Comparison of the binding of glucose and glucose 1-phosphate derivatives to T-state glycogen phosphorylase b

The binding of T-state- and R-state-stabilizing ligands to the catalytic C site of T-state glycogen phosphorylase b has been investigated by crystallographic methods to study the interactions made and the conformational changes that occur at the C site. The compounds studied were alpha-D-glucose, 1, a T-state-stabilizing inhibitor of the enzyme, and the R-state-stabilizing phosphorylated ligands alpha-D-glucose 1-phosphate (2), 2-deoxy-2-fluoro-alpha-D-glucose 1-phosphate (3), and alpha-D-glucose 1-methylenephosphonate (4). The complexes have been refined, giving crystallographic R factors of less than 19%, for data between 8 and 2.3 A. Analysis of the refined structures shows that the glucosyl portions of the phosphorylated ligands bind in the same orientation as glucose and retain most of the interactions formed between glucose and the enzyme. However, the phosphates of the phosphorylated ligands adopt different conformations in each case; the stability of these conformations have been studied by using computational methods to rationalize the different binding modes. Binding of the phosphorylated ligands is accompanied by movement of C-site residues, most notably a shift of a loop out of the C site and toward the exterior of the protein. The C-site alterations do not include movement of Arg569, which has been observed in both the refined complex with 1-deoxy-D-gluco-heptulose 2-phosphate (5) [Johnson, L. N., et al (1990) J. Mol. Biol. 211, 645-661] and in the R-state enzyme [Barford, D. & Johnson, L. N. (1989) Nature 340, 609-616]. Refinement of the ligand complexes has also led to the observation of additional electron density for residues 10-19 at the N-terminus which had not previously been localized in the native structure. The conformation of this stretch of residues is different from that observed in glycogen phosphorylase a.     

Dinosterol side chain biosynthesis in a marine dinoflagellate, Crypthecodinium cohnii

The heterotrophic dinofiagellate, Crypthecodinium cohnii, cultured in a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly synthesized 4α-monomethyl compound dinosterol (4α,23,24-trimethylcholest-22-en-3β-ol). The MS fragmentation pattern indicated that the C-23 methyl group contained three deuterium atoms and was introduced intact by transmethylation from methionine. The C-24 methyl group contained only two deuterium atoms which is consistent with the production of a 24-methylenesterol intermediate which is subsequently reduced to give the 24-methyl side chain. Mechanisms are proposed to account for the production of the dinosterol side chain.     

Potential non-target impact of Microctonus aethiopoides Loan (Hymenoptera: Braconidae) on Cleopus japonicus Wingelmüller (Coleoptera: Curculionidae), a biocontrol agent for putative release to control the butterfly bush Buddleja davidii Franchet in New Zealand

Abstract  Cleopus japonicus Wingelmüller (Coleoptera: Curculionidae) is being considered for release to control buddleia Buddleja davidii in New Zealand. As part of the pre-release testing, Moroccan and Irish biotypes of the solitary endoparasitoid Microctonus aethiopoides Loan (Hymenoptera: Braconidae) were evaluated for potential non-target impacts on adult C. japonicus should release occur. Laboratory experiments evaluated both the behavioural and physiological suitability of C. japonicus to both biotypes of the parasitoid. Parasitoid behavioural attraction was assessed using the pathenogenic bacterium Serratia marcescens (Enterobactereaceae), as an indicator of ovipositor penetration. Physiological suitability was assessed by comparing parasitism of C. japonicus with the natural hosts of the respective parasitoid biotypes. The parasitoid-bacteria study showed that C. japonicus was behaviourally acceptable to both Moroccan and Irish M. aethiopoides , with the two experiments producing 34% and 8% mortality, respectively. Cleopus japonicus did not support development of either Moroccan or Irish M. aethiopoides biotypes. None of the weevils dissected at the end of the experiment contained immature parasitoids. Comparison between unexposed and parasitoid-exposed C. japonicus found no difference in premature mortality during the experiment nor in the number of fully reproductive females at its conclusion. The results of this study predict that should C. japonicus be released, the potential impact of M. aethiopoides on field populations will be negligible.     

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.     

Effect of pedal cadence on the accumulated oxygen deficit, maximal aerobic power and blood lactate transition thresholds of high-performance junior endurance cyclists

In this study we investigated the effect of pedal cadence on the cycling economy, accumulated oxygen deficit (AOD), maximal oxygen consumption (VO2max) and blood lactate transition thresholds of ten high-performance junior endurance cyclists [mean (SD): 17.4 (0.4) years; 183.8 (3.5) cm, 71.56 (3.75) kg]. Cycling economy was measured on three ergometers with the specific cadence requirements of: 90-100 rpm for the road dual chain ring (RDCR90-100 rpm) ergometer, 120-130 rpm for the track dual chain ring (TDCR120-130 rpm) ergometer, and 90-130 rpm for the track single chain ring (TSCR90-130 rpm) ergometer. AODs were then estimated using the regression of oxygen consumption (VO2) on power output for each of these ergometers, in conjunction with the data from a 2-min supramaximal paced effort on the TSCR90-130 rpm ergometer. A regression of VO2 on power output for each ergometer resulted in significant differences (P<0.001) between the slopes and intercepts that produced a lower AOD for the RDCR90-100 rpm [2.79 (0.43) l] compared with those for the TDCR120-130 rpm [4.11 (0.78) l] and TSCR90-130 rpm [4.06 (0.84) l]. While there were no statistically significant VO2max differences (P = 0.153) between the three treatments [RDCR90-100 rpm: 5.31 (0.24) l x min(-1); TDCR120-130 rpm; 5.33 (0.25) 1 x min(-1); TSCR90-130 rpm: 5.44 (0.27) l x min(-1)], all pairwise comparisons of the power output at which VO2max occurred were significantly different (P<0.001). Statistically significant differences were identified between the RDCR90-100 rpm and TDCR120-130 rpm tests for power output (P = 0.003) and blood lactate (P = 0.003) at the lactate threshold (Thla-), and for power output (P = 0.005) at the individual anaerobic threshold (Thiat). Our findings emphasise that pedal cadence specificity is essential when assessing the cycling economy, AOD and blood lactate transition thresholds of high-performance junior endurance cyclists.     

产超广谱β- 内酰胺酶肺炎克雷伯菌耐药性及其基因分型分析

目的:了解新疆独山子地区肺炎克雷伯菌超广谱β-内酰胺酶(ESBLs)的发生率、作为指示剂的五种抗生素的检出情况及ESBLs主要基因型。方法:收集临床分离的148株肺炎克雷伯菌,采用双纸片协同筛选法、NCCLS推荐的表型筛选和确证试验对细菌进行ESBLs产酶株的识别;耐药基因的质粒重组、转化,聚合酶链反应(PCR)扩增阳性产物测序,通过GenBank对序确定基因型。结果:本地区肺炎克雷伯菌ESBLs的分离率达31.1%,头孢曲松(CRO)安曲南(ATM)和头孢噻肟(CTX)头孢泊肟(CPD)、头孢他定(CAZ)作为指示剂检出率分别为97.8%、95.6%、93.4%、76.0%、65.2%;本地区产ESBLs菌株耐药基因型CTX-M-22占54.3%,CTX-M-18占41.3%,TEM61.8%,52.1%的产ESBLs肺炎克雷伯菌SHV耐药基因阳性。结论:CRO、ATM和CTX对检测ESBLs阳性率较高;CTX-M-22、CTX-M-18是本地区产ESBLs菌株的主要基因型。     

Secondary galling: a novel feeding strategy among ‘non‐pollinating’ fig wasps from Ficus curtipes

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A solid-state 31P-NMR investigation of the allosteric transition in glycogen phosphorylase b.

The catalytic role of the cofactor phosphate moiety at the active site of glycogen phosphorylase has been the subject of many investigations including solution-state high-resolution 31P-NMR studies. In this study the pyridoxal phosphate moiety in both the inactive and active forms of microcrystalline phosphorylase b has been investigated by high-resolution 31P magic-angle spinning NMR. The symmetry of the shielding tensor in model compounds at varying degrees of ionization is investigated and the results indicate a marked difference between the dianionic and monoanionic model compounds. Consequently the observed similarity in the principal tensor components describing the shielding tensor of the phosphorus nuclei present at the active site of both the R- and T-state conformations suggests that there is no change in ionization site upon activation in contrast to suggestions based upon isotropic shifts. Since previous relaxation measurements have pointed to the need to consider motional influences in such systems, several plausible models are considered. Subject to the assumption of congruency between the principal axis system describing the shielding interaction and molecular frame determined by the molecular symmetry axes, we conclude that the phosphate cofactor is dianionic in both forms.