Genetic and physical analyses of Caulobacter crescentus trp genes.

Caulobacter crescentus trp mutants were identified from a collection of auxotrophs. Precursor feeding experiments, accumulation studies, and complementation experiments resulted in the identification of six genes corresponding to trpA, trpB, trpC, trpD, trpE, and trpF. Genetic mapping experiments demonstrated that the trp genes were in two clusters, trpCDE and trpFBA, and a 5.4-kilobase restriction fragment from the C. crescentus chromosome was isolated that contained the trpFBA gene cluster. Complementation experiments with clones containing the 5.4-kilobase fragment indicated that trpF was expressed in Escherichia coli and that all three genes were expressed in Pseudomonas putida. This expression was lost in both organisms when the pBR322 tet gene promoter was inactivated, indicating that all three genes were transcribed in the same orientation from the tet promoter. Thus, the C. crescentus promoters do not seem to be expressed in E. coli or P. putida. Complementation of the C. crescentus trp mutants indicated that the tet promoter was not necessary for expression in C. crescentus and suggested that at least two native promoters were present for expression of the trpF, trpB, and trpA genes. Taken together, these results indicate that C. crescentus promoters may have structures that are significantly different from the promoters of other gram-negative species.     

The entire N-terminal half of TatC is involved in twin-arginine precursor binding

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.     

An environmental (pre)history of European fishing: past and future archaeological contributions to sustainable fisheries

This paper explores the past and potential contribution of archaeology to marine historical ecology. The primary focus is European fishing of marine and diadromous taxa, with global comparisons highlighting the wider applicability of archaeological approaches. The review illustrates how study of excavated fish bones, otoliths and shells can inform our understanding of: (a) changes in biogeography, including the previous distribution of lost species; (b) long-term fluctuations in the aquatic environment, including climate change; (c) the intensity of exploitation and other anthropogenic effects; (d) trade, commodification and globalisation. These issues are also relevant to inform fisheries conservation and management targets. Equally important, the long (pre)history of European fishing raises awareness of our ecological heritage debt, owed for centuries of wealth, sustenance and well-being, and for which we share collective responsibility. This debt represents both a loss and a reason for optimism, insofar as it is a reservoir of potential to be filled by careful stewardship of our rivers, lakes, seas and oceans.     

Renaturation of dextranase activity from culture supernatant fluids of Streptococcus sobrinus after sodium dodecylsulfate polyacrylamide gel electrophoresis

A rapid and reproducible method for the assay of individual fractions of the multicomponent dextranase activity of Streptococcus sobrinus after reduction/denaturation and electrophoresis in acrylamide gels is described. Multiple forms of dextranase, possible virulence factors in the formation of dental caries by oral Streptococci (S. mutans, S. sobrinus, S. sanguis, S. cricetus, and S. rattus), have been separated in sodium dodecylsulfate-polyacrylamide gels into which an indicator substrate, blue dextran, has been incorporated, and identified after renaturation to remove the reducing/denaturing agents of the Laemmli buffer system.     

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP)

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.     

Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries

This protocol describes assay development, validation and implementation of automated immobilized metal affinity for phosphochemicals (IMAP)-based fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening (HTS) assays for identification of low-molecular-weight kinase inhibitors. Both procedures are performed in miniaturized kinase reaction volumes and involve the stepwise addition of test or control compounds, enzyme and substrate/ATP. Kinase reactions are stopped by subsequent addition of IMAP-binding buffer. Assay attributes of the IMAP FP and TR-FRET methodologies are described. HTS assays developed using these procedures should result in Z-factors and low assay variability necessary for robust HTS assays. Providing that the required reagents and equipment are available, one scientist should be able to develop a 384-well, miniaturized HTS assay in approximately 6-8 weeks. Specific automated HTS assay conditions will determine the number of assay plates processed in a screening session, but two scientists should expect to process between 100 and 150 assay plates in one 8-h screening day.     

Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans

Subjects with high plasma cholesterol levels exhibit a high production of VLDL apolipoprotein B-100 (apoB-100), suggesting that cholesterol is a mediator for VLDL production. The objective of the study was to examine whether endogenous cholesterol synthesis, reflected by the lathosterol-cholesterol ratio (L-C ratio), affects the secretory rates of different VLDL subfractions. Ten healthy subjects were studied after overnight fasting. During a 10 h primed, constant infusion of 13C-valine (15 micromol/kg/h), enrichment was determined in apoB-100 from ultracentrifugally isolated VLDL-1 and VLDL-2 by gas chromatography mass spectrometry. The synthesis rates of VLDL-1 apoB-100 and VLDL-2 apoB-100, catabolism, and transfer were estimated by compartmental analysis. Mean VLDL-1 apoB-100 pool size was 90 +/- 15 mg, and mean VLDL-2 apoB-100 pool size was 111 +/- 14 mg. Absolute synthesis rate of VLDL-1 apoB-100 was 649 +/- 127 mg/day and 353 +/- 59 mg/day for VLDL-2 apoB-100. There was a strong association between the absolute synthesis rate of VLDL-2 apoB-100 and L-C ratio (r 2 = 0.61, P < 0.01). In contrast, no correlation was observed between L-C ratio and absolute synthesis rate of VLDL-1 apoB-100 (r 2 = 0.302, P = 0.09). In conclusion, these data provide additional support for an independent regulation of VLDL-1 apoB-100 and VLDL-2 apoB-100 production.Endogenous cholesterol synthesis is correlated only with the VLDL-2 apoB-100 production.     

Long‐term phosphite application maintains species assemblages,richness and structure of plant communities invaded by Phytophthora cinnamomi

The impact of the plant pathogen Phytophthora cinnamomi and the fungicide phosphite on species assemblages, richness, abundance and vegetation structure was quantified at three sites in Kwongkan communities in the Southwest Australian Floristic Region. Healthy and diseased vegetation treated with phosphite over 7–16 years was compared with non‐treated healthy and diseased vegetation. After site differences, disease had the greatest effect on species assemblages, species richness and richness within families. Disease significantly reduced cover in the upper and lower shrub layers and increased sedge and bare ground cover. Seventeen of 21 species assessed from the families Ericaceae, Fabaceae, Myrtaceae and Proteaceae were significantly less abundant in non‐treated diseased vegetation. In diseased habitats, phosphite treatment significantly reduced the loss of shrub cover and reduced bare ground and sedge cover. In multivariate analysis of species assemblages, phosphite‐treated diseased plots grouped more closely with healthy plots. Seven of 17 susceptible species were significantly more abundant in phosphite‐treated diseased plots compared with diseased non‐treated plots. The abundance of seven of 10 Phytophthora‐susceptible species was significantly higher along transects in phosphite‐treated vegetation. Comparison of the floristics of healthy non‐treated with healthy‐treated plots showed no significant differences in species assemblages. Of 21 species assessed, three increased in abundance and only one decreased significantly in phosphite‐treated healthy plots. In three Kwongkan communities of the SWAFR, P. cinnamomi had a profound impact on species assemblages, richness, abundance and vegetation structure. There was no evidence of adverse effects of phosphite treatment on phosphorus‐sensitive species, even after fire. Treatment with phosphite enhanced the survival of key susceptible species and mitigated disease‐mediated changes in vegetation structure. In the absence of alternative methods of control in native communities, phosphite will continue to play an important role in the protection of high priority species and communities at risk of extinction due to P. cinnamomi.