Onset of pulsatile pressure causes transiently increased filtration through artery wall

Convective fluid motion through artery walls aids in the transvascular transport of macromolecules. Although many measurements of convective filtration have been reported, they were all obtained under constant transmural pressure. However, arterial pressure in vivo is pulsatile. Therefore, experiments were designed to compare filtration under steady and pulsatile pressure conditions. Rabbit carotid arteries were cannulated and excised from male New Zealand White rabbits anesthetized with pentobarbitol sodium (30 mg/kg i.v. administered). Hydraulic conductance was measured in cannulated excised rabbit carotid arteries at steady pressure. Next, pulsatile pressure trains were applied within the same vessels, and, simultaneously, arterial distension was monitored using Optical coherence tomography (OCT). For each pulse train, the volume of fluid lost through filtration was measured (subtracting volume change due to residual distension) and compared with that predicted from steady pressure measurements. At 60- and 80-mmHg baseline pressures, the experimental filtration volumes were significantly increased compared with those predicted for steady pressure (P < 0.05). OCT demonstrated that the excess fluid volume loss was significantly greater than the volume that would be lost through residual distension (P < 0.05). After 30 s, the magnitude of the excess of fluid loss was reduced. These results suggest that sudden onset of pulsatile pressure may cause changes in arterial interstitial hydration.     

Nicotine's Defensive Function in Nature

Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D₄-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.     

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.     

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.     

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.     

Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M?ssbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.     

Genetic issues in aquatic species management: the shortnose sturgeon (Acipenser brevirostrum) in the southeastern United States

Anadromous species occupy multiple freshwater,estuarine, and marine habitats, which posesspecial challenges in wildlife management. Inparticular, the level of immigration betweendrainages can be a critical factor in thedefinition of management units and the designof population-specific stocking programs. Thesemi-anadromous shortnose sturgeon (Acipenser brevirostrum) is listed as anendangered species under the Endangered SpeciesAct in the United States. To assess populationstructure in this species as a guide toeffective management, tissue samples werecollected from adult specimens (N = 198) fromfive river systems in the southeastern U.S.,and augmented with a sample from New Brunswick,Canada (N = 13), the extreme northern end ofthe species' range. Comparisons of mtDNAcontrol region sequences reveal a shallow genegenealogy and modest, but significant,population structuring (_st = 4.3%or _st = 17.7% when Canadian samplesare included). Populations inhabiting riversystems in the southeastern U.S. are closelyrelated, a pattern consistent with more recentdivergences along evolutionary timeframes. Haplotype diversity is moderate to high in mostdrainages (h = 0.383–1.000), exceptfor the Savannah and Edisto rivers, indicatingthat historical levels of mtDNA diversity mightbe largely intact outside of these drainages. Low mtDNA diversity in the Savannah andneighboring Edisto drainages might stem from anexperimental stocking effort during 1984–1992 that depressed overall genetic diversityin the Savannah and established or augmentedthe Edisto River population with a relativelylimited number of matrilines.     

Oxygen transport capacity in the air-breathing fish,Megalops cyprinoides: compensations for strenuous exercise

Tarpon have high resting or routine hematocrits (Hct) (37.6+/-3.4%) and hemoglobin concentrations (120.6+/-7.3 gl(-1)) that increased significantly following bouts of angling-induced exercise (51.9+/-3.7% and 142.8+/-13.5 gl(-1), respectively). Strenuous exercise was accompanied by an approximately tenfold increase in blood lactate and a muscle metabolite profile indicative of a high energy demand teleost. Routine blood values were quickly restored only when this facultative air-breathing fish was given access to atmospheric air. In vitro studies of oxygen transport capacity, a function of carrying capacity and viscosity, revealed that the optimal Hct range corresponded to that observed in fish under routine behaviour. During strenuous exercise however, further increase in viscosity was largely offset by a pronounced reduction in the shear-dependence of blood which conformed closely to an ideal Newtonian fluid. The mechanism for this behaviour of the erythrocytes appears to involve the activation of surface adrenergic receptors because pre-treatment with propranolol abolished the response. High levels of activity in tarpon living in hypoxic habitats are therefore supported by an elevated Hct with adrenergically mediated viscosity reduction, and air-breathing behaviour that enables rapid metabolic recovery.