Biosynthesis of a novel 3-deoxy-D-manno-oct-2-ulosonic acid-containing outer core oligosaccharide in the lipopolysaccharide of Klebsiella pneumoniae

The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

Evidence for facilitated diffusion of urea across the gill basolateral membrane of the rainbow trout (Oncorhynchus mykiss)

Recent in vivo evidence suggests that the mechanism of branchial urea excretion in the ammoniotelic rainbow trout (Oncorhynchus mykiss) is carrier-mediated. Further characterization of this proposed mechanism was achieved by using an in vitro isolated basolateral membrane vesicle (BLMV) preparation in which isolated gill membranes were used to determine a variety of physiological properties of the transporter. BLMV demonstrated two components of urea uptake, a linear component at concentrations up to 17.5 mmol x l(-1) and a saturable component (K(0.5)=0.35+/-0.01 mmol x l(-1); V(max)=0.14+/-0.02 micromol mg protein(-1) h(-1)) with a Hill constant of 1.35+/-0.18 at low, physiologically relevant urea concentrations (<2 mmol x l(-1)). Saturable uptake of urea at 1 mmol x l(-1) by BLMV was reduced by 88.5% when incubated with 0.25 mmol x l(-1) phloretin, a potent blocker of UT-type facilitated diffusion urea transport mechanisms. BLMV also demonstrated differential handling of urea versus urea analogues at 1 mmol x l(-1) concentrations and total analogue/total urea uptake ratios were 32% for acetamide and 84% for thiourea. Saturable urea uptake at 1 mmol x l(-1) was significantly reduced by almost 100% in the presence of 5 mmol x l(-1) thiourea but was not affected by 5 mmol x l(-1) acetamide or 5 mmol x l(-1) N-methylurea. Lastly, total urea uptake at 1 mmol x l(-1) by BLMV was sensitive to temperatures above and below the temperature of acclimation with a Q(10)>2 suggesting a protein carrier-mediated process. Combined, this evidence indicates that a facilitated diffusion urea transport mechanism is likely present in the basolateral membrane of the rainbow trout gill.     

The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis

We present the complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex. The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol%. There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91%. Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins. There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences. The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site. The companion gene, P102, also has six paralogs. Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family. Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding. There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites. Unlike other mycoplasmas, M. hyopneumoniae contains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation. Thus, it is not clear how M. hyopneumoniae evades the immune response and establishes a chronic infection.     

Activation of human TRPC6 channels by receptor stimulation

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K(0.5) of approximately 10 microm and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30-300 microm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 microm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.     

Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Autosomal dominant FEVR is genetically heterogeneous, but its principal locus, EVR1, is on chromosome 11q13-q23. The gene encoding the Wnt receptor frizzled-4 (FZD4) was recently reported to be the EVR1 gene, but our mutation screen revealed fewer patients harboring mutations than expected. Here, we describe mutations in a second gene at the EVR1 locus, low-density-lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor. This finding further underlines the significance of Wnt signaling in the vascularization of the eye and highlights the potential dangers of using multiple families to refine genetic intervals in gene-identification studies.     

The synergistic effects of stochasticity and dispersal on population densities

Using laboratory experiments, simulation models, and analytical techniques, we examined the impact of dispersal on the mean densities of patchily distributed populations. Even when dispersal leads to no net additions or removals of individuals from a population, it may nonetheless increase mean population densities if the net immigration rate is positive when populations are growing and negative when they are declining. As a model system for exploring this phenomenon, we used the yeastlike fungus Aureobasidium pullulans. In a laboratory experiment, we showed that dispersal can both ensure persistence and increase mean population densities even when dispersal among populations causes no direct addition or loss of fungal cells. From the laboratory data, we constructed a plausible model of A. pullulans dynamics among apple leaves within an orchard. This simulation model demonstrated that the effect of dispersal on mean densities is enhanced by three factors: weak density dependence of the dynamics within populations, high environmental variability affecting population growth rates, and lack of synchrony among the fluctuations of populations. Using an analytical model, we showed that the underlying mechanisms for this phenomenon are general, suggesting that a large effect of dispersal on mean population densities is possible in many natural systems.     

Molecular cytogenetic analysis of recently evolved Tragopogon (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors

Tragopogon mirus and T. miscellus (both 2n = 4x = 24) are recent allotetraploids derived from T. dubius × T. porrifolius and T. dubius × T. pratensis (each 2n = 2x = 12), respectively. The genome sizes of T. mirus are additive of those of its diploid parents, but at least some populations of T. miscellus have undergone genome downsizing. To survey for genomic rearrangements in the allopolyploids, four repetitive sequences were physically mapped. TPRMBO (unit size 160 base pairs [bp]) and TGP7 (532 bp) are tandemly organized satellite sequences isolated from T. pratensis and T. porrifolius, respectively. Fluorescent in situ hybridization to the diploids showed that TPRMBO is a predominantly centromeric repeat on all 12 chromosomes, while TGP7 is a subtelomeric sequence on most chromosome arms. The distribution of tandem repetitive DNA loci (TPRMBO, TGP7, 18S-5.8S-26S rDNA, and 5S rDNA) gave unique molecular karyotypes for the three diploid species, permitting the identification of the parental chromosomes in the polyploids. The location and number of these loci were inherited without apparent changes in the allotetraploids. There was no evidence for major genomic rearrangements in Tragopogon allopolyploids that have arisen multiple times in North America within the last 80 yr.