Edaphic races and phylogenetic taxa in the Lasthenia californica complex (Asteraceae: Heliantheae): an hypothesis of parallel evolution

Lasthenia californica sensu Ornduff consists of two races that differ in their flavonoid pigments and edaphic tolerances. Recent phylogenetic studies of Lasthenia have revealed that members of L. californica sensu Ornduff belong to two phylogenetic species. The relationship of the edaphic races to these new species and to each other is the focus of this study. Characterization of flavonoid profiles and phylogenetic placement of 33 populations demonstrates that races and phylogenetic taxa are not concordant, suggesting that one or both edaphic races evolved in parallel in the two clades. We hypothesize an edaphically linked ecological role for flavonoid differences that first revealed the existence of two races.     

Conformational changes in the multidrug transporter EmrE associated with substrate binding

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP(+)) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP(+) binding; the data showed that one TPP(+) molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP(+) produced well ordered two-dimensional crystals. Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7A resolution. An average native EmrE projection structure was calculated from the c222 and p222(1) crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP(+) bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPP(+) bound at the monomer-monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane alpha-helix.     

B cell deficiency confers protection from renal ischemia reperfusion injury

Recent data have demonstrated a role for CD4(+) cells in the pathogenesis of renal ischemia reperfusion injury (IRI). Identifying engagement of adaptive immune cells in IRI suggests that the other major cell of the adaptive immune response, B cells, may also mediate renal IRI. An established model of renal IRI was used: 30 min of renal pedicle clamping was followed by reperfusion in B cell-deficient ( mu MT) and wild-type mice. Renal function was significantly improved in mu MT mice compared with wild-type mice at 24, 48, and 72 h postischemia. mu MT mice also had significantly reduced tubular injury. Both groups of mice had similar renal phagocyte infiltration postischemia assessed by myeloperoxidase levels and similar levels of CD4(+) T cell infiltration postischemia. Peritubular complement C3d staining was also similar in both groups. To identify the contribution of cellular vs soluble mechanism of action, serum transfer into mu MT mice partially restored ischemic phenotype, but B cell transfers did not. These data are the first demonstration of a pathogenic role for B cells in ischemic acute renal failure, with a serum factor as a potential underlying mechanism of action.     

NF-kappa B hyperactivation has differential effects on the APC function of nonobese diabetic mouse macrophages

Type 1 diabetes is characterized by a chronic inflammatory response resulting in the selective destruction of the insulin-producing beta cells. We have previously demonstrated that dendritic cells (DCs) prepared from nonobese diabetic (NOD) mice, a model for spontaneous type 1 diabetes, exhibit hyperactivation of NF-kappaB resulting in an increased capacity to secrete proinflammatory cytokines and stimulate T cells compared with DCs of nondiabetic strains of mice. In the current study, the activational status of NF-kappaB and its role in regulating the APC function of macrophages (Mphi) prepared from NOD, nonobese resistant (NOR), and BALB/c mice was investigated. Independent of the stimulus, splenic and bone marrow-derived Mphi prepared from NOD mice exhibited increased NF-kappaB activation relative to NOR and BALB/c Mphi. This hyperactivation was detected for different NF-kappaB complexes and correlated with increased IkappaBalpha degradation. Furthermore, increased NF-kappaB activation resulted in an enhanced capacity of NOD vs NOR or BALB/c Mphi to secrete IL-12(p70), TNF-alpha, and IL-1alpha, which was inhibited upon infection with an adenoviral recombinant encoding a modified form of IkappaBalpha. In contrast, elevated NF-kappaB activation had no significant effect on the capacity of NOD Mphi to stimulate CD4(+) or CD8(+) T cells in an Ag-specific manner. These results demonstrate that in addition to NOD DCs, NOD Mphi exhibit hyperactivation of NF-kappaB, which correlates with an increased ability to mediate a proinflammatory response. Furthermore, NF-kappaB influences Mphi APC function by regulating cytokine secretion but not T cell stimulation.     

Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B

The inhibitors okadaic acid (OA), fostriecin (FOS) and cyclosporin A (CsA), were used to investigate the roles of protein phosphatases in regulating exocytosis in rat brain synaptosomes by measuring glutamate release and the release of the styryl dye FM 2-10. Depolarization was induced by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA and FOS produced a similar partial inhibition of KCl- and 0.3 mM 4AP- evoked exocytosis in both assays, but had little effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA had no effect upon KCl- and 0.3 mM 4AP-evoked exocytosis, but significantly enhanced glutamate release but not FM 2-10 dye release evoked by 1 mM 4AP. None of the phosphatase inhibitors changed calcium signals from FURA-2-loaded synaptosomes either before or after depolarization. Pretreatment with 100 nM phorbol 12-myristate 13-acetate abolished the inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP. Taken together, these results show that exocytosis from synaptosomes has a phosphatase-sensitive and phosphatase-insensitive component, and that there are two modes of phosphatase-sensitive exocytosis that can be elicited by different depolarization conditions. Moreover, these two modes are differentially sensitive to phosphatase 2A and 2B.     

Circulation of parasites among fishes from lakes in the Caribou Mountains,Alberta, Canada

Circulation of 24 macroparasite species among 12 species of fish was evaluated within samples of hosts collected from 9 lakes on an isolated plateau in northern Alberta, Canada. Twenty-seven parasite taxa (24 species plus the larval stages of Triaenophorus crassus, T. stizostedionis, and Raphidascaris acus) had the potential to be circulated among hosts. Sixteen parasite taxa were recovered from a single host species within a lake. Of the 11 remaining nonspecialist taxa, 4 were larval stages that matured in fish or birds and 7 were adults. Eight of the 11 cases of circulation among hosts involved lake whitefish, and this host was involved in the transmission of 5 species to piscivorous fishes. Despite evidence for the circulation of 7 taxa among the 4 species of sympatric Salmonidae, 60-99% of all worms were recovered from just 1 species of host. These results indicated that approximately 60% of the parasite taxa that infected fish in these lakes were absolute host specialists. The remaining 40% of parasite taxa had restricted host ranges, with most examples of parasite circulation limited to the 2 species of sympatric coregonid.     

Mismatch uracil glycosylase from Escherichia coli: a general mismatch or a specific DNA glycosylase?

The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U.G mismatches. Subsequently, 3,N4-ethenocytosine (epsilonC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme. The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme. Our results demonstrate that product release by the enzyme is very slow and hence comparing the "steady-state" parameters of the enzyme for different substrates is of limited use. Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions. Although the enzyme excised both epsilonC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis. Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate. This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism.     

Characterization of the heparin binding sites in human apolipoprotein E

Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the C-terminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pK(a) values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan.