Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: The nature of the signal

Summary We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK₁. Assayed in physiological salt solution, the ratio of the concentration of -methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium. In this study, using a cloned line of LLC-PK₁ cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa. By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1mm glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium. Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter. Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism.     

Phylogeny and character evolution of the Neotropical fern genus <Emphasis Type="Italic">Stigmatopteris</Emphasis> (Dryopteridaceae)

We did a phylogenetic analysis on 21 of the 23 species of Stigmatopteris, a neotropical montane genus of wet forest floors. The analysis was based on four plastid markers: rbcL, rps4-trnS, trnG-trnR, and trnL-trnF. Two of the most closely related families to the Dryopteridaceae (Didymochlaenaceae and Hypodematiaceae) and 12 dryopteroid genera were used as outgroups. Eight morphological characters were mapped on the resulting tree. Stigmatopteris was recovered as monophyletic and sister to a clade formed by polystichoid ferns such as Arachniodes, Dryopteris, and Polystichum. Synapomorphies that distinguish Stigmatopteris are 1-pinnate-pinnatifid laminae, visible punctate glands in the mesophyll, the presence of hydathodes, uniseriate cilia on the scale margins, and loss of indusia. Within the genus are two main clades. The first consists of four species characterized by pinnae long-decurrent in at least the distal third of the lamina. The second consists of the remaining 17 species in the analysis and has no known morphological synapomorphies. Nested within this second clade is a subclade of six species endemic to southeastern Brazil. Anastomosing veins, a character often used in keys to distinguish the species, evolved three times within the genus.     

Regio- and enantioselective reduction of methyleneketoesters mediated by Saccharomyces cerevisiae

Methyleneketoesters were readily prepared in high yields by performing a direct -methylenation of the corresponding ketoesters using a previously described protocol. Reactions of ethyl 2-methylene-3-oxo-3-arylpropanoates 2a–c catalyzed by S. cerevisiae were performed with good conversions to give reductions of the CC, CO or both, depending on the reaction conditions and on the substitution of the aryl moiety. Reaction of 3-methylene-2-oxo-4-phenylbutyrate 2d was carried out with free yeast cells and with yeast cells immobilized with calcium alginate, in which the major products resulted from CC and CO bond reduction.

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Field response of rice paddy crop to Azospirillum inoculation: physiology of rhizosphere bacterial communities and the genetic diversity of endophytic bacteria in different parts of the plants

The response of rice plants to the application of inoculant containing two Azospirillum brasilense strains was studied under field conditions. The experiment was performed as three treatments with four replicates in randomized complete blocks arranged as plots of 60 m² in an area on a Vertic Argiudol soil type in the province of Entre Ríos, Argentina. The bacterial rhizosphere community and also the diazotrophic isolates obtained from control and inoculated rice plants were analyzed in relation to their physiology and biological nitrogen fixation (BNF). The MPN of diazotrophs in the rhizosphere varied during the ontogenic cycle. The patterns of distribution of the microbial physiological activities obtained by principal component analysis of community-level physiological profiles (CLPP) showed differences in the utilization of carbon sources by the rhizosphere communities among treatments. Although the analyses of DGGE 16S and nifH profiles have not indicated that the inoculation influenced the genetic diversity of bacterial communities among treatments, they revealed that the banding profiles were altered in different parts of the rice plant by each Azospirillum inoculation treatment. These observations suggest that physiological responses of plant tissues to the inoculation may have occurred. According to agronomic parameters of each treatment, the Azospirillum inoculation increased aerial biomass at the tillering and grain-filling stages. Although the N content accumulated in rice plants increased by 16 and 50 kg ha^?1, the BNF contribution could not be estimated under our experimental conditions by the ¹⁵N balance technique. Based on this field inoculation experiment to rice plants, it is noteworthy that our data suggest that due to Azospirillum inoculation the increase of total N accumulated in rice plants could be a tool to help farmers to improve production and maintain high input of plant residues, providing more organic matter to the soil and guaranteeing sustainability of the system.     

Feral pig population structuring in the rangelands of eastern Australia: applications for designing adaptive management units

Feral pigs (Sus scrofa) are an invasive species in Australia. Their negative impact on conservation values has been demonstrated, and they are controlled in many areas in the rangelands of Australia. However, they are usually controlled over small, often ad hoc management units (MUs), and previous research has revealed that these MUs can be inadequate. Understanding feral pig population structuring can aid in the design of appropriate MUs. This study documents an approach to improving MUs for feral pig control in the rangelands of Australia. Feral pigs from a 500,000 km² region were genotyped with 13 polymorphic markers. Genetic analyses were used to identify population structure. Identified sub-populations were then related to geographical and environmental gradients with geographical information systems, regression analysis and with canonical correspondence analysis. Five sub-populations were identified. These were moderately differentiated, with relatively high-migration rates. Two sub-populations in drier, lower elevation areas overlapped, due to extensive migration, probably along the large, inland rivers and flood plains. Sub-populations in higher rainfall environments appeared less likely to migrate. Sub-population differentiation was also dependant on distance, indicating isolation by distance was present. A case study applying an adaptive MU to a previously controlled area is presented. Generally, however, MUs for feral pig control for natural resource protection and endemic disease eradication in the rangelands should take into account geographical size, but also geographic features, especially major rivers in low-rainfall areas.     

Fam60a defines a variant Sin3a‐Hdac complex in embryonic stem cells required for self‐renewal

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a‐Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3‐positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1‐phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.     

Comparisons of watershed sulfur budgets in southeast Canada and northeast US: new approaches and implications

Most of eastern North America receives elevated levels of atmospheric deposition of sulfur (S) that result from anthropogenic SO₂ emissions from fossil fuel combustion. Atmospheric S deposition has acidified sensitive terrestrial and aquatic ecosystems in this region; however, deposition has been declining since the 1970s, resulting in some recovery in previously acidified aquatic ecosystems. Accurate watershed S mass balances help to evaluate the extent to which atmospheric S deposition is retained within ecosystems, and whether internal cycling sources and biogeochemical processes may be affecting the rate of recovery from decreasing S atmospheric loads. This study evaluated S mass balances for 15 sites with watersheds in southeastern Canada and northeastern US for the period 1985 to 2002. These 15 sites included nine in Canada (Turkey Lakes, ON; Harp Lake, ON; Plastic Lake, ON; Hermine, QC; Lake Laflamme, QC; Lake Clair, QC; Lake Tirasse, QC; Mersey, NS; Moosepit, NS) and six in the US (Arbutus Lake, NY; Biscuit Brook, NY; Sleepers River, VT; Hubbard Brook Experimental Forest, NH; Cone Pond, NH; Bear Brook Watershed, ME). Annual S wet deposition inputs were derived from measured bulk or wet-only deposition and stream export was obtained by combining drainage water fluxes with SO₄ ^2? concentrations. Dry deposition has the greatest uncertainty of any of the mass flux calculations necessary to develop accurate watershed balances, and here we developed a new method to calculate this quantity. We utilized historical information from both the US National Emissions Inventory and the US (CASTNET) and the Canadian (CAPMoN) dry deposition networks to develop a formulation that predicted SO₂ concentrations as a function of SO₂ emissions, latitude and longitude. The SO₂ concentrations were used to predict dry deposition using relationships between concentrations and deposition flux derived from the CASTNET or CAPMoN networks. For the year 2002, we compared the SO₂ concentrations and deposition predictions with the predictions of two continental-scale air quality models, the Community Multiscale Air Quality (CMAQ) model and A Unified Regional Air-quality Modeling System (AURAMS) that utilize complete inventories of emissions and chemical budgets. The results of this comparison indicated that the predictive relationship provides an accurate representation of SO₂ concentrations and S deposition for the region that is generally consistent with these models, and thus provides confidence that our approach could be used to develop accurate watershed S budgets for these 15 sites. Most watersheds showed large net losses of SO₄ ^2? on an annual basis, and the watershed mass balances were grouped into five categories based on the relative value of mean annual net losses or net gains. The net annual fluxes of SO₄ ^2? showed a strong relationship with hydrology; the largest net annual negative fluxes were associated with years of greatest precipitation amount and highest discharge. The important role of catchment hydrology on S budgets suggests implications for future predicted climate change as it affects patterns of precipitation and drought. The sensitivity of S budgets is likely to be greatest in watersheds with the greatest wetland area, which are particularly sensitive to drying and wetting cycles. A small number of the watersheds in this analysis were shown to have substantial S sources from mineral weathering, but most showed evidence of an internal source of SO₄ ^2?, which is likely from the mineralization of organic S stored from decades of increased S deposition. Mobilization of this internal S appears to contribute about 1?C6 kg S ha^?1 year^?1 to stream fluxes at these sites and is affecting the rate and extent of recovery from acidification as S deposition rates have declined in recent years. This internal S source should be considered when developing critical deposition loads that will promote ecosystem recovery from acidification and the depletion of nutrient cations in the northeastern US and southeastern Canada.     

Metabolic Analysis of Wild-type Escherichia coli and a Pyruvate Dehydrogenase Complex (PDHC)-deficient Derivative Reveals the Role of PDHC in the Fermentative Metabolism of Glucose

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of ¹³C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional ¹³C,¹H NMR spectroscopy. Metabolite balancing, biosynthetic ¹³C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO₂ and the calculated CO₂ evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO₂ for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO₂, thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO₂ and H₂) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO₂ generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.     

Transducing particles of Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage-encoded proteins

The relationship between the composition of SaPI1 transducing particles and those of helper phage 80alpha was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80alpha and SaPI1 virions, and all were encoded by 80alpha. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.