mucoprotein synthesis rate was determined in vivo in rats using the flooding dose method with the stable isotope L-[1-13C]valine. Free L-[1-13C]valine enrichments in the intracellular pool were determined by GC-MS. L-[1-13C]valine enrichments into purified mucoproteins or intestinal mucosal proteins were measured by gas chromatography-combustion-isotope ratio mass spectrometry. In rats, we found that the gut mucosa protein synthesis rate (%/day) decreased regularly from duodenum (122%/day) to colon (43%/day). In contrast, mucoprotein fractional synthesis rates were in the same range along the digestive tract, between 112%/day (colon) and 138%/day (ileum).     

Sperm competition and last-male precedence in the honeybee

Five microsatellite loci were used to determine paternities in six Apis mellifera colonies headed by naturally mated queens. The last inseminating males were identified by collecting and genotyping the mating sign left in the genital tract of each queen. Significant differences in paternity frequencies were observed between males, but the proportion of worker and queen offspring sired by the last inseminating drone did not differ significantly from those of other drones. Each male kept his rank of precedence for the different cohorts, although the variance in subfamily proportions decreased over time, most notably in the colony displaying the lowest level of polyandry. These results suggest that, if sperm competition exists in the honeybee, it does not significantly increase the fitness of the last inseminating drone. The spermatozoa of the different inseminating drones are not totally mixed before they reach the spermatheca, in particular when only few males mate with the queen. The weak difference in the subfamily proportions observed between queen and worker samples confirms that nepotistic interactions are rare. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.     

Phytotoxicity and volatile constituents from leaves of Callicarpa japonica Thunb

The essential oil from the leaves of Callicarpa japonica was analyzed by GC-MS, and 84 compounds were identified. The main constituents of the essential oil were spathulenol (18.1%), germacrene B (13.0%), bicyclogermacrene (11.0%), globulol (3.3%), viridiflorol (2.6%), alpha-guaiene (2.3%), and gamma-elemene (2.0%). The essential oil constituents of C. japonica were significantly different from those found in our previous work on Callicarpa americana. The oil of C. japonica was selectively phytotoxic to bentgrass compared to lettuce seeds, with 80-100% growth reduction observed at 0.3 mg/ml.     

The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase

The inhibitory activity of 34 natural products of various structural classes on hydroxyphenylpyruvate dioxygenase (HPPD), the target site for triketone herbicides, and the mode of interaction of selected natural products were investigated. Recombinant HPPD from arabidopsis is sensitive to several classes of natural compounds including, in decreasing order of sensitivity, triketones, benzoquinones, naphthoquinones and anthraquinones. The triketone natural products acted as competitive tight-binding inhibitors, whereas the benzoquinones and naphthoquinones did not appear to bind tightly to HPPD. While these natural products may not have optimal structural features required for in vivo herbicidal activity, the differences in their kinetic behavior suggest that novel classes of HPPD inhibitors may be developed based on their structural backbones.     

Pax genes and eye organogenesis

Pax6 is a highly conserved gene that controls eye development in all species where it has been tested. In spite of this common 'master control regulator', the eyes of different animals are morphologically very different and it is believed that they have evolved independently multiple times through evolution. Recent works looking at eye development in 'primitive' species offer some explanation as to the surprising amount of conservation in genetic and morphogenetic pathways involved in eye development. These studies not only implicate the Pax genes but also the So/Six gene family in playing a crucial ancestral role in visual system development.     

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.ADP.P(i) states accumulate in the steady state [Lionne, C., et al. (1995) FEBS Lett. 364, 59]. We have now studied the kinetics of P(i) release with chemically cross-linked myofibrils that, when adequately cross-linked, appear to be a good model for isometric contraction. By using a method that is specific for free P(i) and rapid quench flow that measures the amount of (A)M.ADP.P(i) states and free P(i), we show that (A)M.ADP.P(i) states predominate which suggests that the overall ATPase is limited by P(i) release kinetics. Therefore, under our experimental conditions with myofibrils prevented from shortening, the concentration of (A)M.ADP states is low, as with rapidly shortening and relaxed myofibrils. This result is difficult to reconcile with the sensitivity of force development in fibers and myofibrils to P(i) which implies interaction of P(i) with an (A)M.ADP state. We discuss two models for accommodating the mechanical and chemical kinetics with reference to the duty cycle in skeletal muscle.