Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

The aim of this work was to investigate the partitioning of imported glucose 6-phosphate (Glc6P) to starch and fatty acids, and to CO₂ via the oxidative pentose phosphate pathway (OPPP) in plastids isolated from developing embryos of oilseed rape (Brassica napus L.). The ability of the isolated plastids to utilize concurrently supplied substrates and the effects of these substrate combinations on the Glc6P partitioning were also assessed. The relative fluxes of carbon from Glc6P to starch, fatty acids, and to CO₂ via the OPPP were close to 2∶1∶1 when Glc6P was supplied alone. Under these conditions NADPH generated via the OPPP was greater than that required by the concurrent rate of fatty acid synthesis. Fatty acid synthesis was unaffected by the presence or absence of exogenous NADH and/or NADPH and the requirement of fatty acid synthesis for reducing power is therefore met entirely by intraplastidial metabolism. When Glc6P was supplied in the presence of either pyruvate or pyruvate and acetate, the total flux from these metabolites to fatty acids was up to threefold greater than that from either Glc6P or pyruvate when they were supplied singly. In these experiments there was little competition between Glc6P and pyruvate in fatty acid synthesis and the flux to starch was unchanged. This implies that the starch and fatty acid biosynthesis pathways did not compete for the exogenously supplied ATP on which they were strongly dependent. When Glc6P and pyruvate were provided together, the NADPH generated by the OPPP pathway was less than that required by the concurrent rate of fatty acid synthesis. This suggests that the metabolism of exogenous Glc6P via the OPPP can contribute to the NADPH demand created during fatty acid synthesis but it also indicates that other intraplastidial sources of reducing power must be available under the in-vitro conditions used.     

Helminth infracommunities in Litoria genimaculata (Amphibia: Anura) from Birthday Creek, and upland rainforest stream in Northern Queensland, Australia

The helminth fauna of Litoria genimaculata, a rainforest frog from northern Queensland, was quantified from 53 adult male frogs collected at monthly intervals between April 1990 and March 1991. The helminth fauna of this species was depauperate (6 species: Mesocoelium sp., Parapolystoma bulliense, Austraplectana sp., Onchocercidae gen. sp., Cosmocerca sp. and an unidentified nematode larva). The most commonly encountered species was P. bulliense, but the intestinal infracommanity was dominated by the digenean Mesocoelium sp. Fifty-five per cent of frogs were infected with only 1 helminth species and only 1 frog had more than 2 species, resulting in low diversity values. These results support previous studies which indicate that amphibians have depauperate helminth communities.     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

Helminths of two anuran species, Atelopus spurrelli (Bufonidae) and Dendrobates histrionicus (Dendrobatidae), from Colombia, South America

Two species of anurans from Colombia, South America, Atelopus spurrelli and Dendrobates histrionicus, were examined for helminths. A. spurrelli was found to harbor three species of Nematoda, adults of Cosmocerca podicipinus and larvae of Physocephalus sp. and Porrocaecum sp. D. histrionicus was found to harbor, in addition to C. podicipinus, Physocephalus sp. and Porrocaecum sp. and cystacanths of three species of Acanthocephala: Centrorhynchus sp., Onicola sp. and Polymorphus sp. A. spurrelli and D. histrionicus represent new host records for these helminths.     

Enterococci as indicators of Lake Michigan recreational water quality: comparison of two methodologies and their impacts on public health regulatory events

The frequency of poor-water-quality advisories issued in Milwaukee and Racine, Wisconsin, in the absence of identifiable sources of contamination brought into question the reliability of the present indicator organism, Escherichia coli. Enteroccoci have been suggested as an alternative to E. coli for freshwater monitoring due to their direct correlation to swimmer-associated gastroenteritis. The purpose of this research was threefold: (i) to explore enterococci as an alternative to E. coli for monitoring freshwater Lake Michigan beaches, (ii) to evaluate the impact of the two indicators on regulatory decisions, and (iii) to compare membrane filtration m-enterococcus agar with indoxyl-beta-D-glucoside to a chemical substrate technique (Enterolert) for the recovery of enterococci. Recreational water samples from Milwaukee (n = 305) and Racine (n = 153) were analyzed for the enumeration of E. coli and enterococci using IDEXX Colilert-18 and Enterolert. Correlation between the indicators was low (R(2) = 0.60 and 0.69). Based on U.S. Environmental Protection Agency bacterial indicator threshold levels of risk for full body immersion, using enterococci would have resulted in 56 additional unsafe-recreational-water-quality advisories compared to the total from using E. coli and the substrate-based methods. A comparison of the two enterococcal methods (n = 124) yielded similar results (R(2) = 0.62). This was further confounded by the frequent inability to verify enterococci from those wells producing fluorescence by the defined substrate test using conventional microbiological methods. These results suggest that further research is necessary regarding the use of defined substrate technology interchangeably with the U.S. Environmental Protection Agency-approved membrane filtration test for the detection of enterococci from fresh surface water.     

Multiplex real-time PCR method to identify Shiga toxin genes stx1 and stx2 and Escherichia coli O157:H7/H- serotype

A multiplex real-time PCR method to simultaneously detect the stx1 and stx2 genes of Shiga toxin-producing Escherichia coli and a unique conserved single-nucleotide polymorphism in the E. coli O157:H7/H- uidA gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were >/= 1.89, and as few as 6 CFU/reaction could be detected.     

Interaction between human respiratory syncytial virus (RSV) M2-1 and P proteins is required for reconstitution of M2-1-dependent RSV minigenome activity

We have investigated protein-protein interactions among the respiratory syncytial virus (RSV) RNA polymerase subunits using affinity chromatography. Here we demonstrate a novel interaction of P and M2-1 proteins. Phosphorylation of either M2-1 or P appears to be dispensable for this interaction. Internal deletions within P mapped the M2-1-binding domain to a region between residues 100 and 120. Alanine-scanning mutagenesis within this region of P revealed that substitution of any one of the three residues, L101, Y102, and F109, prevented both M2-1 and P binding and expression of an M2-1-dependent luciferase reporter gene. However, these same mutations did not prevent the activity of an M2-1-independent chloramphenicol acetyltransferase minigenome, suggesting that these residues of P specifically affect M2-1-P interaction. On the basis of these observations, it is possible that the interaction between RSV M2-1 and P proteins is important for viral replication.     

Remotely sensed landscape heterogeneity as a rapid tool for assessing local biodiversity value in a highly modified New Zealand landscape

The widespread conversion of natural habitats to agricultural land has created a need to integrate intensively managed landscapes into conservation management priorities. However, there are no clearly defined methods for assessing the conservation value of managed landscapes at the local scale. We used remotely sensed landscape heterogeneity as a rapid practical tool for the assessment of local biodiversity value within a predominantly agricultural landscape in Canterbury, New Zealand. Bird diversity was highly significantly correlated with landscape heterogeneity, distance from rivers and the Christchurch central business district, altitude and average annual household income, indicating that remotely sensed landscape heterogeneity is a good predictor of local biodiversity patterns. We discuss the advantages and limitations of using geographic information systems to determine local areas of high conservation value.     

The Arabidopsis ortholog of the 77 kDa subunit of the cleavage stimulatory factor (AtCstF-77) involved in mRNA polyadenylation is an RNA-binding protein

The 77 kDa subunit of the polyadenylation cleavage stimulation factor (CstF77) is important in messenger RNA 3′ end processing. Previously, we demonstrated that AtCstF77 interacts with AtCPSF30, the Arabidopsis ortholog of the 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. In further dissecting this interaction, it was found that the C-terminus of AtCstF77 interacts with AtCPSF30. Remarkably, we also found that the C-terminal domain of AtCstF77 possesses RNA-binding ability. These studies therefore reveal AtCstF77 to be an RNA-binding protein, adding yet another RNA-binding activity to the plant polyadenylation complex. This raises interesting questions as to the means by which RNAs are recognized during mRNA 3′ end formation in plants.

Structured summary:

MINT-7712550: AtCstF77 (uniprotkb:Q8LKG5) binds (MI:0407) to AtCPSF30 (uniprotkb:A9LNK9) by pull down (MI:0096)     

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I: CONSTRAINTS AND FINE-TUNING IN THE EVOLUTION OF A PROTEIN FAMILY*

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His^B5 → Thr markedly destabilizes the hormone (ΔΔG_u 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr^B5 → His (residue 4) specifies a unique structure with native ¹H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His^B5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His^B5 does not significantly enhance thermodynamic stability (ΔΔG_u 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr^B5-insulin is decreased 5-fold, His^B5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr^B5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His^B5 in insulin highlights its critical role in insulin biosynthesis.