Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine.

D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus. In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics. The limiting rate constant for inactivation of the B. sphaericus enzyme is 1.9 min-1, for the B. subilis enzyme it is 0.36 min-1. The number of catalytic events before inactivation is about 450 for the B. sphaericus enzyme and about 800 for the B. subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter. Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles. Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine.     

Nitrate paradigm does not hold up for sugarcane

Modern agriculture is based on the notion that nitrate is the main source of nitrogen (N) for crops, but nitrate is also the most mobile form of N and easily lost from soil. Efficient acquisition of nitrate by crops is therefore a prerequisite for avoiding off-site N pollution. Sugarcane is considered the most suitable tropical crop for biofuel production, but surprisingly high N fertilizer applications in main producer countries raise doubt about the sustainability of production and are at odds with a carbon-based crop. Examining reasons for the inefficient use of N fertilizer, we hypothesized that sugarcane resembles other giant tropical grasses which inhibit the production of nitrate in soil and differ from related grain crops with a confirmed ability to use nitrate. The results of our study support the hypothesis that N-replete sugarcane and ancestral species in the Andropogoneae supertribe strongly prefer ammonium over nitrate. Sugarcane differs from grain crops, sorghum and maize, which acquired both N sources equally well, while giant grass, Erianthus, displayed an intermediate ability to use nitrate. We conclude that discrimination against nitrate and a low capacity to store nitrate in shoots prevents commercial sugarcane varieties from taking advantage of the high nitrate concentrations in fertilized soils in the first three months of the growing season, leaving nitrate vulnerable to loss. Our study addresses a major caveat of sugarcane production and affords a strong basis for improvement through breeding cultivars with enhanced capacity to use nitrate as well as through agronomic measures that reduce nitrification in soil.     

Wide variation in ploidy level and genome size in a New Zealand freshwater snail with coexisting sexual and asexual lineages

Natural animal populations are rarely screened for ploidy-level variation at a scale that allows detection of potentially important aberrations of common ploidy patterns. This type of screening can be especially important for the many mixed sexual/asexual systems in which sexuals are presumed to be dioecious diploids and asexuals are assumed to be triploid and all-female. For example, elevation of ploidy level above triploidy can be a source of genetic variation and raises the possibility of gene flow among ploidy levels and to asexual lineages. We used flow cytometry and mtDNA sequencing to characterize ploidy level and genome size in Potamopyrgus antipodarum, a New Zealand freshwater snail where obligate sexual (presumed diploid and dioecious) and obligate apomictic asexual (presumed triploid and nearly all female) individuals frequently coexist. We documented the widespread occurrence and multiple origins of polyploid males and individuals with >3× ploidy, and find that both are likely to be descended from asexual females. Our survey also suggested the existence of extensive variation in genome size. The discovery of widespread variation in ploidy level and genome size in such a well-studied system highlights the importance of broad, extensive, and ecologically representative sampling in uncovering ploidy level and genome-size variation in natural populations.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI₂) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI₂ is inactivated under physiological conditions it has not been possible to demonstrate specific PGI₂ binding to the rat stomach. Therefore a stable PGI₂ analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE₂ nor 6 keto PGF_1α displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 × 10⁻¹¹ M and 7.1 × 10⁻⁸ M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI₂ was less potent than unlabelled Iloprost in displacing ³H-Iloprost from its binding site. The addition of PGE₂ to the incubation medium resulted in an increase in ³H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI₂-like agents but not for either PGE₂ or 6 keto PGF_1α.     

Essentiality of Glu-48 of ribulose bisphosphate carboxylase/oxygenase as demonstrated by site-directed mutagenesis

Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or ligand binding. However, the Glu-48 mutant is severely deficient in catalytic activity, exhibiting a kcat only 0.05% that of wild-type enzyme. These results demonstrate that Glu-48 is positioned at the active site and suggest that it serves a functional role. In conjunction with previous studies, the discovery of essentiality of Glu-48 argues that the active site is located at an interface between subunits.     

Properties of Venezuelan Equine Encephalomyelitis Virus Grown In Vivo

One intracerebral passage of either the parent egg seed (PES) or an attenuated variant (10t) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus in young adult mice produced progeny that were no longer differentiated unequivocally on the basis of plaque size. Plaques averaging about 2 mm in diameter, which was somewhat smaller than those formed by the PES virus and larger than those of the 10t strain, were formed by both strains. Seven serial passages of the PES virus in mouse brain failed to alter its virulence appreciably. In contrast, passage in mouse brain progressively changed the properties of the attenuated 10t strain. A substrain was isolated that possessed virulence similar to that of the PES virus and formed small plaques similar to those of the 10t strain. These findings showed a unique dissociation between the plaque size and virulence of the 10t strain. The new substrain differed from the PES virus and the 10t strain in its capacity for growth in mouse tissues after intraperitoneal inoculation. The substrain multiplied poorly in splenic tissue, which supports growth of the PES and 10t strains, but grew to high titers in the brain, which does not support appreciable growth of the 10t strain.     

Point-of-care biosensor systems for cancer diagnostics/prognostics

With the growing number of fatalities resulting from the 100 or so cancer-related diseases, new enabling tools are required to provide extensive molecular profiles of patients to guide the clinician in making viable diagnosis and prognosis. Unfortunately with cancer-related diseases, there is not one molecular marker that can provide sufficient information to assist the clinician in making effective prognoses or even diagnoses. Indeed, large panels of markers must typically be evaluated that cut across several different classes (mutations in certain gene fragments--DNA; over/under-expression of gene activity as monitored by messenger RNAs; the amount of proteins present in serum or circulating tumor cells). The classical biosensor format (dipstick approach for monitoring the presence of a single element) is viewed as a valuable tool in many bioassays, but possesses numerous limitations in cancer due primarily to the single element nature of these sensing platforms. As such, if biosensors are to become valuable tools in the arsenal of the clinician to manage cancer patients, new formats are required. This review seeks to provide an overview of the current thinking on molecular profiling for diagnosis and prognosis of cancers and also, provide insight into the current state-of-the-art in the biosensor field and new strategies that must be considered to bring this important technology into the cancer field.     

Synthesis and evaluation of thiazepines as interleukin-1beta converting enzyme (ICE) inhibitors

A series of monocyclic thiazepine inhibitors of interleukin-1beta converting enzyme (ICE) were synthesized in eight steps from commercially available intermediates. In vitro biological evaluation showed the thiazepines to be moderately potent ICE inhibitors, with the most active compound exhibiting an IC50 value of 30 nM in an enzyme inhibition assay. Compounds of this class possessed good selectivity against the related enzymes caspase-3 and caspase-8.