Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

Aptamer-based multiplexed proteomic technology for biomarker discovery

Background

The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.

Methodology/Principal Findings

We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.

Conclusions/Significance

We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.     

Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.     

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Chromosomal Organization of Rrna Operons in Bacillus Subtilis

Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion.     

Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild-type strain

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap⁺, sfa⁺, pil⁺, hly⁺ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were sub-cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (Nal^R) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA⁺ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal-coated latex beads. CBA mice (N =100) were challenged transurethrally with 10⁵, 10⁶, 10⁷, or 10⁹ cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro-pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.     

A framework for modelling soil structure dynamics induced by biological activity

Soil degradation is a worsening global phenomenon driven by socio‐economic pressures, poor land management practices and climate change. A deterioration of soil structure at timescales ranging from seconds to centuries is implicated in most forms of soil degradation including the depletion of nutrients and organic matter, erosion and compaction. New soil–crop models that could account for soil structure dynamics at decadal to centennial timescales would provide insights into the relative importance of the various underlying physical (e.g. tillage, traffic compaction, swell/shrink and freeze/thaw) and biological (e.g. plant root growth, soil microbial and faunal activity) mechanisms, their impacts on soil hydrological processes and plant growth, as well as the relevant timescales of soil degradation and recovery. However, the development of such a model remains a challenge due to the enormous complexity of the interactions in the soil–plant system. In this paper, we focus on the impacts of biological processes on soil structure dynamics, especially the growth of plant roots and the activity of soil fauna and microorganisms. We first define what we mean by soil structure and then review current understanding of how these biological agents impact soil structure. We then develop a new framework for modelling soil structure dynamics, which is designed to be compatible with soil–crop models that operate at the soil profile scale and for long temporal scales (i.e. decades, centuries). We illustrate the modelling concept with a case study on the role of root growth and earthworm bioturbation in restoring the structure of a severely compacted soil.     

Passive exposure to tobacco smoke: saliva cotinine concentrations in a representative population sample of non-smoking schoolchildren.

Saliva cotinine concentrations in 569 non-smoking schoolchildren were strongly related to the smoking habits of their parents. When neither parent smoked the mean concentration was 0.44 ng/ml, rising to 3.38 ng/ml when both parents were cigarette smokers. Mothers'' smoking had a stronger influence than did fathers'' (p less than 0.01). In addition, there was a small independent effect of number of siblings who smoked (p less than 0.01). The dose of nicotine received from fathers'' smoking was estimated as equivalent to the active smoking of 30 cigarettes a year, that from mothers'' smoking as equivalent to smoking 50 cigarettes a year, and that from both parents smoking as equivalent to smoking 80 cigarettes a year. This unsolicited burden may be prolonged throughout childhood and poses a definite risk to health.