Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway

RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.     

An apterous scelionid wasp in mid-Cretaceous Burmese amber (Hymenoptera: Scelionidae)

A remarkably specialized parasitoid wasp of the family Scelionidae (Platygastroidea) is described and figured from mid-Cretaceous (Cenomanian) amber of the Hukawng Valley in northern Myanmar. Geoscelio mckellari Engel and Huang, gen. et sp. nov., is unique for its combination of a compact body, 12 antennal flagellomeres, a 1-2-2 tibial spur formula, a distinct malar sulcus, deeply impressed notauli, complete reduction of the wings, and basal crenulae on the metasomal terga and sterna, among many other features, and is placed within a separate tribe, Geoscelionini Engel and Huang, trib. nov. This is the first flightless species of Platygastroidea known from the Mesozoic, and its affinities with other Mesozoic and extant lineages are discussed.     

Bioaccumulation of Metals in Cultured Carp (Cyprinus carpio) from Lake Chapala,Mexico

Biological Trace Element Research - Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis....     

Killed Saccharomyces cerevisiae Protects Against Lethal Challenge of Cryptococcus grubii

Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 × 10⁷; 2S, 1.2 × 10⁸; 3S, 2.4 × 10⁸) began 28 (×3), 35 (×4), or 42 (×6) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S×4, or 2S reduced brain CFU; liver or spleen CFU was reduced by S×4 or 2S. In a more lethal second experiment, 2S×6, 2S, or 3S×4 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S×6, 2S, or 3S×4 most efficacious. Dose size appears more important than the number of doses: Regimens >S were superior, and 2S and 2S×6 were equivalent. 2S and 3S were equivalent, suggesting doses >2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine.